A Genome-Wide Characterization of MicroRNA Genes in Maize

MicroRNAs (miRNAs) are small, non-coding RNAs that play essential roles in plant growth, development, and stress response. We conducted a genome-wide survey of maize miRNA genes, characterizing their structure, expression, and evolution. Computational approaches based on homology and secondary structure modeling identified 150 high-confidence genes within 26 miRNA families. For 25 families, expression was verified by deep-sequencing of small RNA libraries that were prepared from an assortment of maize tissues. PCR–RACE amplification of 68 miRNA transcript precursors, representing 18 families conserved across several plant species, showed that splice variation and the use of alternative transcriptional start and stop sites is common within this class of genes. Comparison of sequence variation data from diverse maize inbred lines versus teosinte accessions suggest that the mature miRNAs are under strong purifying selection while the flanking sequences evolve equivalently to other genes. Since maize is derived from an ancient tetraploid, the effect of whole-genome duplication on miRNA evolution was examined. We found that, like protein-coding genes, duplicated miRNA genes underwent extensive gene-loss, with ∼35% of ancestral sites retained as duplicate homoeologous miRNA genes. This number is higher than that observed with protein-coding genes. A search for putative miRNA targets indicated bias towards genes in regulatory and metabolic pathways. As maize is one of the principal models for plant growth and development, this study will serve as a foundation for future research into the functional roles of miRNA genes

The ABC130 barrel module prototyping programme for the ATLAS strip tracker

For the Phase-II Upgrade of the ATLAS Detector, its Inner Detector, consisting of silicon pixel, silicon strip and transition radiation sub-detectors, will be replaced with an all new 100 % silicon tracker, composed of a pixel tracker at inner radii and a strip tracker at outer radii. The future ATLAS strip tracker will include 11,000 silicon sensor modules in the central region (barrel) and 7,000 modules in the forward region (end-caps), which are foreseen to be constructed over a period of 3.5 years. The construction of each module consists of a series of assembly and quality control steps, which were engineered to be identical for all production sites. In order to develop the tooling and procedures for assembly and testing of these modules, two series of major prototyping programs were conducted: an early program using readout chips designed using a 250 nm fabrication process (ABCN-25) and a subsequent program using a follow-up chip set made using 130 nm processing (ABC130 and HCC130 chips). This second generation of readout chips was used for an extensive prototyping program that produced around 100 barrel-type modules and contributed significantly to the development of the final module layout. This paper gives an overview of the components used in ABC130 barrel modules, their assembly procedure and findings resulting from their tests.Comment: 82 pages, 66 figure

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Operation and performance of the ATLAS semiconductor tracker in LHC Run 2

The semiconductor tracker (SCT) is one of the tracking systems for charged particles in the ATLAS detector. It consists of 4088 silicon strip sensor modules. During Run 2 (2015–2018) the Large Hadron Collider delivered an integrated luminosity of 156 fb-1 to the ATLAS experiment at a centre-of-mass proton-proton collision energy of 13 TeV. The instantaneous luminosity and pile-up conditions were far in excess of those assumed in the original design of the SCT detector. Due to improvements to the data acquisition system, the SCT operated stably throughout Run 2. It was available for 99.9% of the integrated luminosity and achieved a data-quality efficiency of 99.85%. Detailed studies have been made of the leakage current in SCT modules and the evolution of the full depletion voltage, which are used to study the impact of radiation damage to the modules

Upregulation of HSPB8 as potential therapeutic approach in familial and sporadic ALS

