Usability of eyetracking computer systems and impact on psychological wellbeing in patients with advanced amyotrophic lateral sclerosis.

Restrictions in communicative abilities are well known in patients with amyotrophic lateral sclerosis (ALS), but only few approaches in terms of evaluation of supportive technologies have been made. We aimed to assess the use and perceived usability of eye-tracking computer devices (ETCS) of severely impacted patients with ALS in an independent, direct manner and relate it to psychological well-being. ETCS enable active communication and social participation in the quadriplegic and anarthric disease state. Therefore, ETCS-based versions of widely used psychosocial questionnaires (ADI-12, SeiQoL-DW, WHO-5) as well as structured questions on communicative functioning and ETCS usage were developed to assess ALS patients, their next of kin and professional caregivers. Eleven patients (ALSFRS-R: 5.3 ± 5.9; ALS duration: 6.5 ± 3.8 years, range 1‒12; 82% invasively ventilated), nine next of kin and 10 professional caregivers could be assessed. Patients reported a mean use of their personal ETCS of 9.1 h per d (range 0.5‒16), with a high user satisfaction, preservation of communicative abilities and subjective indispensability of the ETCS. ETCS use was associated with higher psychological well-being. Next of kin and professional caregivers also nominated some critical aspect, which remains to be clarified. Our results strengthen the evidence that preserved mental autonomy influences psychological well-being in ALS and might even modify disease course and end-of-life-decisions in ALS.

Eye-tracking-based assessment suggests preserved well-being in locked-in patients.

We assessed quality of life (QoL) and psychological well-being in patients with amyotrophic lateral sclerosis-induced locked-in state and their next of kin in a fully unbiased manner using eye-tracking computer systems. Eleven of 30 screened patients and 9 next of kin completed study procedures. Patients reported good QoL, which appeared to be at the cost of the QoL of their next of kin. Next of kin rated their own or patients' QoL similarly, but they identified different areas as important as compared with patients. Our results are of importance for the discussion of end-of-life decisions and the evaluation of patients' presumed wishes as well as for psychosocial interventions. Ann Neurol 2017;81:310-315.

The mono-ADP-ribosyltransferases Alt and ModB of bacteriophage T4: target proteins identified.

Infection of Escherichia coli by bacteriophage T4 leads to the expression of three phage mono-ADP-ribosyltransferases (namely, Alt, ModA, and ModB), each of which modifies a distinct group of host proteins. To improve understanding of these interactions and their consequences for the T4 replication cycle, we used high-resolution two-dimensional gel electrophoresis and mass-spectrometry to identify some of the putative target proteins ADP-ribosylated in vitro by Alt (total approximately 27) and ModB (total approximately 8). E. coli trigger factor and the elongation factor EF-Tu were 2 targets of ModB action, and these proteins were among the 10 identified as targets of Alt, hinting that these factors are involved in phage replication.

ModA and ModB, two ADP-ribosyltransferases encoded by bacteriophage T4: catalytic properties and mutation analysis.

Bacteriophage T4 encodes three ADP-ribosyltransferases, Alt, ModA, and ModB. These enzymes participate in the regulation of the T4 replication cycle by ADP-ribosylating a defined set of host proteins. In order to obtain a better understanding of the phage-host interactions and their consequences for regulating the T4 replication cycle, we studied cloning, overexpression, and characterization of purified ModA and ModB enzymes. Site-directed mutagenesis confirmed that amino acids, as deduced from secondary structure alignments, are indeed decisive for the activity of the enzymes, implying that the transfer reaction follows the Sn1-type reaction scheme proposed for this class of enzymes. In vitro transcription assays performed with Alt- and ModA-modified RNA polymerases demonstrated that the Alt-ribosylated polymerase enhances transcription from T4 early promoters on a T4 DNA template, whereas the transcriptional activity of ModA-modified polymerase, without the participation of T4-encoded auxiliary proteins for middle mode or late transcription, is reduced. The results presented here support the conclusion that ADP-ribosylation of RNA polymerase and of other host proteins allows initial phage-directed mRNA synthesis reactions to escape from host control. In contrast, subsequent modification of the other cellular target proteins limits transcription from phage early genes and participates in redirecting transcription to phage middle and late genes.

Bacteriophage T4 alpha-glucosyltransferase: a novel interaction with gp45 and aspects of the catalytic mechanism.

The bacteriophage T4 alpha- and beta-glucosyltransferases (AGT and BGT) catalyse the transfer of glucose from uridine diphosphoglucose to 5-hydroxymethyl cytosine of T4 DNA in an alpha- and beta-conformation, respectively. Following the 3D structure of BGT and a secondary structure alignment of AGT and BGT, we performed a site-directed mutagenesis of AGT. A two-domain structure was deduced, with an open substrate-free and a closed substrate-bound conformation. We also identified specific amino acids involved in DNA binding. The identification of a protein-protein interaction of AGT and gp45 which is a part of the T4 replication complex supports the idea that T4 DNA is alpha-glucosylated immediately after synthesis. BGT then glucosylates those hydroxymethyl cytosines not previously served by AGT.

