Formation and Toxicity of Soluble Polyglutamine Oligomers in Living Cells

Aggregation and cytotoxicity of mutant proteins containing an expanded number of polyglutamine (polyQ) repeats is a hallmark of several diseases, including Huntington's disease (HD). Within cells, mutant Huntingtin (mHtt) and other polyglutamine expansion mutant proteins exist as monomers, soluble oligomers, and insoluble inclusion bodies (IBs). Determining which of these forms constitute a toxic species has proven difficult. Recent studies support a role for IBs as a cellular coping mechanism to sequester levels of potentially toxic soluble monomeric and oligomeric species of mHtt.When fused to a fluorescent reporter (GFP) and expressed in cells, the soluble monomeric and oligomeric polyglutamine species are visually indistinguishable. Here, we describe two complementary biophysical fluorescence microscopy techniques to directly detect soluble polyglutamine oligomers (using Htt exon 1 or Htt(ex1)) and monitor their fates in live cells. Photobleaching analyses revealed a significant reduction in the mobilities of mHtt(ex1) variants consistent with their incorporation into soluble microcomplexes. Similarly, when fused to split-GFP constructs, both wildtype and mHtt(ex1) formed oligomers, as evidenced by the formation of a fluorescent reporter. Only the mHtt(ex1) split-GFP oligomers assembled into IBs. Both FRAP and split-GFP approaches confirmed the ability of mHtt(ex1) to bind and incorporate wildtype Htt into soluble oligomers. We exploited the irreversible binding of split-GFP fragments to forcibly increase levels of soluble oligomeric mHtt(ex1). A corresponding increase in the rate of IBs formation and the number formed was observed. Importantly, higher levels of soluble mHtt(ex1) oligomers significantly correlated with increased mutant cytotoxicity, independent of the presence of IBs.Our study describes powerful and sensitive tools for investigating soluble oligomeric forms of expanded polyglutamine proteins, and their impact on cell viability. Moreover, these methods should be applicable for the detection of soluble oligomers of a wide variety of aggregation prone proteins

Single-Cell Census of Mechanosensitive Channels in Living Bacteria

Bacteria are subjected to a host of different environmental stresses. One such insult occurs when cells encounter changes in the osmolarity of the surrounding media resulting in an osmotic shock. In recent years, a great deal has been learned about mechanosensitive (MS) channels which are thought to provide osmoprotection in these circumstances by opening emergency release valves in response to membrane tension. However, even the most elementary physiological parameters such as the number of MS channels per cell, how MS channel expression levels influence the physiological response of the cells, and how this mean number of channels varies from cell to cell remain unanswered. In this paper, we make a detailed quantitative study of the expression of the mechanosensitive channel of large conductance (MscL) in different media and at various stages in the growth history of bacterial cultures. Using both quantitative fluorescence microscopy and quantitative Western blots our study complements earlier electrophysiology-based estimates and results in the following key insights: i) the mean number of channels per cell is much higher than previously estimated, ii) measurement of the single-cell distributions of such channels reveals marked variability from cell to cell and iii) the mean number of channels varies under different environmental conditions. The regulation of MscL expression displays rich behaviors that depend strongly on culturing conditions and stress factors, which may give clues to the physiological role of MscL. The number of stress-induced MscL channels and the associated variability have far reaching implications for the in vivo response of the channels and for modeling of this response. As shown by numerous biophysical models, both the number of such channels and their variability can impact many physiological processes including osmoprotection, channel gating probability, and channel clustering

Gender Nonconformity During Adolescence:Links with Stigma, Sexual Minority Status, and Psychosocial Outcomes

Both gender nonconformity and sexual minority status during adolescence are associated with elevated levels of victimization and harassment, experiences that have serious consequences for adolescent psychosocial outcomes. While gender nonconformity and sexual minority status reflect separate constructs, they are associated because (1) sexual minority youth report higher levels of gender nonconformity and (2) gender nonconformity is frequently used to attribute sexual minority status by others. Following from classic stigma theory, the current chapter focuses on the role of gender nonconformity in explaining variation in social exclusion and victimization among both sexual minority and sexual majority youth. Of particular interest is the potential for gender nonconformity to mediate or moderate the association between sexual minority status and individual mental health and wellbeing outcomes. Gender differences will also be discussed, focusing on differences between girls and boys in the links between sexual minority status, gender nonconformity, experiences of victimization, and negative psychosocial outcomes. Additionally, the emerging literature on conceptualizing gender nonconformity among trans and non-binary youth will be addressed. Finally, the current chapter will finish with a discussion of how and why gender nonconformity must be taken into consideration in the development of programs aimed at reducing homophobia among adolescent populations

