Thermal proteome profiling (TPP) allows for the unbiased detection of drug – target protein engagements in vivo. Traditionally, one cell type is used for TPP studies, with the risk of missing important differentially expressed target proteins. The use of whole organisms would circumvent this problem. Zebrafish embryos are amenable to such an approach. Here, we used TPP on whole zebrafish embryo lysate to identify protein targets of napabucasin, a compound that may affect Signal transducer and activator of transcription 3 (Stat3) signaling through an ill-understood mechanism. In zebrafish embryos, napabucasin induced developmental defects consistent with inhibition of Stat3 signaling. TPP profiling showed no distinct shift in Stat3 upon napabucasin treatment, but effects were detected on the oxidoreductase, Pora, which might explain effects on Stat3 signaling. Interestingly, thermal stability of several aldehyde dehydrogenases (Aldhs) was affected. Moreover, napabucasin activated ALDH enzymatic activity in vitro. Aldhs have crucial roles in retinoic acid metabolism and functionally we validated napabucasin-mediated activation of the retinoic acid pathway in zebrafish in vivo. We conclude that TPP profiling in whole zebrafish embryo lysate is feasible and facilitates direct correlation of in vivo effects of small molecule drugs with their protein targets.

The goal of clinical proteomics is to identify, quantify, and characterize proteins in body fluids or tissue to assist diagnosis, prognosis, and treatment of patients. In this way, it is similar to more mature omics technologies, such as genomics, that are increasingly applied in biomedicine. We argue that, similar to those fields, proteomics also faces ethical issues related to the kinds of information that is inherently obtained through sample measurement, although their acquisition was not the primary purpose. Specifically, we demonstrate the potential to identify individuals both by their characteristic, individual-specific protein levels and by variant peptides reporting on coding single nucleotide polymorphisms. Furthermore, it is in the nature of blood plasma proteomics profiling that it broadly reports on the health status of an individual – beyond the disease under investigation. Finally, we show that private and potentially sensitive information, such as ethnicity and pregnancy status, can increasingly be derived from proteomics data. Although this is potentially valuable not only to the individual, but also for biomedical research, it raises ethical questions similar to the incidental findings obtained through other omics technologies. We here introduce the necessity of - and argue for the desirability for - ethical and human rights-related issues to be discussed within the proteomics community. Those thoughts are more fully developed in our accompanying manuscript. Appreciation and discussion of ethical aspects of proteomic research will allow for deeper, better-informed, more diverse, and, most importantly, wiser guidelines for clinical proteomics.

Alpha-1-acid glycoprotein (AGP) is an acute phase glycoprotein in blood, which is primarily synthetized in the liver and whose biological role is not completely understood. It consists of 45% carbohydrates that are present in the form of five N-linked complex glycans. AGP N-glycosylation was shown to be changed in many different diseases and some changes appear to be disease-specific, thus it has a great diagnostic and prognostic potential. However, AGP glycosylation was mainly analyzed in small cohorts and without detailed site-specific glycan information. Here, we developed a cost-effective method for a high-throughput and site-specific N-glycosylation LC-MS analysis of AGP which can be applied on large cohorts, aid in search for novel disease biomarkers and enable better understanding of AGP’s role and function in health and disease. The method does not require isolation of AGP with antibodies and affinity chromatography, but AGP is enriched by acid precipitation from 5 μl of bloodplasma in a 96 well format. After trypsinization, AGP glycopeptides are purified using a hydrophilic interaction chromatography based solid-phase extraction and analyzed by RP-LC-ESI-MS. We used our method to show for the first time that AGP N-glycan profile is stable in healthy individuals (14 individuals in 3 time points), which is a requirement for evaluation of its diagnostic potential. Furthermore, we tested our method on a population including individuals with registered hyperglycemia in critical illness (59 cases and 49 controls), which represents a significantly increased risk of developing type 2 diabetes. Individuals at higher risk of diabetes presented increased N-glycan branching on AGP’s second glycosylation site and lower sialylation of N-glycans on AGP’s third and AGP1’s fourth glycosylation site. Although this should be confirmed on a larger prospective cohort, it indicates that site-specific AGP N-glycan profile could help distinguish individuals who are at risk of type 2 diabetes.

Proportionality in the Conduct of Hostilities: The Incidental Harm Side of the Assessment Research paper sysadmin 6 December 2018

Clarification of international humanitarian law is important in ensuring compliance with the rule of proportionality, but a culture of compliance within armed forces and groups is also crucial.

