Subgenomic replicons of the flavivirus Kunjin: construction and applications

Several Kunjin virus (KUN) subgenomic replicons containing large deletions in the structural region (C-prM-E) and in the 3' untranslated region (3'UTR) of the genome have been constructed. Replicon RNA Delta ME with 1,987 nucleotides deleted (from nucleotide 417 [in codon 108] in the C gene to nucleotide 2403 near the carboxy terminus of the E gene, inclusive) and replicon RNA C20rep with 2,247 nucleotides deleted (from nucleotide 157 [in codon 20] in C to nucleotide 2403) replicated efficiently in electroporated BHK21 cells. A further deletion from C20rep of 53 nucleotides, reducing the coding sequence in core protein to two codons (C2rep RNA), resulted in abolishment of RNA replication. Replicon Delta ME/76 with a deletion of 76 nucleotides in the 3'UTR of Delta ME RNA (nucleotides 10423 to 10498) replicated efficiently, whereas replicon Delta ME/352 with a larger deletion of 352 nucleotides (nucleotides 10423 to 10774), including two conserved sequences RCS3 and CS3, was significantly inhibited in RNA replication. To explore the possibility of using a reporter gene assay to monitor synthesis of the positive strand and the negative strand of KUN RNA, we inserted a chloramphenicol acetyltransferase (CAT) gene into the 3'UTR of Delta ME/76 RNA under control of the internal ribosomal entry site (IRES) of encephalomyelocarditis virus RNA in both plus (Delta ME/76CAT[+])- and minus (Delta ME/76CAT[-])-sense orientations. Although insertion of the IRES-CAT cassette in the plus-sense orientation resulted in a significant (10- to 20-fold) reduction of RNA replication compared to that of the parental Delta ME/76 RNA, CAT expression was readily detected in electroporated BHK cells. No CAT expression was detected after electroporation of RNA containing the IRES-CAT cassette inserted in the minus-sense orientation despite its apparently more efficient replication (similar to that of Delta ME/76 RNA); this result indicated that KUN negative-strand RNA was probably not released from its template after synthesis. Replacement of the CAT gene in the Delta ME/76CAT(+) RNA with the neomycin gene (Neo) enabled selection and recovery of a BHK cell culture in which the majority of cells were continuously expressing the replicon RNA for 41 days (nine passages) without apparent cytopathic effect. The constructed KUN replicons should provide valuable tools to study flavivirus RNA replication as well as providing possible vectors for a long-lasting and noncytopathic RNA virus expression system

Landscape preservation under post‐European settlement alluvium in the south‐eastern Australian tablelands, inferred from portable OSL reader data

Human land‐use changes leading to widespread erosion and gully incision have been well studied, but the effects that erosion and sediment mixing, which accompany the deposition of post‐(European) settlement alluvium (PSA), have in valley bottoms and wetlands receive considerably less attention. PSA overlying pre‐disturbance swampy meadow (SM) wetland sediments is commonly exposed along incised stream channel gully walls throughout the south‐eastern Australian Tablelands, providing an ideal setting in which to assess and understand better how PSA deposition affects valley bottoms and the wetland environments that often occupy them. Portable optically stimulated luminescence (pOSL) reader data were measured on bulk sediment samples from SM‐PSA stratigraphies at 16 locations throughout the south‐eastern Australian Tablelands to assess the effects of erosion and sediment mixing at the SM‐PSA boundary. Trends of pOSL data with depth at each profile were used in conjunction with visual profile descriptions to identify the stratigraphic boundary between SM and PSA sediment and to infer the degree of valley bottom erosion and sediment mixing during PSA deposition. At most sites, SM sediments experienced minimal, if any, disturbance during PSA deposition, and we refer to these as non‐eroded sites. Many sites, however, experienced a significant degree of erosion and sediment mixing – eroded sites – often corresponding to visually diffuse sedimentary boundaries between the two stratigraphic units. Our findings demonstrate that SM landscapes in the Tablelands can be preserved with minimal disturbance under PSA at non‐eroded sites and are preserved beneath a mixing zone at all eroded sites. Copyright © 2016 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134129/1/esp3942.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134129/2/esp3942_am.pd

Insights Into Aboriginal Australian Mortuary Practices: Perspectives From Ancient DNA

