Rapid Peptide Cyclization Inspired by the Modular Logic of Nonribosomal Peptide Synthetases.

Nonribosomal cyclic peptides (NRcPs) are structurally complex natural products and a vital pool of therapeutics, particularly antibiotics. Their structural diversity arises from the ability of the multidomain enzyme assembly lines, nonribosomal peptide synthetases (NRPSs), to utilize bespoke nonproteinogenic amino acids, modify the linear peptide during elongation, and catalyze an array of cyclization modes, e.g., head to tail, side chain to tail. The study and drug development of NRcPs are often limited by a lack of easy synthetic access to NRcPs and their analogues, with selective macrolactamization being a major bottleneck. Herein, we report a generally applicable chemical macrocyclization method of unprecedented speed and selectivity. Inspired by biosynthetic cyclization, it combines the deprotected linear biosynthetic precursor peptide sequence with a highly reactive C-terminus to produce NRcPs and analogues in minutes. The method was applied to several NRcPs of varying sequences, ring sizes, and cyclization modes including rufomycin, colistin, and gramicidin S with comparable success. We thus demonstrate that the linear order of modules in NRPS enzymes that determines peptide sequence encodes the key structural information to produce peptides conformationally biased toward macrocyclization. To fully exploit this conformational bias synthetically, a highly reactive C-terminal acyl azide is also required, alongside carefully balanced pH and solvent conditions. This allows for consistent, facile cyclization of exceptional speed, selectivity, and atom efficiency. This exciting macrolactamization method represents a new enabling technology for the biosynthetic study of NRcPs and their development as therapeutics.

The human gut symbiont Ruminococcus gnavus shows specificity to blood group A antigen during mucin glycan foraging: Implication for niche colonisation in the gastrointestinal tract.

The human gut symbiont Ruminococcus gnavus displays strain-specific repertoires of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity network analysis identified strain-specific differences in blood-group endo-ß-1,4-galactosidase belonging to the GH98 family. We determined the substrate and linkage specificities of GH98 from R. gnavus ATCC 29149, RgGH98, against a range of defined oligosaccharides and glycoconjugates including mucin. We showed by HPAEC-PAD and LC-FD-MS/MS that RgGH98 is specific for blood group A tetrasaccharide type II (BgA II). Isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR confirmed RgGH98 affinity for blood group A over blood group B and H antigens. The molecular basis of RgGH98 strict specificity was further investigated using a combination of glycan microarrays, site-directed mutagenesis, and X-ray crystallography. The crystal structures of RgGH98 in complex with BgA trisaccharide (BgAtri) and of RgGH98 E411A with BgA II revealed a dedicated hydrogen network of residues, which were shown by site-directed mutagenesis to be critical to the recognition of the BgA epitope. We demonstrated experimentally that RgGH98 is part of an operon of 10 genes that is overexpresssed in vitro when R. gnavus ATCC 29149 is grown on mucin as sole carbon source as shown by RNAseq analysis and RT-qPCR confirmed RgGH98 expression on BgA II growth. Using MALDI-ToF MS, we showed that RgGH98 releases BgAtri from mucin and that pretreatment of mucin with RgGH98 confered R. gnavus E1 the ability to grow, by enabling the E1 strain to metabolise BgAtri and access the underlying mucin glycan chain. These data further support that the GH repertoire of R. gnavus strains enable them to colonise different nutritional niches in the human gut and has potential applications in diagnostic and therapeutics against infection.

Electroreductive Radical Borylation of Unactivated (Hetero)Aryl Chlorides Without Light by Using Cumulene-Based Redox Mediators.

Single-electron transfer (SET) plays a critical role in many chemical processes, from organic synthesis to environmental remediation. However, the selective reduction of inert substrates (Ep/2 <-2 V vs Fc/Fc+ ), such as ubiquitous electron-neutral and electron-rich (hetero)aryl chlorides, remains a major challenge. Current approaches largely rely on catalyst photoexcitation to reach the necessary deeply reducing potentials or suffer from limited substrate scopes. Herein, we demonstrate that cumulenes-organic molecules with multiple consecutive double bonds-can function as catalytic redox mediators for the electroreductive radical borylation of (hetero)aryl chlorides at relatively mild cathodic potentials (approximately -1.9 V vs. Ag/AgCl) without the need for photoirradiation. Electrochemical, spectroscopic, and computational studies support that step-wise electron transfer from reduced cumulenes to electron-neutral chloroarenes is followed by thermodynamically favorable mesolytic cleavage of the aryl radical anion to generate the desired aryl radical intermediate. Our findings will guide the development of other sustainable, purely electroreductive radical transformations of inert molecules using organic redox mediators.