Summary

• Military operations are taking place with increasing frequency in densely populated areas. Such operations result in loss of life and harm to civilians, as well as damage to civilian objects, (including infrastructure providing essential services). In order to protect civilians, it is imperative that armed forces and groups comply with the rules of international humanitarian law on the conduct of hostilities, including the rule of proportionality.
• The rule of proportionality prohibits attacks which may be expected to cause incidental loss of civilian life, injury to civilians, damage to civilian objects or a combination thereof, which would be excessive in relation to the concrete and direct military advantage anticipated. This research paper analyses the key steps that belligerents must take to give effect to the rule, with a particular focus on one side of proportionality assessments – the expected incidental harm.
• Those undertaking proportionality assessments before or during an attack must consider whether the expected harm will be caused by the attack, and whether that harm could be expected (that is, was it reasonably foreseeable).
• For the purpose of proportionality assessments, injury to civilians includes disease, and there is no reason in principle to exclude mental harm, even though it is currently challenging to identify and quantify it. Damage to civilian objects includes damage to elements of the natural environment.
• Once the incidental harm to be considered has been identified, a value or weight must be assigned to it. This is then balanced against the value or weight of the military advantage anticipated from the attack to determine whether the harm would be excessive.
• In the determination of whether the expected incidental harm would be excessive compared to the anticipated military advantage, ‘excessive’ is a wide but not indeterminate standard.
• Belligerents should develop methodologies so that those planning and deciding attacks are provided with all necessary information on expected incidental harm, and to assist them in assigning weight to the incidental harm to be considered.
• If it becomes apparent that the rule of proportionality will be contravened, the attack in question must be cancelled or suspended.
• Clarification of the law is important in ensuring compliance with the rule of proportionality, but a culture of compliance within armed forces and groups, inculcated by their leaders, is also crucial.

DNA polymerase from bacteriophage T7 undergoes large, substrate-induced conformational changes that are thought to account for high replication fidelity, but prior studies were adversely affected by mutations required to construct a Cys-lite variant needed for site-specific fluorescence labeling. Here we have optimized the direct incorporation of a fluorescent un-natural amino acid, (7-hydroxy-4-coumarin-yl)-ethylglycine, using orthogonal amber suppression machinery in Escherichia coli. MS methods verify that the unnatural amino acid is only incorporated at one position with minimal background. We show that the single fluorophore provides a signal to detect nucleotide-induced conformational changes through equilibrium and stopped-flow kinetic measurements of correct nucleotide binding and incorporation. Pre-steady-state chemical quench methods show that the kinetics and fidelity of DNA replication catalyzed by the labeled enzyme are largely unaffected by the unnatural amino acid. These advances enable rigorous analysis to establish the kinetic and mechanistic basis for high-fidelity DNA replication.

Four U.S. Marines remain hospitalized after an attack near Hamid Karzai International Airport in Kabul, Afghanistan, last month as service members were evacuating foreign nationals and refugees.

The U.S. Army is investigating the suspected suicides of three soldiers based at New York's Fort Drum.

This positive personality trait linked to more suicidal thoughts and suicide itself.

The two states are the first in 20 years to add such a requirement.

School administrators in some states are caught up in tensions about who gets the final say about when they can reopen their buildings and what precautions they should take to protect their communities.

School administrators in some states are caught up in tensions about who gets the final say about when they can reopen their buildings and what precautions they should take to protect their communities.

As foliage darkens in the fall, the pigments within the plant matter break down and transform

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Genie Tech Genie Complete, designed in SolidWorks, sets new tone for non-surgical electrotherapy treatments

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The softball camp in Hungary was not originally planned. But there in the 38-degree (100° F) heat, prayers and decisions opened lives to the Lord.

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To make up for lost time, instructional leaders will need to streamline curricula and offer "just-in-time" support. These steps can help.

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DOVER, DEL. (Sept. 16, 2024) – The Delaware Coalition for Injury Prevention’s Falls Prevention Team asks Delawareans to reduce broken bones, head injuries, and disabilities by preventing falls. Governor John Carney and Lieutenant Governor Bethany Hall-Long proclaimed Sep. 23 to 27, 2024 as Falls Prevention Awareness Week. A fall can impact a person’s mobility, functionality, and […]

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DEPARTMENT OF AGRICULTURE: Office of the Secretary