Topology of molecular machines of the endoplasmic reticulum: a compilation of proteomics and cytological data

The endoplasmic reticulum (ER) is a key organelle of the secretion pathway involved in the synthesis of both proteins and lipids destined for multiple sites within and without the cell. The ER functions to both co- and post-translationally modify newly synthesized proteins and lipids and sort them for housekeeping within the ER and for transport to their sites of function away from the ER. In addition, the ER is involved in the metabolism and degradation of specific xenobiotics and endogenous biosynthetic products. A variety of proteomics studies have been reported on different subcompartments of the ER providing an ER protein dictionary with new data being made available on many protein complexes of relevance to the biology of the ER including the ribosome, the translocon, coatomer proteins, cytoskeletal proteins, folding proteins, the antigen-processing machinery, signaling proteins and proteins involved in membrane traffic. This review examines proteomics and cytological data in support of the presence of specific molecular machines at specific sites or subcompartments of the ER

An ecological future for weed science to sustain crop production and the environment. A review

Sustainable strategies for managing weeds are critical to meeting agriculture's potential to feed the world's population while conserving the ecosystems and biodiversity on which we depend. The dominant paradigm of weed management in developed countries is currently founded on the two principal tools of herbicides and tillage to remove weeds. However, evidence of negative environmental impacts from both tools is growing, and herbicide resistance is increasingly prevalent. These challenges emerge from a lack of attention to how weeds interact with and are regulated by the agroecosystem as a whole. Novel technological tools proposed for weed control, such as new herbicides, gene editing, and seed destructors, do not address these systemic challenges and thus are unlikely to provide truly sustainable solutions. Combining multiple tools and techniques in an Integrated Weed Management strategy is a step forward, but many integrated strategies still remain overly reliant on too few tools. In contrast, advances in weed ecology are revealing a wealth of options to manage weedsat the agroecosystem levelthat, rather than aiming to eradicate weeds, act to regulate populations to limit their negative impacts while conserving diversity. Here, we review the current state of knowledge in weed ecology and identify how this can be translated into practical weed management. The major points are the following: (1) the diversity and type of crops, management actions and limiting resources can be manipulated to limit weed competitiveness while promoting weed diversity; (2) in contrast to technological tools, ecological approaches to weed management tend to be synergistic with other agroecosystem functions; and (3) there are many existing practices compatible with this approach that could be integrated into current systems, alongside new options to explore. Overall, this review demonstrates that integrating systems-level ecological thinking into agronomic decision-making offers the best route to achieving sustainable weed management

A Participatory Approach to Assessing the Climate-Smartness of Agricultural Interventions: The Lushoto Case

The concept of climate-smart agriculture (CSA) is gaining momentum across the globe. However, it is not specific on what should be covered under its three pillars—productivity, resilience and mitigation. Consequently, CSA encompasses many different agricultural practices/technologies, making it difficult to prioritise CSA objectives. Firstly, there is a lack of clear and workable criteria as well as methods for assessing the climate-smartness of interventions. Secondly, little information exists about the impact of the various interventions already promoted as CSA, especially in the developing world. Finally, CSA prioritisation does not take into account stakeholders’ perspectives to ensure that the interventions are applicable, suitable and of high adoption-potential. Here, we describe a new participatory protocol for assessing the climate-smartness of agricultural interventions in smallholder practices. This identifies farm-level indicators (and indices) for the food security and adaptation pillars of CSA. It also supports the participatory scoring of indicators, enabling baseline and future assessments of climate-smartness to be made. The protocol was tested among 72 farmers implementing a variety of CSA interventions in the climate-smart village of Lushoto, Tanzania. Farmers especially valued interventions that improved soil fertility and structure, reduced surface runoff, and reclaimed degraded land due to the positive impacts on yield and off-season crop agriculture. Mostly, the CSA interventions increased animal production, food production, consumption and income. The protocol is easy to adapt to different regions and farming systems and allows for the better prioritisation of interventions. But we recommend that CSA is adopted as part of a monitoring, evaluation and learning process