Paleogenetics is a relatively new and promising field that has the potential to provide new information about past Indigenous social systems, including insights into the complexity of burial practices. We present results of the first ancient DNA (aDNA) investigation into traditional mortuary practices among Australian Aboriginal people with a focus on North-East Australia. We recovered mitochondrial and Y chromosome sequences from five ancestral Aboriginal Australian remains that were excavated from the Flinders Island group in Cape York, Queensland. Two of these individuals were sampled from disturbed beach burials, while the other three were from bundle burials located in rock shelters. Genomic analyses showed that individuals from all three rock shelter burials and one of the two beach burials had a close genealogical relationship to contemporary individuals from communities from Cape York. In contrast the remaining male individual, found buried on the beach, had a mitochondrial DNA sequence that suggested that he was not from this location but that he was closely related to people from central Queensland or New South Wales. In addition, this individual was associated with a distinctive burial practice to the other four people. It has been suggested that traditionally non-locals or lower status individuals were buried on beaches. Our findings suggest that theories put forward about beach burials being non-local, or less esteemed members of the community, can potentially be resolved through analyses of uniparental genomic data. Generally, these results support the suggestion often derived from ethnohistoric accounts that inequality in Indigenous Australian mortuary practices might be based on the status, sex, and/or age of individuals and may instead relate to place of geographic origin. There is, however, some departure from the traditional ethnohistoric account in that complex mortuary internments were also offered to female individuals of the community, with genomic analyses helping to confirm that the gender of one of the rockshelter internments was that of a young female

Assignment of the Human and Mouse Prion Protein Genes to Homologous Chromosomes

Purified preparations of scrapie prions contain one major macromolecule, designated prion protein (PrP). Genes encoding PrP are found in normal animals and humans but not within the infectious particles. The PrP gene was assigned to human chromosome 20 and the corresponding mouse chromosome 2 using somatic cell hybrids. In situ hybridization studies mapped the human PrP gene to band 20p12→pter. Our results should lead to studies of genetic loci syntenic with the PrP gene, which may play a role in the pathogenesis of prion diseases or other degenerative neurologic disorders

Understanding communication pathways to foster community engagement for health improvement in North West Pakistan

Background: This paper describes the community engagement process undertaken to ascertain the focus, development and implementation of an intervention to improve iodised salt consumption in rural communities in North West Pakistan. The Jirga is a traditional informal structure, which gathers men respected within their community and acts in a governing and decision making capacity in the Pukhtoon culture. The Jirga system had a dual purpose for the study; to access men from the community to discuss the importance of iodised salt, and as an engagement process for the intervention. Methods: A number of qualitative data collection activities were undertaken, with Jirga members and their wives, male and female outreach workers and two groups of women, under and over forty years old. The aim of these were to highlight the communication channels and levers of influence on health behaviour, which were multiple and complex and all needed to be taken into consideration in order to ensure successful and locally sensitive community engagement. Results: Communication channels are described within local families and the communities around them. The key influential role of the Jirga is highlighted as linked both to the standing of its members and the community cohesion ethos that it embodies. Engaging Jirga members in discussions about iodised salt was key in designing an intervention that would activate the most influential levers to decision making in the community. Gendered decision making-processes within the household have been highlighted as restricting women’s autonomy. Whilst in one respect our data confirm this, a more complex hierarchy of decisional power has been highlighted, whereby the concept of ‘wisdom’, an amalgamation of age, experience and education, presents important possibilities. Community members with the least autonomy are the youngest uneducated females, who rely on a web of socially and culturally determined ways to influence decision-making. Conclusions: The major lines of communication and influence in the local community described are placed within the wider literature on community engagement in health improvement. The process of maximisation of local cultural knowledge as part of a community engagement effort is one that has application well beyond the particular setting of this study

Phenotypic heterogeneity and genetic modification of P102L inherited prion disease in an international series