Self-acetylation at the active site of phosphoenolpyruvate carboxykinase (PCK1) controls enzyme activity.

Acetylation is known to regulate the activity of cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme in gluconeogenesis, by promoting the reverse reaction of the enzyme (converting phosphoenolpyruvate to oxaloacetate). It is also known that the histone acetyltransferase p300 can induce PCK1 acetylation in cells, but whether that is a direct or indirect function was not known. Here we initially set out to determine whether p300 can acetylate directly PCK1 in vitro. We report that p300 weakly acetylates PCK1, but surprisingly, using several techniques including protein crystallization, mass spectrometry, isothermal titration calorimetry, saturation-transfer difference nuclear magnetic resonance and molecular docking, we found that PCK1 is also able to acetylate itself using acetyl-CoA independently of p300. This reaction yielded an acetylated recombinant PCK1 with a 3-fold decrease in kcat without changes in Km for all substrates. Acetylation stoichiometry was determined for 14 residues, including residues lining the active site. Structural and kinetic analyses determined that site-directed acetylation of K244, located inside the active site, altered this site and rendered the enzyme inactive. In addition, we found that acetyl-CoA binding to the active site is specific and metal dependent. Our findings provide direct evidence for acetyl-CoA binding and chemical reaction with the active site of PCK1 and suggest a newly discovered regulatory mechanism of PCK1 during metabolic stress.

Fucosidases from the human gut symbiont Ruminococcus gnavus.

The availability and repartition of fucosylated glycans within the gastrointestinal tract contributes to the adaptation of gut bacteria species to ecological niches. To access this source of nutrients, gut bacteria encode α-L-fucosidases (fucosidases) which catalyze the hydrolysis of terminal α-L-fucosidic linkages. We determined the substrate and linkage specificities of fucosidases from the human gut symbiont Ruminococcus gnavus. Sequence similarity network identified strain-specific fucosidases in R. gnavus ATCC 29149 and E1 strains that were further validated enzymatically against a range of defined oligosaccharides and glycoconjugates. Using a combination of glycan microarrays, mass spectrometry, isothermal titration calorimetry, crystallographic and saturation transfer difference NMR approaches, we identified a fucosidase with the capacity to recognize sialic acid-terminated fucosylated glycans (sialyl Lewis X/A epitopes) and hydrolyze α1-3/4 fucosyl linkages in these substrates without the need to remove sialic acid. Molecular dynamics simulation and docking showed that 3'-Sialyl Lewis X (sLeX) could be accommodated within the binding site of the enzyme. This specificity may contribute to the adaptation of R. gnavus strains to the infant and adult gut and has potential applications in diagnostic glycomic assays for diabetes and certain cancers.

Self-Correcting Method for the Measurement of Free Calcium and Magnesium Concentrations by 1H NMR.

A method for the direct measurement of free Ca2+ and Mg2+ concentrations in the range of 1-100 mM by NMR spectroscopy is demonstrated. The method automatically corrects for the effect of ionic strength on the activity of the species in solution and works satisfactorily even when significant concentrations of competitive ions are present. The method requires only the measurement of the 1H chemical shifts of our reporter ligands, glycolate and sulfoacetate, and is easily implemented using NMR imaging techniques. As a proof of concept, we extract the thermodynamic binding constants and conformer distributions of analyte ligands using an in situ ion gradient. Existing approaches for the measurement of free Ca2+ or Mg2+ concentrations by NMR operate only at very low ion concentrations or else require careful recalibration for different sample conditions. By providing the free Ca2+ or Mg2+ concentrations, the proposed methodology significantly enhances the information obtainable via NMR investigations of ion-responsive systems.

Effectiveness of the tobacco tactics program for psychiatric inpatient veterans: an implementation study.

BACKGROUND: The objective of this study was to evaluate the effectiveness of the inpatient, nurse-administered Tobacco Tactics program for patients admitted for psychiatric conditions in two Veterans Affairs (VA) hospitals compared to a control hospital. METHODS: This is a subgroup analysis of data from the inpatient tobacco tactics effectiveness trial, which was a longitudinal, pre- post-nonrandomized comparison design with 6-month follow-up in the three large Veterans Integrated Service Networks (VISN) 11 hospitals. RESULTS: Six-month self-reported quit rates for patients admitted for psychiatric conditions increased from 3.5% pre-intervention to 10.2% post-intervention compared to a decrease in self-reported quit rates in the control hospital (12% pre-intervention to 1.6% post-intervention). There was significant improvement in self-reported quit rates for the pre- versus post-intervention time periods in the Detroit and Ann Arbor intervention sites compared to the Indianapolis control site (P=0.01) and cotinine results were in the same direction. CONCLUSION: The implementation of the Tobacco Tactics intervention has the potential to significantly decrease smoking and smoking-related morbidity and mortality among smokers admitted to VA hospitals for psychiatric disorders.

