BRAHMA ATPase of the SWI/SNF Chromatin Remodeling Complex Acts as a Positive Regulator of Gibberellin-Mediated Responses in Arabidopsis

SWI/SNF chromatin remodeling complexes perform a pivotal function in the regulation of eukaryotic gene expression. Arabidopsis (Arabidopsis thaliana) mutants in major SWI/SNF subunits display embryo-lethal or dwarf phenotypes, indicating their critical role in molecular pathways controlling development and growth. As gibberellins (GA) are major positive regulators of plant growth, we wanted to establish whether there is a link between SWI/SNF and GA signaling in Arabidopsis. This study revealed that in brm-1 plants, depleted in SWI/SNF BRAHMA (BRM) ATPase, a number of GA-related phenotypic traits are GA-sensitive and that the loss of BRM results in markedly decreased level of endogenous bioactive GA. Transcriptional profiling of brm-1 and the GA biosynthesis mutant ga1-3, as well as the ga1-3/brm-1 double mutant demonstrated that BRM affects the expression of a large set of GA-responsive genes including genes responsible for GA biosynthesis and signaling. Furthermore, we found that BRM acts as an activator and directly associates with promoters of GA3ox1, a GA biosynthetic gene, and SCL3, implicated in positive regulation of the GA pathway. Many GA-responsive gene expression alterations in the brm-1 mutant are likely due to depleted levels of active GAs. However, the analysis of genetic interactions between BRM and the DELLA GA pathway repressors, revealed that BRM also acts on GA-responsive genes independently of its effect on GA level. Given the central position occupied by SWI/SNF complexes within regulatory networks controlling fundamental biological processes, the identification of diverse functional intersections of BRM with GA-dependent processes in this study suggests a role for SWI/SNF in facilitating crosstalk between GA-mediated regulation and other cellular pathways

Mast Cell Diseases in Practice and Research: Issues and Perspectives Raised by Patients and Their Recommendations to the Scientific Community and Beyond

Background: Since 2010, patients and physicians have collaborated to understand unmet needs of patients with mast cell diseases, incorporating mastocytosis and mast cell activation disorders, which include mast cell activation syndromes. Objective: This Open Innovation in Science project aims to expand understanding of the needs of patients affected by mast cell diseases, and encourage global communication among patient advocacy groups, physicians, researchers, industry, and government. A major aim is to support the scientific community's efforts to improve diagnosis, management, therapy, and patients’ quality of life by addressing unmet needs. Methods: In collaboration with mast cell disease specialists, 13 patient advocacy groups from 12 countries and regions developed lists of top patient needs. A core team of leaders from patient advocacy groups collected and analyzed the data and proposed possible actions to address patient needs. Results: Findings identified similarities and differences among participating countries in unmet needs between patients with mastocytosis and those with mast cell activation syndromes. Issues emphasized struggles relating to the nature and rarity of mast cell diseases, their impact on quality of life, the diagnostic process, access to appropriate care, more effective treatment, and the need for research. Conclusions: Solutions vary across countries because situations differ, in particular regarding the existence of and access to centers of excellence and reference centers. Multifaceted mast cell activation syndrome barriers necessitate innovative approaches to improve access to appropriate care. The outcomes of this project should greatly support scientists and clinicians in their efforts to improve diagnosis, management, and treatment of patients with mastocytosis and mast cell activation disorders.The authors thank Tania Bray, Jan Hempstead, Heather Mayne, Joanne Mulder-Brambleby, and Irene Wilson for their supporting contributions, and all patients and families affected by MCDs, who shared their needs and concerns for development of this project. Authors involved in study conception and design were P. Valent, S.V. Jennings, C.C. Finnerty, J.S. Hobart, M. Martín-Martínez, K.A. Sinclair, V.M. Slee, J. Agopian, C. Akin, I. Álvarez-Twose, P. Bonadonna, A.A. Bowman, K. Brockow, H. Bumbea, C. de Haro, J.S. Fok, K. Hartmann, N. Hegmann, O. Hermine, M. Kalisiak, C.H. Katelaris, J. Kurz, P. Marcis, D. Mayne, D. Mendoza, A. Moussy, G. Mudretzkyj, N. Nidelea Vaia, M. Niedoszytko, H. Oude Elberink, A. Orfao, D.H. Radia, S. Rosenmeier, E. Ribada, W. Schinhofen, J. Schwaab, F. Siebenhaar, M. Triggiani, G. Tripodo, R. Velazquez, Y. Wielink, F. Wimazal, T. Yigit, and C. Zubrinich. Authors involved in acquisition and review of data were S.V. Jennings, C.C. Finnerty, J.S. Hobart, M. Martín-Martínez, K.A. Sinclair, V.M. Slee, J. Agopian, C. Akin, I. Álvarez-Twose, P. Bonadonna, A.A. Bowman, K. Brockow, H. Bumbea, C. de Haro, J.S. Fok, K. Hartmann, N. Hegmann, O. Hermine, M. Kalisiak, C.H. Katelaris, J. Kurz, P. Marcis, D. Mayne, D. Mendoza, A. Moussy, G. Mudretzkyj, N. Nidelea Vaia, M. Niedoszytko, H. Oude Elberink, A. Orfao, D.H. Radia, S. Rosenmeier, E. Ribada, W. Schinhofen, J. Schwaab, F. Siebenhaar, M. Triggiani, G. Tripodo, R. Velazquez, Y. Wielink, F. Wimazal, T. Yigit, C. Zubrinich, and P. Valent. The Core Group (analysis and interpretation of data and drafting of the manuscript) include S.V. Jennings, C.C. Finnerty, J.S. Hobart, M. Martín-Martínez, K.A. Sinclair, and V.M. Slee. Critical revision was performed by S.V. Jennings, C.C. Finnerty, J.S. Hobart, M. Martín-Martínez, K.A. Sinclair, V.M. Slee, J. Agopian, C. Akin, I. Álvarez-Twose, P. Bonadonna, A.A. Bowman, K. Brockow, H. Bumbea, C. de Haro, J.S. Fok, K. Hartmann, N. Hegmann, O. Hermine, M. Kalisiak, C.H. Katelaris, J. Kurz, P. Marcis, D. Mayne, D. Mendoza, A. Moussy, G. Mudretzkyj, N. Nidelea Vaia, M. Niedoszytko, H. Oude Elberink, A. Orfao, D.H. Radia, S. Rosenmeier, E. Ribada, W. Schinhofen, J. Schwaab, F. Siebenhaar, M. Triggiani, G. Tripodo, R. Velazquez, Y. Wielink, F Wimazal, T. Yigit, C. Zubrinich, and P. Valent

