Global Profiling and Inhibition of Protein Lipidation in Vector and Host Stages of the Sleeping Sickness Parasite Trypanosoma brucei

The enzyme N-myristoyltransferase (NMT) catalyzes the essential fatty acylation of substrate proteins with myristic acid in eukaryotes and is a validated drug target in the parasite Trypanosoma brucei, the causative agent of African trypanosomiasis (sleeping sickness). N-Myristoylation typically mediates membrane localization of proteins and is essential to the function of many. However, only a handful of proteins are experimentally validated as N-myristoylated in T. brucei. Here, we perform metabolic labeling with an alkyne-tagged myristic acid analogue, enabling the capture of lipidated proteins in insect and host life stages of T. brucei. We further compare this with a longer chain palmitate analogue to explore the chain length-specific incorporation of fatty acids into proteins. Finally, we combine the alkynyl-myristate analogue with NMT inhibitors and quantitative chemical proteomics to globally define N-myristoylated proteins in the clinically relevant bloodstream form parasites. This analysis reveals five ARF family small GTPases, calpain-like proteins, phosphatases, and many uncharacterized proteins as substrates of NMT in the parasite, providing a global view of the scope of this important protein modification and further evidence for the crucial and pleiotropic role of NMT in the cell

Polyamine stress at high pH in Escherichia coli K-12

BACKGROUND: Polyamines such as spermine and spermidine are required for growth of Escherichia coli; they interact with nucleic acids, and they bind to ribosomes. Polyamines block porins and decrease membrane permeability, activities that may protect cells in acid. At high concentrations, however, polyamines impair growth. They impair growth more severely at high pH, probably due to their increased uptake as membrane-permeant weak bases. The role of pH is critical in understanding polyamine stress. RESULTS: The effect of polyamines was tested on survival of Escherichia coli K-12 W3110 in extreme acid or base (pH conditions outside the growth range). At pH 2, 10 mM spermine increased survival by 2-fold, and putrescine increased survival by 30%. At pH 9.8, however, E. coli survival was decreased 100-fold by 10 mM spermine, putrescine, cadaverine, or spermidine. At pH 8.5, spermine decreased the growth rate substantially, whereas little effect was seen at pH 5.5. Spermidine required ten-fold higher concentrations to impair growth. On proteomic 2-D gels, spermine and spermidine caused differential expression of 31 different proteins. During log-phase growth at pH 7.0, 1 mM spermine induced eight proteins, including PykF, GlpK, SerS, DeaD, OmpC and OmpF. Proteins repressed included acetate-inducible enzymes (YfiD, Pta, Lpd) as well as RapA (HepA), and FabB. At pH 8.5, spermine induced additional proteins: TnaA, OmpA, YrdA and NanA (YhcJ) and also repressed 17 proteins. Four of the proteins that spermine induced (GlpK, OmpA, OmpF, TnaA) and five that were repressed (Lpd, Pta, SucB, TpiA, YfiD) show similar induction or repression, respectively, in base compared to acid. Most of these base stress proteins were also regulated by spermidine, but only at ten-fold higher concentration (10 mM) at high pH (pH 8.5). CONCLUSION: Polyamines increase survival in extreme acid, but decrease E. coli survival in extreme base. Growth inhibition by spermine and spermidine requires neutral or higher pH. At or above pH 7, spermine and spermidine regulate specific proteins, many of which are known to be regulated by base stress. High pH amplifies polyamine stress; and naturally occurring polyamines may play an important role in base stress

Descriptive and Substantive Representation in Congress: Evidence from 80,000 Congressional Inquiries

A vast literature debates the efficacy of descriptive representation in legislatures. Though studies argue it influences how communities are represented through constituency service, they are limited since legislators’ service activities are unobserved. Using Freedom of Information Act (FOIA) requests, we collected 88,000 records of communication between members of the U.S. Congress and federal agencies during the 108th–113th Congresses. These legislative interventions allow us to examine members’ “follow‐through” with policy implementation. We find that women, racial/ethnic minorities, and veterans are more likely to work on behalf of constituents with whom they share identities. Including veterans offers leverage in understanding the role of political cleavages and shared experiences. Our findings suggest that shared experiences operate as a critical mechanism for representation, that a lack of political consensus is not necessary for substantive representation, and that the causal relationships identified by experimental work have observable implications in the daily work of Congress.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/150606/1/ajps12443-sup-0001-SuppMat.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150606/2/ajps12443.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150606/3/ajps12443_am.pd