Effectiveness of the tobacco tactics program in the Department of Veterans Affairs.

PURPOSE: The purpose was to determine the effectiveness of the Tobacco Tactics program in three Veterans Affairs hospitals. METHODS: In this effectiveness trial, inpatient nurses were educated to provide the Tobacco Tactics intervention in Ann Arbor and Detroit, while Indianapolis was the control site (N = 1,070). Smokers were surveyed and given cotinine tests. The components of the intervention included nurse counseling, brochure, DVD, manual, pharmaceuticals, 1-800-QUIT-NOW card, and post-discharge telephone calls. RESULTS: There were significant improvements in 6-month quit rates in the pre- to post-intervention time periods in Ann Arbor (p = 0.004) and Detroit (p < 0.001) compared to Indianapolis. Pre- versus post-intervention quit rates were 4 % compared to 13 % in Detroit, were similar (6 %) pre- and post-intervention in Ann Arbor, and dropped from 26 % to 12 % in Indianapolis. CONCLUSION: The Tobacco Tactics program, which meets the Joint Commission standards that apply to all inpatient smokers, has the potential to significantly decrease smoking among Veterans.

Fast Quantitative Validation of 3D Models of Low-Affinity Protein-Ligand Complexes by STD NMR Spectroscopy.

Low-affinity protein-ligand interactions are important for many biological processes, including cell communication, signal transduction, and immune responses. Structural characterization of these complexes is also critical for the development of new drugs through fragment-based drug discovery (FBDD), but it is challenging due to the low affinity of fragments for the binding site. Saturation transfer difference (STD) NMR spectroscopy has revolutionized the study of low-affinity receptor-ligand interactions enabling binding detection and structural characterization. Comparison of relaxation and exchange matrix calculations with 1H STD NMR experimental data is essential for the validation of 3D structures of protein-ligand complexes. In this work, we present a new approach based on the calculation of a reduced relaxation matrix, in combination with funnel metadynamics MD simulations, that allows a very fast generation of experimentally STD-NMR-validated 3D structures of low-affinity protein-ligand complexes.

MOFganic Chemistry: Challenges and Opportunities for Metal-Organic Frameworks in Synthetic Organic Chemistry.

Metal-organic frameworks (MOFs) are porous, crystalline solids constructed from organic linkers and inorganic nodes that have been widely studied for applications in gas storage, chemical separations, and drug delivery. Owing to their highly modular structures and tunable pore environments, we propose that MOFs have significant untapped potential as catalysts and reagents relevant to the synthesis of next-generation therapeutics. Herein, we outline the properties of MOFs that make them promising for applications in synthetic organic chemistry, including new reactivity and selectivity, enhanced robustness, and user-friendly preparation. In addition, we outline the challenges facing the field and propose new directions to maximize the utility of MOFs for drug synthesis. This perspective aims to bring together the organic and MOF communities to develop new heterogeneous platforms capable of achieving synthetic transformations that cannot be replicated by homogeneous systems.

Factors influencing fluid intake behavior among kidney stone formers.

PURPOSE: We determined factors influencing the behavior of patients with kidney stones in the prevention of recurrent stones. MATERIALS AND METHODS: Patients with stones from an academic and a community practice were recruited for key informant interviews and focus groups. Groups were guided based on the framework of the health belief model. Content analysis was done on transcriptions using qualitative data analysis software. RESULTS: Key informant interviews were completed with 16 patients and with a total of 29 subjects in 5 focus groups. Content analysis revealed that patients were highly motivated to prevent stones. The minimum level of perceived benefit for adopting the behavior change varied among patients and the behaviors proposed. An important strategy to increase fluid intake was insuring availability with containers. Patients were more consistently confident in the ability to increase fluid, in contrast to ingesting medicine or changing the diet. While barriers to increasing fluid were multifactorial among individuals, the barriers aligned into 3 progressive stages that were associated with distinct patient characteristics. Stage 1 barriers included not knowing the benefits of fluid or not remembering to drink. Stage 2 barriers included disliking the taste of water, lack of thirst and lack of availability. Stage 3 barriers included the need to void frequently and related workplace disruptions. CONCLUSIONS: Patients with kidney stones are highly motivated to prevent recurrence and were more amenable to fluid intake change than to another dietary or pharmaceutical intervention. Barriers preventing fluid intake success aligned into 3 progressive stages. Tailoring fluid intake counseling based on patient stage may improve fluid intake behavior.