Rapid Sampling of Molecular Motions with Prior Information Constraints

Proteins are active, flexible machines that perform a range of different functions. Innovative experimental approaches may now provide limited partial information about conformational changes along motion pathways of proteins. There is therefore a need for computational approaches that can efficiently incorporate prior information into motion prediction schemes. In this paper, we present PathRover, a general setup designed for the integration of prior information into the motion planning algorithm of rapidly exploring random trees (RRT). Each suggested motion pathway comprises a sequence of low-energy clash-free conformations that satisfy an arbitrary number of prior information constraints. These constraints can be derived from experimental data or from expert intuition about the motion. The incorporation of prior information is very straightforward and significantly narrows down the vast search in the typically high-dimensional conformational space, leading to dramatic reduction in running time. To allow the use of state-of-the-art energy functions and conformational sampling, we have integrated this framework into Rosetta, an accurate protocol for diverse types of structural modeling. The suggested framework can serve as an effective complementary tool for molecular dynamics, Normal Mode Analysis, and other prevalent techniques for predicting motion in proteins. We applied our framework to three different model systems. We show that a limited set of experimentally motivated constraints may effectively bias the simulations toward diverse predicates in an outright fashion, from distance constraints to enforcement of loop closure. In particular, our analysis sheds light on mechanisms of protein domain swapping and on the role of different residues in the motion

Evaluation of high-affinity phenyltetrahydroisoquinoline aldoximes, linked through anti-triazoles, as reactivators of phosphylated cholinesterases