Oxygen limitation modulates pH regulation of catabolism and hydrogenases, multidrug transporters, and envelope composition in Escherichia coli K-12

BACKGROUND: In Escherichia coli, pH regulates genes for amino-acid and sugar catabolism, electron transport, oxidative stress, periplasmic and envelope proteins. Many pH-dependent genes are co-regulated by anaerobiosis, but the overall intersection of pH stress and oxygen limitation has not been investigated. RESULTS: The pH dependence of gene expression was analyzed in oxygen-limited cultures of E. coli K-12 strain W3110. E. coli K-12 strain W3110 was cultured in closed tubes containing LBK broth buffered at pH 5.7, pH 7.0, and pH 8.5. Affymetrix array hybridization revealed pH-dependent expression of 1,384 genes and 610 intergenic regions. A core group of 251 genes showed pH responses similar to those in a previous study of cultures grown with aeration. The highly acid-induced gene yagU was shown to be required for extreme-acid resistance (survival at pH 2). Acid also up-regulated fimbriae (fimAC), periplasmic chaperones (hdeAB), cyclopropane fatty acid synthase (cfa), and the "constitutive" Na+/H+ antiporter (nhaB). Base up-regulated core genes for maltodextrin transport (lamB, mal), ATP synthase (atp), and DNA repair (recA, mutL). Other genes showed opposite pH responses with or without aeration, for example ETS components (cyo,nuo, sdh) and hydrogenases (hya, hyb, hyc, hyf, hyp). A hypF strain lacking all hydrogenase activity showed loss of extreme-acid resistance. Under oxygen limitation only, acid down-regulated ribosome synthesis (rpl,rpm, rps). Acid up-regulated the catabolism of sugar derivatives whose fermentation minimized acid production (gnd, gnt, srl), and also a cluster of 13 genes in the gadA region. Acid up-regulated drug transporters (mdtEF, mdtL), but down-regulated penicillin-binding proteins (dacACD, mreBC). Intergenic regions containing regulatory sRNAs were up-regulated by acid (ryeA, csrB, gadY, rybC). CONCLUSION: pH regulates a core set of genes independently of oxygen, including yagU, fimbriae, periplasmic chaperones, and nhaB. Under oxygen limitation, however, pH regulation is reversed for genes encoding electron transport components and hydrogenases. Extreme-acid resistance requires yagU and hydrogenase production. Ribosome synthesis is down-regulated at low pH under oxygen limitation, possibly due to the restricted energy yield of catabolism. Under oxygen limitation, pH regulates metabolism and transport so as to maximize alternative catabolic options while minimizing acidification or alkalinization of the cytoplasm

Evolution dynamics of a dense frozen Rydberg gas to plasma

Dense samples of cold Rydberg atoms have previously been observed to spontaneously evolve to a plasma, despite the fact that each atom may be bound by as much as 100 cm−1. Initially, ionization is caused by blackbody photoionization and Rydberg-Rydberg collisions. After the first electrons leave the interaction re- gion, the net positive charge traps subsequent electrons. As a result, rapid ionization starts to occur after 1 μs caused by electron-Rydberg collisions. The resulting cold plasma expands slowly and persists for tens of microseconds. While the initial report on this process identified the key issues described above, it failed to resolve one key aspect of the evolution process. Specifically, redistribution of population to Rydberg states other than the one initially populated was not observed, a necessary mechanism to maintain the energy balance in the system. Here we report new and expanded observations showing such redistribution and confirming theoretical predictions concerning the evolution to a plasma. These measurements also indicate that, for high n states of purely cold Rydberg samples, the initial ionization process which leads to electron trapping is one involving the interactions between Rydberg atoms

