Distinct conformational behaviors of four mammalian dual‐flavin reductases (cytochrome P450 reductase, methionine synthase reductase, neuronal nitric oxide synthase, endothelial nitric oxide synthase) determine their unique catalytic profiles

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109651/1/febs13073.pd

Distinct Conformational Behaviors of Four Mammalian Dual-Flavin Reductases (Cytochrome P450 Reductase, Methionine Synthase Reductase, Neuronal Nitric Oxide Synthase, Endothelial Nitric Oxide Synthase) Determine Their Unique Catalytic Profiles

Multidomain enzymes often rely on large conformational motions to function. However, the conformational setpoints, rates of domain motions and relationships between these parameters and catalytic activity are not well understood. To address this, we determined and compared the conformational setpoints and the rates of conformational switching between closed unreactive and open reactive states in four mammalian diflavin NADPH oxidoreductases that catalyze important biological electron transfer reactions: cytochrome P450 reductase, methionine synthase reductase and endothelial and neuronal nitric oxide synthase. We used stopped-flow spectroscopy, single turnover methods and a kinetic model that relates electron flux through each enzyme to its conformational setpoint and its rates of conformational switching. The results show that the four flavoproteins, when fully-reduced, have a broad range of conformational setpoints (from 12% to 72% open state) and also vary 100-fold with respect to their rates of conformational switching between unreactive closed and reactive open states (cytochrome P450 reductase \u3e neuronal nitric oxide synthase \u3e methionine synthase reductase \u3e endothelial nitric oxide synthase). Furthermore, simulations of the kinetic model could explain how each flavoprotein can support its given rate of electron flux (cytochrome c reductase activity) based on its unique conformational setpoint and switching rates. The present study is the first to quantify these conformational parameters among the diflavin enzymes and suggests how the parameters might be manipulated to speed or slow biological electron flux

Teaching Cultural Competency through Global Health Education at Weill Cornell Medicine

Background: Educating medical students to better understand the complexities of cultural competence, the social determinants and environmental determinants of health that are important and integral components of the medical school curriculum. Methods: In 2014, Weill Cornell Medicine (WCM) implemented a new curriculum, the adoption of which provided the means to enhance an existing global health program, informally introduced in 2009, and to address the issues of cultural competency. In this article, we share WCM’s experience in building and expanding its global health curriculum. Results: A hallmark of our program is the successful collaboration between students and faculty to create a multi-disciplinary global health program that incorporates electives, clinical field placement, and collaborative research. Conclusion: Key lessons learned through our experience include the necessity for strong faculty-student collaboration, full support from the administration, and building global partnerships. Our example could be a useful guide for other medical schools seeking to establish a global health education curriculum.&nbsp

Iterative Near-Term Ecological Forecasting: Needs, Opportunities, And Challenges

Two foundational questions about sustainability are “How are ecosystems and the services they provide going to change in the future?” and “How do human decisions affect these trajectories?” Answering these questions requires an ability to forecast ecological processes. Unfortunately, most ecological forecasts focus on centennial-scale climate responses, therefore neither meeting the needs of near-term (daily to decadal) environmental decision-making nor allowing comparison of specific, quantitative predictions to new observational data, one of the strongest tests of scientific theory. Near-term forecasts provide the opportunity to iteratively cycle between performing analyses and updating predictions in light of new evidence. This iterative process of gaining feedback, building experience, and correcting models and methods is critical for improving forecasts. Iterative, near-term forecasting will accelerate ecological research, make it more relevant to society, and inform sustainable decision-making under high uncertainty and adaptive management. Here, we identify the immediate scientific and societal needs, opportunities, and challenges for iterative near-term ecological forecasting. Over the past decade, data volume, variety, and accessibility have greatly increased, but challenges remain in interoperability, latency, and uncertainty quantification. Similarly, ecologists have made considerable advances in applying computational, informatic, and statistical methods, but opportunities exist for improving forecast-specific theory, methods, and cyberinfrastructure. Effective forecasting will also require changes in scientific training, culture, and institutions. The need to start forecasting is now; the time for making ecology more predictive is here, and learning by doing is the fastest route to drive the science forward

Vaccine breakthrough hypoxemic COVID-19 pneumonia in patients with auto-Abs neutralizing type I IFNs