Prevalence of amyotrophic lateral sclerosis (ALS), United States, 2016.

Objective: To estimate the prevalence of amyotrophic lateral sclerosis (ALS) in the United States for 2016 using data from the National ALS Registry (Registry). Established in 2009, the Registry collects data on ALS patients in the U.S. to better describe the epidemiology of ALS, examine risk factors such as environmental and occupational exposures, and characterize the demographics of those living with the disease. Methods: To identify adult prevalent cases of ALS, the Registry compiles data from three national administrative databases (maintained by the Centers for Medicare and Medicaid Services, the Veterans Health Administration, and the Veterans Benefits Administration). To ascertain cases not necessarily included in these databases and to better understand risk-factors associated with ALS and disease progression, the Registry also includes data collected from patients who voluntarily enroll via a web portal to complete online surveys. Results: In 2016, the Registry conservatively identified 16,424 adult persons who met the Registry definition of ALS for an age-adjusted prevalence rate of 5.2 per 100,000 U.S. population. The pattern of patient characteristics (e.g., age, sex, and race/ethnicity) has not changed from previous Registry reports. Overall, ALS was more common among whites, males, and persons aged 60-69 years. The age groups with the lowest number of ALS cases were persons aged 18-39 years. Males had a higher prevalence rate of ALS than females overall and across all data sources. Conclusions: Data collected by the National ALS Registry are being used to better describe the epidemiology and demographics of ALS in the U.S.

Cross-reactivity of glycan-reactive HIV-1 broadly neutralizing antibodies with parasite glycans.

The HIV-1 Envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs). Env is heavily glycosylated with host-derived N-glycans, and many bnAbs bind to, or are dependent upon, Env glycans for neutralization. Although glycan-binding bnAbs are frequently detected in HIV-infected individuals, attempts to elicit them have been unsuccessful because of the poor immunogenicity of Env N-glycans. Here, we report cross-reactivity of glycan-binding bnAbs with self- and non-self N-glycans and glycoprotein antigens from different life-stages of Schistosoma mansoni. Using the IAVI Protocol C HIV infection cohort, we examine the relationship between S. mansoni seropositivity and development of bnAbs targeting glycan-dependent epitopes. We show that the unmutated common ancestor of the N332/V3-specific bnAb lineage PCDN76, isolated from an HIV-infected donor with S. mansoni seropositivity, binds to S. mansoni cercariae while lacking reactivity to gp120. Overall, these results present a strategy for elicitation of glycan-reactive bnAbs which could be exploited in HIV-1 vaccine development.

Practicing internal medicine onboard the USNS COMFORT in the aftermath of the Haitian earthquake.

On 12 January 2010, a 7.0-magnitude earthquake devastated the island nation of Haiti, leading to the world's largest humanitarian effort in over 6 decades. The catastrophe caused massive destruction of homes and buildings and overwhelmed the Haitian health care system. The United States responded immediately with a massive relief effort, sending U.S. military forces and civilian volunteers to Haiti's aid and providing a tertiary care medical center aboard the USNS COMFORT hospital ship. The COMFORT offered sophisticated medical care to a geographically isolated population and helped to transfer resource-intensive patients from other treatment facilities. Working collaboratively with the surgical staff, ancillary services, and nursing staff, internists aboard the COMFORT were integral to supporting the mission of the hospital ship and provided high-level care to the casualties. This article provides the perspective of the U.S. Navy internists who participated in the initial response to the Haitian earthquake disaster onboard the COMFORT.

NleB/SseK-catalyzed arginine-glycosylation and enteropathogen virulence are finely tuned by a single variable position contiguous to the catalytic machinery.