Evolution dynamics of a dense frozen Rydberg gas to plasma

Dense samples of cold Rydberg atoms have previously been observed to spontaneously evolve to a plasma, despite the fact that each atom may be bound by as much as 100 cm−1. Initially, ionization is caused by blackbody photoionization and Rydberg-Rydberg collisions. After the first electrons leave the interaction re- gion, the net positive charge traps subsequent electrons. As a result, rapid ionization starts to occur after 1 μs caused by electron-Rydberg collisions. The resulting cold plasma expands slowly and persists for tens of microseconds. While the initial report on this process identified the key issues described above, it failed to resolve one key aspect of the evolution process. Specifically, redistribution of population to Rydberg states other than the one initially populated was not observed, a necessary mechanism to maintain the energy balance in the system. Here we report new and expanded observations showing such redistribution and confirming theoretical predictions concerning the evolution to a plasma. These measurements also indicate that, for high n states of purely cold Rydberg samples, the initial ionization process which leads to electron trapping is one involving the interactions between Rydberg atoms

Interplay between liver and blood stages of Plasmodium infection dictates malaria severity via γδ T cells and IL-17-promoted stress erythropoiesis

© 2023 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Plasmodium replicates within the liver prior to reaching the bloodstream and infecting red blood cells. Because clinical manifestations of malaria only arise during the blood stage of infection, a perception exists that liver infection does not impact disease pathology. By developing a murine model where the liver and blood stages of infection are uncoupled, we showed that the integration of signals from both stages dictated mortality outcomes. This dichotomy relied on liver stage-dependent activation of Vγ4+ γδ T cells. Subsequent blood stage parasite loads dictated their cytokine profiles, where low parasite loads preferentially expanded IL-17-producing γδ T cells. IL-17 drove extra-medullary erythropoiesis and concomitant reticulocytosis, which protected mice from lethal experimental cerebral malaria (ECM). Adoptive transfer of erythroid precursors could rescue mice from ECM. Modeling of γδ T cell dynamics suggests that this protective mechanism may be key for the establishment of naturally acquired malaria immunity among frequently exposed individuals.We would like to acknowledge Freddy Frischknecht (Integrative Parasitology Center for Infectious Diseases, Heidelberg) for providing the Plasmodium berghei lisp2− parasite line, Immo Prinz (Hannover Medical School, Hannover) for providing genetically modified mouse lines, Ana Parreira (iMM-JLA, Portugal) and Geoff McFadden’s lab (School of BioSciences, University of Melbourne, Australia) for mosquito rearing and infection with Plasmodium parasites, Helena Pinheiro (iMM-JLA, Portugal) for assistance with graphical design, Inês Bento and Miguel Prudêncio for critically reviewing this manuscript, and the Flow Cytometry and Rodent Facilities teams (iMM-JLA, Portugal) for their assistance. Work at iMM-JLA was supported by Fundação para a Ciência e a Tecnologia. Portugal (PTDC/MED-IMU/28664/2017) and the “La caixa” Banking Foundation, Spain (HR17-00264-PoEMM) grants attributed to Â.F.C. and M.M.M., respectively. Work at the Department of Microbiology and Immunology, The University of Melbourne, Australia, was funded by the National Health and Medical Research Council, Australia (1113293, 1154457) and the Australian Research Council, Australia (CE140100011). Â.F.C., S.M., J.L.G., M.I.M., R.M.R., and K.S. were supported by Fundação para a Ciência e a Tecnologia, Portugal (DL57/2016/CP1451/CT0004, DL57/2016/CP1451/CT0010, PD/BD/139053/2018, PD/BD/135454/2017, PTDC/MAT-APL/31602/2017, and CEECIND/00697/2018, respectively), P.L. was supported by Conselho Nacional de Desenvolvimento Científico e Tenológico, Brazil (SN/CGEFO/CNPQ 201801/2015-9), and A.T.T. was supported in part by Alfred P. Sloan Foundation Fellowship (FG-2020-12949).info:eu-repo/semantics/publishedVersio

Development and validation of a 30-day mortality index based on pre-existing medical administrative data from 13,323 COVID-19 patients: The Veterans Health Administration COVID-19 (VACO) Index.

