Search

We present automated continuous evolution (ACE), a platform for the hands-free directed evolution of biomolecules. ACE pairs OrthoRep, a genetic system for continuous targeted mutagenesis of user-selected genes in vivo, with eVOLVER, a scalable and automated continuous culture device for precise, multiparameter regulation of growth conditions. By implementing real-time feedback-controlled tuning of selection stringency with eVOLVER, genes of interest encoded on OrthoRep autonomously traversed multimutation adaptive pathways to reach desired functions, including drug resistance and improved enzyme activity. The durability, scalability, and speed of biomolecular evolution with ACE should be broadly applicable to protein engineering as well as prospective studies on how selection parameters and schedules shape adaptation.

Accurate analysis of gene expression in human tissues using RNA sequencing is dependent on the quality of source material. One major source of variation in mRNA quality is post-mortem time. While it is known that individual transcripts show differential post-mortem stability, few studies have directly and comprehensively analyzed mRNA stability following death, and in particular the extent to which tissue- and species-specific factors influence post-mortem mRNA stability are poorly understood. This knowledge is particularly important for ocular tissues studies, where tissues obtained post-mortem are frequently used for research or therapeutic applications. To directly investigate this question, we profiled mRNA levels in both neuroretina and retinal pigment epithelium (RPE) from mouse and baboon over a series of post-mortem intervals. We found substantial changes in gene expression as early as 15 minutes in the mouse and as early as three hours in the baboon eye tissues. Importantly, our findings demonstrate both tissue- and species- specific patterns of RNA metabolism, by identifying a set of genes that are either rapidly degraded or very stable in both species and/or tissues. Taken together, the data from this study lay the foundation for understanding RNA regulation post-mortem and provide novel insights into RNA metabolism in the tissues of the mammalian eye.

While protein aggregation is a hallmark of many neurodegenerative diseases, acquiring structural information on protein aggregates inside live cells remains challenging. Traditional microscopy does not provide structural information on protein systems. Routinely used fluorescent protein tags, such as Green Fluorescent Protein (GFP), might perturb native structures. Here, we report a counter‐propagating mid‐infrared photothermal imaging approach enabling mapping of secondary structure of protein aggregates in live cells modeling Huntington's disease. By comparing mid‐infrared photothermal spectra of label‐free and GFP‐tagged huntingtin inclusions, we demonstrate that GFP fusions indeed perturb the secondary structure of aggregates. By implementing spectra with small spatial step for dissecting spectral features within sub‐micrometer distances, we reveal that huntingtin inclusions partition into a β‐sheet‐rich core and a ɑ‐helix‐rich shell. We further demonstrate that this structural partition exists only in cells with the [RNQ+] prion state, while [rnq−] cells only carry smaller β‐rich non‐toxic aggregates. Collectively, our methodology has the potential to unveil detailed structural information on protein assemblies in live cells, enabling high‐throughput structural screenings of macromolecular assemblies. [ABSTRACT FROM AUTHOR]

The importance of autonomous weapon systems (AWS) is particularly apparent in contemporary conflicts, in which all parties are striving to expand the autonomy of these systems, particularly in offensive contexts. The traditional understanding is that the deployment of AWS poses significant threats to civilian life, as well as a range of delicate legal concerns. The narrow understanding is evident in the principal drawbacks of AWS in the absence of human supervision, which results in unpredictable and unreliable outputs, particularly during the targeting and engagement stages, leading to a clear breach of international humanitarian law (IHL). This creates a legal vacuum regarding the attribution of criminal responsibility. This research paper aims to characterise AWS and the implications of their potential deployment, with the primary goal of better identifying and understanding emerging legal concerns. Furthermore, the authors seek to offer a comprehensive understanding of the concept of autonomy, which is internationally accepted. Additionally, they explore the validity of the statement that AWS create an accountability gap relating to the International Criminal Court (ICC) Statute by analysing various types of AWS that are categorised in this study and deployed in modern wars. While the ICC Statute does not explicitly address all issues raised by using AWS, the study presents an interpretation that criminal responsibility could be assigned to all individuals involved in the use of AWS, especially in thorny situations. This study employs an analytical approach to examine and analyse traditional theories of criminology and the ICC Statute in relation to the ability to attribute individual criminal responsibility when AWS may be used. This study aims to provide a new perspective on criminal legal rules that are compatible with modern technology. [ABSTRACT FROM AUTHOR]

Cyberspaces can be significantly influenced by simple tools and tactics, and offer cost-effective solutions for states to achieve their objectives. However, it can also be used for conducting cyberwarfare, and its effects remain in gray areas. The spectrum of cyberwarfare activities ranges from minor web disruptions to crippling attacks on critical national infrastructures. Nevertheless, cyberattacks present a unique challenge in applying well-established humanitarian legal principles, particularly regarding the distinction between civilian and military targets. This is because of the interconnectedness and shared infrastructure of cyberspace between civilian and military entities, which blurs the lines between combatants and civilian populations. This article seeks to shift the general perception of the problems associated with this manner of conducting hostilities by building on the learning of the Russian-Ukrainian conflict. Furthermore, the authors examine the cyber activities that occurred during the conflict and the legal and ethical challenges that arise from classifying these activities and applying international law. They try to distinguish cyber activities as an act of force or armed attack, with a focus on determining the criteria that played a role in this classification, in light of Articles 2 (4) and 51 of the UN Charter. With reference to the United Nations (UN) Charter and the principles of self-defence, the concept of attack is scrutinised. Furthermore, the article addresses the principles of distinction and proportionality in relation to qualified cyber activities as an armed attack in the same conflict, emphasising the importance of the principle of proportionality in assessing cyber warfare. [ABSTRACT FROM AUTHOR]

Patient satisfaction is essential to the patient-centered approach in health services delivery, but little is known about satisfaction with physiotherapy services in Afghanistan. This study evaluated patients' satisfaction with physiotherapy services and related factors in this country. This study was conducted on 420 recipients of a physiotherapy center in Afghanistan. A questionnaire was used to collect data that measured satisfaction in 4 dimensions: treatment process, logistics, organizational factors, and overall. The scale of the questionnaire was 5-point Likert. The highest possible score was 100. Statistics methods included Spearman's correlation coefficient, Mann-Whitney and Kruskal-Wallis tests for univariate analysis, and Linear Regression for multivariate analysis. The average overall satisfaction score was 84.82 ± 13.24. Among the demographic variables, the relationship between education level and overall satisfaction score was significant. The findings of the linear regression model showed that the 3 dimensions of satisfaction, including the treatment process, logistics, and organizational factors, had statistically significant relationships with overall satisfaction. The level of satisfaction was high in all dimensions. To maximize satisfaction, reducing waiting time and better communication with patients, as components of logistics and organizational factors, are suggested. Patients also need to receive feedback from physiotherapists that are an essential components of the treatment process dimension. [ABSTRACT FROM AUTHOR]