Proteostasis and movement disorders: Parkinson's disease and amyotrophic lateral sclerosis

Parkinson's disease (PD) is a movement disorder that afflicts over one million in the U.S.; amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) is less prevalent but also has a high incidence. The two disorders sometimes present together, making a comparative study of interest. Both ALS and PD are neurodegenerative diseases, and are characterized by the presence of intraneuronal inclusions; however, different classes of neurons are affected and the primary protein in the inclusions differs between the diseases, and in some cases is different in distinct forms of the same disease. These observations might suggest that the more general approach of proteostasis pathway alteration would be a powerful one in treating these disorders. Examining results from human genetics and studies in model organisms, as well as from biochemical and biophysical characterization of the proteins involved in both diseases, we find that most instances of PD can be considered as arising from the misfolding, and self-association to a toxic species, of the small neuronal protein α-synuclein, and that proteostasis strategies are likely to be of value for this disorder. For ALS, the situation is much more complex and less clear-cut; the available data are most consistent with a view that ALS may actually be a family of disorders, presenting similarly but arising from distinct and nonoverlapping causes, including mislocalization of some properly folded proteins and derangement of RNA quality control pathways. Applying proteostasis approaches to this disease may require rethinking or broadening the concept of what proteostasis means.     

White to brown fat phenotypic switch induced by genetic and environmental activation of a hypothalamic-adipocyte axis

Living in an enriched environment with complex physical and social stimulation leads to improved cognitive and metabolic health. In white fat, enrichment induced the upregulation of the brown fat cell fate determining gene Prdm16, brown fat-specific markers, and genes involved in thermogenesis and β-adrenergic signaling. Moreover, pockets of cells with prototypical brown fat morphology and high UCP1 levels were observed in the white fat of enriched mice associated with resistance to diet-induced obesity. Hypothalamic overexpression of BDNF reproduced the enrichment-associated activation of the brown fat gene program and lean phenotype. Inhibition of BDNF signaling by genetic knockout or dominant-negative trkB reversed this phenotype. Our genetic and pharmacologic data suggest a mechanism whereby induction of hypothalamic BDNF expression in response to environmental stimuli leads to selective sympathoneural modulation of white fat to induce "browning" and increased energy dissipation.     

Improvement of a Potential Anthrax Therapeutic by Computational Protein Design

Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.     

There is no trade-off between speed and force in a dynamic lever system

Lever systems within a skeleton transmit force with a capacity determined by the mechanical advantage, A. A is the distance from input force to a joint, divided by the distance from the joint to the output force. A lever with a relatively high A in static equilibrium has a great capacity to generate force but moves a load over a small distance. Therefore, the geometry of a skeletal lever presents a trade-off between force and speed under quasi-static conditions. The present study considers skeletal dynamics that do not assume static equilibrium by modelling kicking by a locust leg, which is powered by stored elastic energy. This model predicts that the output force of this lever is proportional to A, but its maximum speed is independent of A. Therefore, no trade-off between force and velocity exists in a lever system with spring-mass dynamics. This demonstrates that the motion of a skeleton depends on the major forces that govern its dynamics and cannot be inferred from skeletal geometry alone.     

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids ¹. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes ². Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 10⁸ TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line ^{3, 4}. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.     

Age distribution of cases of 2009 (H1N1) pandemic influenza in comparison with seasonal influenza

Introduction

Several aspects of the epidemiology of 2009 (H1N1) pandemic influenza have not been accurately determined. We sought to study whether the age distribution of cases differs in comparison with seasonal influenza.

Methods

We searched for official, publicly available data through the internet from different countries worldwide on the age distribution of cases of influenza during the 2009 (H1N1) pandemic influenza period and most recent seasonal influenza periods. Data had to be recorded through the same surveillance system for both compared periods.