Several data suggest that accumulation of aggregated proteins (mutated SOD1, TDP-43 and FUS/TLS) plays an important role in motor neuronal cell death occurring in Amyotrophic Lateral Sclerosis (ALS). Protein aggregation results from the formation of aberrant conformations (misfolding) of aggregate-prone proteins, some of which have been found mutated in the familial forms of ALS. The removal of misfolded (aggregated) proteins is operated by the cells via two major degradative systems the ubiquitin-proteasome pathway (UPP) and the autophagy. Both systems require the assistance of intracellular chaperons. The molecular chaperones recognize and bind misfolded proteins, preventing their aggregation and facilitating their degradation, thus exerting neuroprotective functions. In this project, we will focus on the chaperone HSPB8, which forms a stable complex with the co-chaperone Bag3. Overexpression of HSPB8 (and Bag3) prevents aggregation of mutated SOD1 and TDP-43, associated with familial and sporadic ALS, by increasing their degradation via autophagy, an essential process for aggregate-prone protein clearance and neuronal survival. Thus, HSPB8 (and Bag3) may help motor neurons to cope with misfolded TDP-43 and SOD1 by either directly targeting them to the autophagic vacuoles for degradation and/or restoring/boosting the autophagy flux. Interestingly, deregulated autophagy is amongst the causes for motor neuron diseases (MNDs), further pointing to a link between protein aggregation, protein quality control, HSPB8-Bag3 and autophagy. Besides, mislocalization/aggregation of TDP-43 and FUS/TLS to the mRNA containing cytoplasmic stress granules (SGs) alters RNA metabolism and has been suggested as pathomechanism contributing to ALS. Our preliminary data indicate that HSPB8 is recruited to SG, where it colocalizes with TDP-43. Therefore, HSPB8, by either preventing the mislocalization/aggregation of mutated TDP-43 and FUS to SG and/or by targeting them for degradation may also contribute to disease amelioration by avoiding impairment of specific RNA translation/processing. The hypothesis that HSPB8 may exert essential functions for motor neuron viability is further supported by the observation that HSPB8 is upregulated in surviving motor neurons in both an ALS mouse model and in human ALS tissues, as well as by the finding that mutated forms of HSPB8 (which are found in aggregates) cause dominant hereditary motor neuropathy. In this project, we will investigate the hypothesis that upregulation of HSPB8 (and Bag3) may protect against ALS, using both motor neuronal cells and the mutated SOD1, TDP-43 and FUS/TLS based Drosophila models of ALS. We will also perform a drug screening to find compounds able to induce HSPB8 expression specifically in motor neurons. This work will provide insights in the role of HSPB8 as modulator of ALS and will identify specific sites of action of HSPB8 whose modulation may also represent new therapeutic targets for ALS

Upregulation of HSPB8 as potential therapeutic approach in familial and sporadic ALS

Several data suggest that accumulation of aggregated proteins (mutated SOD1, TDP-43 and FUS/TLS) plays an important role in motor neuronal cell death occurring in Amyotrophic Lateral Sclerosis (ALS). Protein aggregation results from the formation of aberrant conformations (misfolding) of aggregate-prone proteins, some of which have been found mutated in the familial forms of ALS. The removal of misfolded (aggregated) proteins is operated by the cells via two major degradative systems the ubiquitin-proteasome pathway (UPP) and the autophagy. Both systems require the assistance of intracellular chaperons. The molecular chaperones recognize and bind misfolded proteins, preventing their aggregation and facilitating their degradation, thus exerting neuroprotective functions. In this project, we will focus on the chaperone HSPB8, which forms a stable complex with the co-chaperone Bag3. Overexpression of HSPB8 (and Bag3) prevents aggregation of mutated SOD1 and TDP-43, associated with familial and sporadic ALS, by increasing their degradation via autophagy, an essential process for aggregate-prone protein clearance and neuronal survival. Thus, HSPB8 (and Bag3) may help motor neurons to cope with misfolded TDP-43 and SOD1 by either directly targeting them to the autophagic vacuoles for degradation and/or restoring/boosting the autophagy flux. Interestingly, deregulated autophagy is amongst the causes for motor neuron diseases (MNDs), further pointing to a link between protein aggregation, protein quality control, HSPB8-Bag3 and autophagy. Besides, mislocalization/aggregation of TDP-43 and FUS/TLS to the mRNA containing cytoplasmic stress granules (SGs) alters RNA metabolism and has been suggested as pathomechanism contributing to ALS. Our preliminary data indicate that HSPB8 is recruited to SG, where it colocalizes with TDP-43. Therefore, HSPB8, by either preventing the mislocalization/aggregation of mutated TDP-43 and FUS to SG and/or by targeting them for degradation may also contribute to disease amelioration by avoiding impairment of specific RNA translation/processing. The hypothesis that HSPB8 may exert essential functions for motor neuron viability is further supported by the observation that HSPB8 is upregulated in surviving motor neurons in both an ALS mouse model and in human ALS tissues, as well as by the finding that mutated forms of HSPB8 (which are found in aggregates) cause dominant hereditary motor neuropathy. In this project, we will investigate the hypothesis that upregulation of HSPB8 (and Bag3) may protect against ALS, using both motor neuronal cells and the mutated SOD1, TDP-43 and FUS/TLS based Drosophila models of ALS. We will also perform a drug screening to find compounds able to induce HSPB8 expression specifically in motor neurons. This work will provide insights in the role of HSPB8 as modulator of ALS and will identify specific sites of action of HSPB8 whose modulation may also represent new therapeutic targets for ALS