The largest kindred with inherited prion disease P102L, historically Gerstmann-Sträussler-Scheinker syndrome, originates from central England, with émigrés now resident in various parts of the English-speaking world. We have collected data from 84 patients in the large UK kindred and numerous small unrelated pedigrees to investigate phenotypic heterogeneity and modifying factors. This collection represents by far the largest series of P102L patients so far reported. Microsatellite and genealogical analyses of eight separate European kindreds support multiple distinct mutational events at a cytosine-phosphate diester-guanidine dinucleotide mutation hot spot. All of the smaller P102L kindreds were linked to polymorphic human prion protein gene codon 129M and were not connected by genealogy or microsatellite haplotype background to the large kindred or each other. While many present with classical Gerstmann-Sträussler-Scheinker syndrome, a slowly progressive cerebellar ataxia with later onset cognitive impairment, there is remarkable heterogeneity. A subset of patients present with prominent cognitive and psychiatric features and some have met diagnostic criteria for sporadic Creutzfeldt-Jakob disease. We show that polymorphic human prion protein gene codon 129 modifies age at onset: the earliest eight clinical onsets were all MM homozygotes and overall age at onset was 7 years earlier for MM compared with MV heterozygotes (P = 0.02). Unexpectedly, apolipoprotein E4 carriers have a delayed age of onset by 10 years (P = 0.02). We found a preponderance of female patients compared with males (54 females versus 30 males, P = 0.01), which probably relates to ascertainment bias. However, these modifiers had no impact on a semi-quantitative pathological phenotype in 10 autopsied patients. These data allow an appreciation of the range of clinical phenotype, modern imaging and molecular investigation and should inform genetic counselling of at-risk individuals, with the identification of two genetic modifiers

Two decades of numerical modelling to understand long term fluvial archives: Advances and future perspectives

The development and application of numerical models to investigate fluvial sedimentary archives has increased during the last decades resulting in a sustained growth in the number of scientific publications with keywords, 'fluvial models', 'fluvial process models' and 'fluvial numerical models'. In this context we compile and review the current contributions of numerical modelling to the understanding of fluvial archives. In particular, recent advances, current limitations, previous unexpected results and future perspectives are all discussed. Numerical modelling efforts have demonstrated that fluvial systems can display non-linear behaviour with often unexpected dynamics causing significant delay, amplification, attenuation or blurring of externally controlled signals in their simulated record. Numerical simulations have also demonstrated that fluvial records can be generated by intrinsic dynamics without any change in external controls. Many other model applications demonstrate that fluvial archives, specifically of large fluvial systems, can be convincingly simulated as a function of the interplay of (palaeo) landscape properties and extrinsic climate, base level and crustal controls. All discussed models can, after some calibration, produce believable matches with real world systems suggesting that equifinality - where a given end state can be reached through many different pathways starting from different initial conditions and physical assumptions - plays an important role in fluvial records and their modelling. The overall future challenge lies in the development of new methodologies for a more independent validation of system dynamics and research strategies that allow the separation of intrinsic and extrinsic record signals using combined fieldwork and modelling

Climate, people and faunal succession on Java, Indonesia: evidence from Song Gupuh

Song Gupuh, a partially collapsed cave in the Gunung Sewu Limestones of East Java, Indonesia, contains over 16 m of deposits with a faunal sequence spanning some 70 ka. Major changes in the range of animals represented show the impact of climate change and humans. The Terminal Pleistocene and Early Holocene was a period of maximum biodiversity. Human use of Song Gupuh and other cave sites in the region also intensified significantly from ca. 12 ka, together with a new focus on exploitation of small-bodied species (macaque monkeys and molluscs), the first evidence for import of resources from the coast, and use of bone and shell tools. Human activity, especially after the onset of the Neolithic around 2.6 ka, subsequently contributed to a progressive loss of many species from the area, including tapir, elephant, Malayan bear, rhino and tiger, and this extinction process is continuing. We conclude by discussing the biogeographical significance of Song Gupuh in the context of other sites in Java (e.g. Punung, Wajak) and further afield (e.g. Liang Bua)

Acute dosing of latrepirdine (Dimebon), a possible Alzheimer therapeutic, elevates extracellular amyloid-beta levels in vitro and in vivo.