Results

For 2009 pandemic influenza versus recent influenza seasons, in USA, visits for influenza-like illness to sentinel providers were more likely to involve the age groups of 5–24, 25–64 and 0–4 years compared with the reference group of >64 years [odds ratio (OR) (95% confidence interval (CI)): 2.43 (2.39–2.47), 1.66 (1.64–1.69), and 1.51 (1.48–1.54), respectively]. Pediatric deaths were less likely in the age groups of 2–4 and <2 years than the reference group of 5–17 years [OR (95% CI): 0.46 (0.25–0.85) and 0.49 (0.30–0.81), respectively]. In Australia, notifications for laboratory-confirmed influenza were more likely in the age groups of 10–19, 5–9, 20–44, 45–64 and 0–4 years than the reference group of >65 years [OR (95% CI): 7.19 (6.67–7.75), 5.33 (4.90–5.79), 5.04 (4.70–5.41), 3.12 (2.89–3.36) and 1.89 (1.75–2.05), respectively]. In New Zealand, consultations for influenza-like illness by sentinel providers were more likely in the age groups of <1, 1–4, 35–49, 5–19, 20–34 and 50–64 years than the reference group of >65 years [OR (95% CI): 2.38 (1.74–3.26), 1.99 (1.62–2.45), 1.57 (1.30–1.89), 1.57 (1.30–1.88), 1.40 (1.17–1.69) and 1.39 (1.14–1.70), respectively].

Conclusions

The greatest increase in influenza cases during 2009 (H1N1) pandemic influenza period, in comparison with most recent seasonal influenza periods, was seen for school-aged children, adolescents, and younger adults.     

Bacterial Communities Associated with the Leaves and the Roots of Arabidopsis thaliana

Diverse communities of bacteria inhabit plant leaves and roots and those bacteria play a crucial role for plant health and growth. Arabidopsis thaliana is an important model to study plant pathogen interactions, but little is known about its associated bacterial community under natural conditions. We used 454 pyrosequencing to characterize the bacterial communities associated with the roots and the leaves of wild A. thaliana collected at 4 sites; we further compared communities on the outside of the plants with communities in the endophytic compartments. We found that the most heavily sequenced bacteria in A. thaliana associated community are related to culturable species. Proteobacteria, Actinobacteria, and Bacteroidetes are the most abundant phyla in both leaf and root samples. At the genus level, sequences of Massilia and Flavobacterium are prevalent in both samples. Organ (leaf vs root) and habitat (epiphytes vs endophytes) structure the community. In the roots, richness is higher in the epiphytic communities compared to the endophytic compartment (P = 0.024), while the reverse is true for the leaves (P = 0.032). Interestingly, leaf and root endophytic compartments do not differ in richness, diversity and evenness, while they differ in community composition (P = 0.001). The results show that although the communities associated with leaves and roots share many bacterial species, the associated communities differ in structure.     

Bayesian spatial modeling of moose count data: increasing estimator efficiency and exploring ecological hypotheses

Moose management throughout much of Alaska and Canada relies on aerial count data, which are commonly collected using the geospatial population estimator (GSPE) protocol. The GSPE uses a model-based analytical approach and finite-population block kriging to estimate abundance from a collection of sampled survey units. Widespread implementation and well-documented analytical software have resulted in reliable estimates of moose abundance, density, and composition across a large proportion of their range. Analysis is conducted almost exclusively using the GSPE software, which fits a fixed model structure to data collected within a single year. The downside of this approach to analysis is that the fixed model structure is inefficient for estimation, leading to more field effort than would otherwise be necessary to achieve a desired level of estimator precision. We developed a more easily modified and flexible Bayesian spatial general additive model approach (BSG) that accommodates spatial and temporal covariates (e.g., habitat characteristics, trend), multiple survey events, prior information, and incomplete detection. Using a series of 6 GSPE surveys conducted in Yukon-Charley Rivers National Preserve, Alaska, USA, from 2003–2019, we established the equivalence of the 2 approaches under similar model structures. We then extended the BSG to demonstrate how a more comprehensive approach to analysis can affect estimator precision and be used to assess ecological relationships. The precision of annual abundance estimators from the BSG were improved by an average of 43% over those based on the standard GSPE analysis, highlighting the very real costs of assuming a fixed (i.e., suboptimal) model structure. The population increased at a rate of 2.3%/year (95% CrI = 0.8–3.8%), and the increase was largely explained by a parallel increase in wildfire extent (i.e., high quality moose habitat). These results suggest that our approach could be used to increase estimator efficiency or decrease future survey costs without any modifications to the basic protocol. While modification of the GSPE software is possible, practitioners may find the BSG approach more convenient for quickly developing model structures for a particular application, thereby allowing them to extract more information from existing and future datasets.