BACKGROUND: Available COVID-19 mortality indices are limited to acute inpatient data. Using nationwide medical administrative data available prior to SARS-CoV-2 infection from the US Veterans Health Administration (VA), we developed the VA COVID-19 (VACO) 30-day mortality index and validated the index in two independent, prospective samples. METHODS AND FINDINGS: We reviewed SARS-CoV-2 testing results within the VA between February 8 and August 18, 2020. The sample was split into a development cohort (test positive between March 2 and April 15, 2020), an early validation cohort (test positive between April 16 and May 18, 2020), and a late validation cohort (test positive between May 19 and July 19, 2020). Our logistic regression model in the development cohort considered demographics (age, sex, race/ethnicity), and pre-existing medical conditions and the Charlson Comorbidity Index (CCI) derived from ICD-10 diagnosis codes. Weights were fixed to create the VACO Index that was then validated by comparing area under receiver operating characteristic curves (AUC) in the early and late validation cohorts and among important validation cohort subgroups defined by sex, race/ethnicity, and geographic region. We also evaluated calibration curves and the range of predictions generated within age categories. 13,323 individuals tested positive for SARS-CoV-2 (median age: 63 years; 91% male; 42% non-Hispanic Black). We observed 480/3,681 (13%) deaths in development, 253/2,151 (12%) deaths in the early validation cohort, and 403/7,491 (5%) deaths in the late validation cohort. Age, multimorbidity described with CCI, and a history of myocardial infarction or peripheral vascular disease were independently associated with mortality-no other individual comorbid diagnosis provided additional information. The VACO Index discriminated mortality in development (AUC = 0.79, 95% CI: 0.77-0.81), and in early (AUC = 0.81 95% CI: 0.78-0.83) and late (AUC = 0.84, 95% CI: 0.78-0.86) validation. The VACO Index allows personalized estimates of 30-day mortality after COVID-19 infection. For example, among those aged 60-64 years, overall mortality was estimated at 9% (95% CI: 6-11%). The Index further discriminated risk in this age stratum from 4% (95% CI: 3-7%) to 21% (95% CI: 12-31%), depending on sex and comorbid disease. CONCLUSION: Prior to infection, demographics and comorbid conditions can discriminate COVID-19 mortality risk overall and within age strata. The VACO Index reproducibly identified individuals at substantial risk of COVID-19 mortality who might consider continuing social distancing, despite relaxed state and local guidelines

Using a Non-Image-Based Medium-Throughput Assay for Screening Compounds Targeting N-myristoylation in Intracellular Leishmania Amastigotes

We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC50 values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania Nmyristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors

A Novel Semi-Supervised Methodology for Extracting Tumor Type-Specific MRS Sources in Human Brain Data

BackgroundThe clinical investigation of human brain tumors often starts with a non-invasive imaging study, providing information about the tumor extent and location, but little insight into the biochemistry of the analyzed tissue. Magnetic Resonance Spectroscopy can complement imaging by supplying a metabolic fingerprint of the tissue. This study analyzes single-voxel magnetic resonance spectra, which represent signal information in the frequency domain. Given that a single voxel may contain a heterogeneous mix of tissues, signal source identification is a relevant challenge for the problem of tumor type classification from the spectroscopic signal.Methodology/Principal FindingsNon-negative matrix factorization techniques have recently shown their potential for the identification of meaningful sources from brain tissue spectroscopy data. In this study, we use a convex variant of these methods that is capable of handling negatively-valued data and generating sources that can be interpreted as tumor class prototypes. A novel approach to convex non-negative matrix factorization is proposed, in which prior knowledge about class information is utilized in model optimization. Class-specific information is integrated into this semi-supervised process by setting the metric of a latent variable space where the matrix factorization is carried out. The reported experimental study comprises 196 cases from different tumor types drawn from two international, multi-center databases. The results indicate that the proposed approach outperforms a purely unsupervised process by achieving near perfect correlation of the extracted sources with the mean spectra of the tumor types. It also improves tissue type classification.Conclusions/SignificanceWe show that source extraction by unsupervised matrix factorization benefits from the integration of the available class information, so operating in a semi-supervised learning manner, for discriminative source identification and brain tumor labeling from single-voxel spectroscopy data. We are confident that the proposed methodology has wider applicability for biomedical signal processing