BACKGROUND: Recent reports suggest that latrepirdine (Dimebon, dimebolin), a retired Russian antihistamine, improves cognitive function in aged rodents and in patients with mild to moderate Alzheimer's disease (AD). However, the mechanism(s) underlying this benefit remain elusive. AD is characterized by extracellular accumulation of the amyloid-beta (Abeta) peptide in the brain, and Abeta-lowering drugs are currently among the most popular anti-amyloid agents under development for the treatment of AD. In the current study, we assessed the effect of acute dosing of latrepirdine on levels of extracellular Abeta using in vitro and in vivo experimental systems. RESULTS: We evaluated extracellular levels of Abeta in three experimental systems, under basal conditions and after treatment with latrepirdine. Mouse N2a neuroblastoma cells overexpressing Swedish APP were incubated for 6 hr in the presence of either vehicle or vehicle + latrepirdine (500pM-5 muM). Synaptoneurosomes were isolated from TgCRND8 mutant APP-overexpressing transgenic mice and incubated for 0 to 10 min in the absence or presence of latrepirdine (1 muM or 10 muM). Drug-naïve Tg2576 Swedish mutant APP overexpressing transgenic mice received a single intraperitoneal injection of either vehicle or vehicle + latrepirdine (3.5 mg/kg). Picomolar to nanomolar concentrations of acutely administered latrepirdine increased the extracellular concentration of Abeta in the conditioned media from Swedish mutant APP-overexpressing N2a cells by up to 64% (p = 0.01), while a clinically relevant acute dose of latrepirdine administered i.p. led to an increase in the interstitial fluid of freely moving APP transgenic mice by up to 40% (p = 0.01). Reconstitution of membrane protein trafficking and processing is frequently inefficient, and, consistent with this interpretation, latrepirdine treatment of isolated TgCRND8 synaptoneurosomes involved higher concentrations of drug (1-10 muM) and led to more modest increases in extracellular Abeta(x-42 )levels (+10%; p = 0.001); of note, however, was the observation that extracellular Abeta(x-40 )levels did not change. CONCLUSIONS: Here, we report the surprising association of acute latrepirdine dosing with elevated levels of extracellular Abeta as measured in three independent neuron-related or neuron-derived systems, including the hippocampus of freely moving Tg2576 mice. Given the reported association of chronic latrepirdine treatment with improvement in cognitive function, the effects of chronic latrepirdine treatment on extracellular Abeta levels must now be determined.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Dietary composition modulates brain mass and solubilizable Aβ levels in a mouse model of aggressive Alzheimer's amyloid pathology

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Objective Alzheimer's disease (AD) is a progressive neurodegenerative disease of the central nervous system (CNS). Recently, an increased interest in the role diet plays in the pathology of AD has resulted in a focus on the detrimental effects of diets high in cholesterol and fat and the beneficial effects of caloric restriction. The current study examines how dietary composition modulates cerebral amyloidosis and neuronal integrity in the TgCRND8 mouse model of AD. Methods From 4 wks until 18 wks of age, male and female TgCRND8 mice were maintained on one of four diets: (1) reference (regular) commercial chow; (2) high fat/low carbohydrate custom chow (60 kcal% fat/30 kcal% protein/10 kcal% carbohydrate); (3) high protein/low carbohydrate custom chow (60 kcal% protein/30 kcal% fat/10 kcal% carbohydrate); or (4) high carbohydrate/low fat custom chow (60 kcal% carbohydrate/30 kcal% protein/10 kcal% fat). At age 18 wks, mice were sacrificed, and brains studied for (a) wet weight; (b) solubilizable Aβ content by ELISA; (c) amyloid plaque burden; (d) stereologic analysis of selected hippocampal subregions. Results Animals receiving a high fat diet showed increased brain levels of solubilizable Aβ, although we detected no effect on plaque burden. Unexpectedly, brains of mice fed a high protein/low carbohydrate diet were 5% lower in weight than brains from all other mice. In an effort to identify regions that might link loss of brain mass to cognitive function, we studied neuronal density and volume in hippocampal subregions. Neuronal density and volume in the hippocampal CA3 region of TgCRND8 mice tended to be lower in TgCRND8 mice receiving the high protein/low carbohydrate diet than in those receiving the regular chow. Neuronal density and volume were preserved in CA1 and in the dentate gyrus. Interpretation Dissociation of Aβ changes from brain mass changes raises the possibility that diet plays a role not only in modulating amyloidosis but also in modulating neuronal vulnerability. However, in the absence of a study of the effects of a high protein/low carbohydrate diet on nontransgenic mice, one cannot be certain how much, if any, of the loss of brain mass exhibited by high protein/low carbohydrate diet-fed TgCRND8 mice was due to an interaction between cerebral amyloidosis and diet. Given the recent evidence that certain factors favor the maintenance of cognitive function in the face of substantial structural neuropathology, we propose that there might also exist factors that sensitize brain neurons to some forms of neurotoxicity, including, perhaps, amyloid neurotoxicity. Identification of these factors could help reconcile the poor clinicopathological correlation between cognitive status and structural neuropathology, including amyloid pathology.Published versio