Bacteriophage T4 genome.

Phage T4 has provided countless contributions to the paradigms of genetics and biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene products. T4 biology and its genomic sequence provide the best-understood model for modern functional genomics and proteomics. Variations on gene expression, including overlapping genes, internal translation initiation, spliced genes, translational bypassing, and RNA processing, alert us to the caveats of purely computational methods. The T4 transcriptional pattern reflects its dependence on the host RNA polymerase and the use of phage-encoded proteins that sequentially modify RNA polymerase; transcriptional activator proteins, a phage sigma factor, anti-sigma, and sigma decoy proteins also act to specify early, middle, and late promoter recognition. Posttranscriptional controls by T4 provide excellent systems for the study of RNA-dependent processes, particularly at the structural level. The redundancy of DNA replication and recombination systems of T4 reveals how phage and other genomes are stably replicated and repaired in different environments, providing insight into genome evolution and adaptations to new hosts and growth environments. Moreover, genomic sequence analysis has provided new insights into tail fiber variation, lysis, gene duplications, and membrane localization of proteins, while high-resolution structural determination of the "cell-puncturing device," combined with the three-dimensional image reconstruction of the baseplate, has revealed the mechanism of penetration during infection. Despite these advances, nearly 130 potential T4 genes remain uncharacterized. Current phage-sequencing initiatives are now revealing the similarities and differences among members of the T4 family, including those that infect bacteria other than Escherichia coli. T4 functional genomics will aid in the interpretation of these newly sequenced T4-related genomes and in broadening our understanding of the complex evolution and ecology of phages-the most abundant and among the most ancient biological entities on Earth.

Crystallization and preliminary crystallographic study of a ternary complex between the T4 phage beta-glucosyltransferase, uridine diphosphoglucose and a DNA fragment containing an abasic site.

A base-flipping phenomenon has been established for DNA methyltransferases and for DNA base-excision repair glycosylases and is likely to prove general for enzymes that need access to DNA bases to undergo chemical reaction. T4 phage beta-glucosyltransferase (BGT) is a good candidate for this novel mechanism. In order to confirm this, BGT was crystallized with an abasic site-containing DNA and uridine diphosphoglucose (UDP-glucose). The crystallization strategy is described. A complete data set was collected at 1.8 A resolution on a Cu Kalpha rotating-anode X-ray source. Molecular replacement was performed and the initial electron-density maps clearly show bound DNA.

SAR directed design and synthesis of novel beta(1-4)-glucosyltransferase inhibitors and their in vitro inhibition studies.

This paper describes SAR directed design and synthesis of novel beta(1-4)-glucosyltransferase (BGT) inhibitors. The designed inhibitors 1-5 provide conformational mimicry of the transition-state in glucosyltransfer reactions. The compounds were tested for in vitro inhibitory activity against (BGT) and the inhibition kinetics were examined. Three of the designed molecules were found to be potential inhibitors of BGT having IC50 values in micromolar (microM) range. Useful structure-activity relationships were established, which provide guidelines for the design of future generations of inhibitors of BGT.

T4 early promoter strength probed in vivo with unribosylated and ADP-ribosylated Escherichia coli RNA polymerase: a mutation analysis.

The consensus sequence of T4 early promoters differs in length, sequence and degree of conservation from that of Escherichia coli sigma(70) promoters. The enzyme interacting with these promoters, and transcribing the T4 genome, is native host RNA polymerase, which is increasingly modified by the phage-encoded ADP-ribosyltransferase, Alt. T4 early transcription is a very active process, possibly out-competing host transcription. The much stronger T4 promoters enhance viral transcription by a factor of at least two and the Alt-catalysed ADP-ribosylation of the host enzyme triggers an additional enhancement, again by a factor of about two. To address the question of which promoter elements contribute to the increasing transcriptional activity directed towards phage genes, the very strong E. coli promoter, Ptac, was sequentially mutated towards the sequence of the T4 early promoter consensus. Second, mutations were introduced into the highly conserved regions of the T4 early promoter, P8.1. The co-occurrence of the promoter-encoding plasmid pKWIII and vector pTKRI, which expresses Alt in E. coli, constitutes a test system that allows comparison of the transcriptional activities of phage and bacterial promoters, in the presence of native, or alternatively ADP-ribosylated RNA polymerase. Results reveal that T4 early promoters exhibit a bipartite structure, capable of strong interaction with both types of RNA polymerase. The -10, -16, -42 and -52 regions are important for transcript initiation with the native polymerase. To facilitate acceleration of transcription, the ADP-ribosylated enzyme requires not only the integrity of the -10, -16 and -35 regions, but also that of position -33, and even more importantly, maintenance of the upstream promoter element at position -42. The latter positions introduced into the E. coli Ptac promoter render this mutant promoter responsive to Alt-ADP-ribosylated RNA polymerase, like T4 early promoters.