Life-threatening `breakthrough' cases of critical COVID-19 are attributed to poor or waning antibody response to the SARS- CoV-2 vaccine in individuals already at risk. Pre-existing autoantibodies (auto-Abs) neutralizing type I IFNs underlie at least 15% of critical COVID-19 pneumonia cases in unvaccinated individuals; however, their contribution to hypoxemic breakthrough cases in vaccinated people remains unknown. Here, we studied a cohort of 48 individuals ( age 20-86 years) who received 2 doses of an mRNA vaccine and developed a breakthrough infection with hypoxemic COVID-19 pneumonia 2 weeks to 4 months later. Antibody levels to the vaccine, neutralization of the virus, and auto- Abs to type I IFNs were measured in the plasma. Forty-two individuals had no known deficiency of B cell immunity and a normal antibody response to the vaccine. Among them, ten (24%) had auto-Abs neutralizing type I IFNs (aged 43-86 years). Eight of these ten patients had auto-Abs neutralizing both IFN-a2 and IFN-., while two neutralized IFN-omega only. No patient neutralized IFN-ss. Seven neutralized 10 ng/mL of type I IFNs, and three 100 pg/mL only. Seven patients neutralized SARS-CoV-2 D614G and the Delta variant (B.1.617.2) efficiently, while one patient neutralized Delta slightly less efficiently. Two of the three patients neutralizing only 100 pg/mL of type I IFNs neutralized both D61G and Delta less efficiently. Despite two mRNA vaccine inoculations and the presence of circulating antibodies capable of neutralizing SARS-CoV-2, auto-Abs neutralizing type I IFNs may underlie a significant proportion of hypoxemic COVID-19 pneumonia cases, highlighting the importance of this particularly vulnerable population

Determining the halo mass scale where galaxies lose their gas

A major question in galaxy formation is how the gas supply that fuels activity in galaxies is modulated by their environment. We use spectroscopy of a set of well characterized clusters and groups at $0.410.4$) of these old galaxies with weak [OII] emission. We use line ratios and compare to studies of local early type galaxies to conclude that this gas is likely excited by post-AGB stars and hence represents a diffuse gas component in the galaxies. For cluster and group galaxies the fraction with EW([OII])$>5$\AA\ is $f_{[OII]}=0.08^{+0.03}_{-0.02}$ and $f_{[OII]}=0.06^{+0.07}_{-0.04}$ respectively. For field galaxies we find $f_{[OII]}=0.27^{+0.07}_{-0.06}$, representing a 2.8$\sigma$ difference between the [OII] fractions for old galaxies between the different environments. We conclude that a population of old galaxies in all environments has ionized gas that likely stems from stellar mass loss. In the field galaxies also experience gas accretion from the cosmic web and in groups and clusters these galaxies have had their gas accretion shut off by their environment. Additionally, galaxies with emission preferentially avoid the virialized region of the cluster in position-velocity space. We discuss the implications of our results, among which is that gas accretion shutoff is likely effective at group halo masses (log~${\cal M}/$\msol$>12.8$) and that there are likely multiple gas removal processes happening in dense environments

Rarely Acquired Type II-A CRISPR-Cas Spacers Mediate Anti-Viral Immunity Through the Targeting of a Non-Canonical PAM Sequence

Viruses that infect prokaryotes, called bacteriophages or phages, are thought to outnumber bacteria by a ratio of 10:1 and provide a constant threat to their hosts. In response, bacteria have evolved numerous defense mechanisms, attacking all major steps of the phage infection and replication cycle. In turn, phages have evolved their own counter defenses. One phage defense system, only recently characterized, has proved revolutionary for our understanding of prokaryote-phage dynamics and our ability to edit genomes of nearly any cell type. CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes) functions as an adaptive immune system by identifying and degrading specific sequences in invading phage nucleic acids. CRISPR loci consist of repeat sequences interspersed with short fragments of phage DNA (spacers). The cas genes code for proteins that regulate 1) the acquisition of new spacers during phage infection (“immunization”) and 2) the use of spacer transcripts to identify and degrade complementary foreign DNA sequences (protospacers) to prevent infection (“immunity”). In the Streptococcus pyogenes type II-A CRISPR-Cas system, the protospacer targets on the viral genome are followed by a conserved “N-G-G” DNA motif, known as the protospacer adjacent motif (PAM). The PAM identifies the phage DNA as “foreign” and triggers the Cas nuclease, Cas9, to cleave the target phage DNA. Most of the spacers present in bacterial populations that survive phage infection target protospacers flanked by N-G-G PAMs. As a consequence, the great majority of acquired spacers target such sequences. However, there is a small fraction of acquired spacers that target non- canonical PAMs. Whether these “non-canonical” spacers originate through accidental acquisition of phage sequences and/or provide efficient defense has been unknown. In the present body of work, we found that many of the identified non-canonical spacers match phage target regions flanked by a PAM of N-A-G-G. Despite being scarcely present in bacterial populations, these “NAGG spacers” provide substantial immunity in vivo and generate RNA guides that support robust DNA cleavage by Cas9 in vitro. Moreover, during phage infection competition assays, bacteria harboring NAGG spacers are similarly fit as, and sometimes even outcompete, those organisms carrying spacers targeting canonical AGG sequences. In contrast, when we tested the acquisition efficiency of NAGG spacers, we found that they are infrequently incorporated into the CRISPR array, several orders of magnitude less often than AGG spacers. We therefore conclude that, while NAGG PAMs facilitate robust targeting of phage DNA, these particular non-canonical sequences are selected against during spacer acquisition. Thus, they seem to operate under different mechanisms during each stage of phage defense. Our findings demonstrate that NAGG PAMs can mediate efficient immunity against invading phages, and at times, result in immune levels previously only seen with canonical PAMs. This indicates a lesser degree of stringency in PAM recognition by Cas9 than is commonly thought. Additionally, our results reveal unexpected, and hitherto unappreciated, differences in PAM recognition during the spacer acquisition versus targeting stages of the type II-A CRISPR-Cas immune response, suggesting a different role of Cas9-PAM interactions in these stages. Together, this work furthers our understanding of how bacteria generate, store, and use “memories” of interactions with foreign DNA, thereby driving the co-evolution of phage, bacteria, and in some cases, human hosts. Moreover, because of CRISPR-Cas9’s many biotechnological applications, deciphering the biological relevance of non-canonical PAMs to Cas9 also has wide-ranging implications, from modulating the specificity and efficiency of gene editing, to tracking the recording of cell memory, to developing potentially life-saving phage therapies that combat antibiotic resistance