Acetylcholinesterase (AChE) is a pivotal enzyme in neurotransmission. Its inhibition leads to cholinergic crises and could ultimately result in death. A related enzyme, butyrylcholinesterase (BChE), may act in the CNS as a co-regulator in terminating nerve impulses and is a natural plasma scavenger upon exposure to organophosphate (OP) nerve agents that irreversibly inhibit both enzymes. With the aim of improving reactivation of cholinesterases phosphylated by nerve agents sarin, VX, cyclosarin, and tabun, ten phenyltetrahydroisoquinoline (PIQ) aldoximes were synthesized by Huisgen 1,3 dipolar cycloaddition between alkyne- and azide-building blocks. The PIQ moiety may serve as a peripheral site anchor positioning the aldoxime moiety at the AChE active site. In terms of evaluated dissociation inhibition constants, the aldoximes could be characterized as high-affinity ligands. Nevertheless, high binding affinity of these oximes to AChE or its phosphylated conjugates did not assure rapid and selective AChE reactivation. Rather, potential reactivators of phosphylated BChE, with its enlarged acyl pocket, were identified, especially in case of cyclosarin, where the reactivation rates of the lead reactivator was 100- and 6-times that of 2-PAM and HI-6, respectively. Nevertheless, the return of the enzyme activity was affected by the nerve agent conjugated to catalytic serine, which highlights the lack of the universality of reactivators with respect to both the target enzyme and OP structure

Click Chemistry-Facilitated Structural Diversification of Nitrothiazoles, Nitrofurans, and Nitropyrroles Enhances Antimicrobial Activity against Giardia lamblia

Giardia lamblia is an important and ubiquitous cause of diarrheal disease. The primary agents in the treatment of giardiasis are nitroheterocyclic drugs, particularly the imidazoles metronidazole and tinidazole and the thiazole nitazoxanide. Although these drugs are generally effective, treatment failures occur in up to 20% of cases, and resistance has been demonstrated in vivo and in vitro Prior work had suggested that side chain modifications of the imidazole core can lead to new effective 5-nitroimidazole drugs that can combat nitro drug resistance, but the full potential of nitroheterocycles other than imidazole to yield effective new antigiardial agents has not been explored. Here, we generated derivatives of two clinically utilized nitroheterocycles, nitrothiazole and nitrofuran, as well as a third heterocycle, nitropyrrole, which is related to nitroimidazole but has not been systematically investigated as an antimicrobial drug scaffold. Click chemistry was employed to synthesize 442 novel nitroheterocyclic compounds with extensive side chain modifications. Screening of this library against representative G. lamblia strains showed a wide spectrum of in vitro activities, with many of the compounds exhibiting superior activity relative to reference drugs and several showing >100-fold increase in potency and the ability to overcome existing forms of metronidazole resistance. The majority of new compounds displayed no cytotoxicity against human cells, and several compounds were orally active against murine giardiasis in vivo These findings provide additional impetus for the systematic development of nitroheterocyclic compounds with nonimidazole cores as alternative and improved agents for the treatment of giardiasis and potentially other infectious agents

Click chemistry-facilitated comprehensive identification of proteins adducted by antimicrobial 5-nitroimidazoles for discovery of alternative drug targets against giardiasis.

Giardiasis and other protozoan infections are major worldwide causes of morbidity and mortality, yet development of new antimicrobial agents with improved efficacy and ability to override increasingly common drug resistance remains a major challenge. Antimicrobial drug development typically proceeds by broad functional screens of large chemical libraries or hypothesis-driven exploration of single microbial targets, but both strategies have challenges that have limited the introduction of new antimicrobials. Here, we describe an alternative drug development strategy that identifies a sufficient but manageable number of promising targets, while reducing the risk of pursuing targets of unproven value. The strategy is based on defining and exploiting the incompletely understood adduction targets of 5-nitroimidazoles, which are proven antimicrobials against a wide range of anaerobic protozoan and bacterial pathogens. Comprehensive adductome analysis by modified click chemistry and multi-dimensional proteomics were applied to the model pathogen Giardia lamblia to identify dozens of adducted protein targets common to both 5'-nitroimidazole-sensitive and -resistant cells. The list was highly enriched for known targets in G. lamblia, including arginine deiminase, α-tubulin, carbamate kinase, and heat shock protein 90, demonstrating the utility of the approach. Importantly, over twenty potential novel drug targets were identified. Inhibitors of two representative new targets, NADP-specific glutamate dehydrogenase and peroxiredoxin, were found to have significant antigiardial activity. Furthermore, all the identified targets remained available in resistant cells, since giardicidal activity of the respective inhibitors was not impacted by resistance to 5'-nitroimidazoles. These results demonstrate that the combined use of click chemistry and proteomics has the potential to reveal alternative drug targets for overcoming antimicrobial drug resistance in protozoan parasites