BYK191023 (2-[2-(4-methoxy-pyridin-2-yl)-ethyl]-3himidazo[4,5-b]pyridine) is an NADPH- and time-dependent irreversible inhibitor of inducible nitric-oxide synthase

ABSTRACT Imidazopyridine derivates were recently shown to be a novel class of selective and arginine-competitive inhibitors of inducible nitric-oxide synthase (iNOS), and 2-[2-(4-methoxypyridin-2-yl)-ethyl]-3H-imidazo [4,5-b]pyridine (BYK191023) was found to have very high selectivity in enzymatic and cellular models (Mol Pharmacol 69: 328 -337, 2006). Here, we show that BYK191023 irreversibly inactivates murine iNOS in an NADPH-and time-dependent manner, whereas it acts only as a reversible L-arginine-competitive inhibitor in the absence of NADPH or during anaerobic preincubation. Time-dependent irreversible inhibition by BYK191023 could also be demonstrated in intact cells using the RAW macrophage or iNOSoverexpressing human embryonic kidney 293 cell lines. The mechanism of BYK191023 inhibition in the presence of NADPH was studied using spectral, kinetic, chromatographic, and radioligand binding methods. BYK191023-bound iNOS was spectrally indistinguishable from L-arginine-bound iNOS, pointing to an interaction of BYK191023 with the catalytic center of the enzyme. [ 3 H]BYK191023 was recovered quantitatively from irreversibly inactivated iNOS, and no inhibitor metabolite was detected by high-performance liquid chromatography (HPLC). Size exclusion chromatography revealed only about 20% iNOS dissociation into monomers. Furthermore, HPLC and spectrophotometric analysis showed that the irreversible inhibition was associated with loss of heme from iNOS and a reduced ability to form the distinctive ferrous heme-CO complex (cytochrome P450). Thus, enzyme inactivation is mainly caused by heme loss, and it occurs in the inhibitorbound enzyme in the presence of electron flux from NADPH. NO is a highly diffusible biological messenger generated by nitric-oxide synthases (EC1.14.13.39; NOS), a family of enzymes that catalyze the NADPH-dependent conversion of L-arginine to L-citrulline and NO in a two-step proces

A Holocene Sediment Record of Phosphorus Accumulation in Shallow Lake Harris, Florida (USA) Offers New Perspectives on Recent Cultural Eutrophication

<div><p>We studied a complete Holocene sediment record from shallow (z_max = 9.7 m) Lake Harris, Florida (USA) to infer the historical development of the lake and its current eutrophic status. We used ²¹⁰Pb and ¹⁴C to date the 5.9-m sediment sequence (core LH-6-13) and determined accumulation rates for bulk sediment, organic matter, calcium carbonate, phosphorus fractions and biogenic silica fractions. The chronology of changes in sediment characteristics for LH-6-13 is consistent with the general paleoenvironmental framework established by core studies from other Florida lakes. Lake Harris began to fill with water in the early Holocene, ca. 10,680 cal a BP. A shift from carbonate-dominated to organic-rich sediments ca. 5,540 cal a BP corresponds to a transition to wetter climate in the middle Holocene. A rapid increase in diatom biogenic silica concentrations and accumulation rates ca. 2,600 cal a BP signals that the lake had deepened to its modern limnetic state. In LH-6-13, an up-core decrease in rates of accumulation for several sediment variables indicates time-course oligotrophication of the lake through the Holocene. In near-surface sediments, abrupt increases in the accumulation rates of these same variables indicate progressive cultural eutrophication after ca. AD 1900. Comparison of the modern state of Lake Harris to its condition 50–100 years ago provides a measure of the impact of recent cultural eutrophication. Because the pre-disturbance trajectory of this lake was one of oligotrophication, the true impact of cultural eutrophication is even greater than what is inferred from the changes over the past century.</p></div