NleB/SseK effectors are arginine-GlcNAc-transferases expressed by enteric bacterial pathogens that modify host cell proteins to disrupt signaling pathways. While the conserved Citrobacter rodentium NleB and E. coli NleB1 proteins display a broad selectivity towards host proteins, Salmonella enterica SseK1, SseK2, and SseK3 have a narrowed protein substrate selectivity. Here, by combining computational and biophysical experiments, we demonstrate that the broad protein substrate selectivity of NleB relies on Tyr284NleB/NleB1, a second-shell residue contiguous to the catalytic machinery. Tyr284NleB/NleB1 is important in coupling protein substrate binding to catalysis. This is exemplified by S286YSseK1 and N302YSseK2 mutants, which become active towards FADD and DR3 death domains, respectively, and whose kinetic properties match those of enterohemorrhagic E. coli NleB1. The integration of these mutants into S. enterica increases S. enterica survival in macrophages, suggesting that better enzymatic kinetic parameters lead to enhanced virulence. Our findings provide insights into how these enzymes finely tune arginine-glycosylation and, in turn, bacterial virulence. In addition, our data show how promiscuous glycosyltransferases preferentially glycosylate specific protein substrates.

FUT8-Directed Core Fucosylation of N-glycans Is Regulated by the Glycan Structure and Protein Environment.

FUT8 is an essential α-1,6-fucosyltransferase that fucosylates the innermost GlcNAc of N-glycans, a process called core fucosylation. In vitro, FUT8 exhibits substrate preference for the biantennary complex N-glycan oligosaccharide (G0), but the role of the underlying protein/peptide to which N-glycans are attached remains unclear. Here, we explored the FUT8 enzyme with a series of N-glycan oligosaccharides, N-glycopeptides, and an Asn-linked oligosaccharide. We found that the underlying peptide plays a role in fucosylation of paucimannose (low mannose) and high-mannose N-glycans but not for complex-type N-glycans. Using saturation transfer difference (STD) NMR spectroscopy, we demonstrate that FUT8 recognizes all sugar units of the G0 N-glycan and most of the amino acid residues (Asn-X-Thr) that serve as a recognition sequon for the oligosaccharyltransferase (OST). The largest STD signals were observed in the presence of GDP, suggesting that prior FUT8 binding to GDP-ß-l-fucose (GDP-Fuc) is required for an optimal recognition of N-glycans. We applied genetic engineering of glycosylation capacities in CHO cells to evaluate FUT8 core fucosylation of high-mannose and complex-type N-glycans in cells with a panel of well-characterized therapeutic N-glycoproteins. This confirmed that core fucosylation mainly occurs on complex-type N-glycans, although clearly only at selected glycosites. Eliminating the capacity for complex-type glycosylation in cells (KO mgat1) revealed that glycosites with complex-type N-glycans when converted to high mannose lost the core Fuc. Interestingly, however, for erythropoietin that is uncommon among the tested glycoproteins in efficiently acquiring tetra-antennary N-glycans, two out of three N-glycosites obtained Fuc on the high-mannose N-glycans. An examination of the N-glycosylation sites of several protein crystal structures indicates that core fucosylation is mostly affected by the accessibility and nature of the N-glycan and not by the nature of the underlying peptide sequence. These data have further elucidated the different FUT8 acceptor substrate specificities both in vitro and in vivo in cells, revealing different mechanisms for promoting core fucosylation.

Fucosyltransferase-specific inhibition via next generation of fucose mimetics.

The ability to custom-modify cell surface glycans holds great promise for treatment of a variety of diseases. We propose a glycomimetic of l-fucose that markedly inhibits the creation of sLeX by FTVI and FTVII, but has no effect on creation of LeX by FTIX. Our findings thus indicate that selective suppression of sLex display can be achieved, and STD-NMR studies surprisingly reveal that the mimetic does not compete with GDP-fucose at the enzymatic binding site.

Large-scale automated machine reading discovers new cancer-driving mechanisms.

PubMed, a repository and search engine for biomedical literature, now indexes >1 million articles each year. This exceeds the processing capacity of human domain experts, limiting our ability to truly understand many diseases. We present Reach, a system for automated, large-scale machine reading of biomedical papers that can extract mechanistic descriptions of biological processes with relatively high precision at high throughput. We demonstrate that combining the extracted pathway fragments with existing biological data analysis algorithms that rely on curated models helps identify and explain a large number of previously unidentified mutually exclusive altered signaling pathways in seven different cancer types. This work shows that combining human-curated 'big mechanisms' with extracted 'big data' can lead to a causal, predictive understanding of cellular processes and unlock important downstream applications.

Cognitive behaviour therapy for specific phobia of vomiting (Emetophobia): A pilot randomized controlled trial.

This is the first randomised controlled trial to evaluate a protocol for cognitive behaviour therapy (CBT) for a Specific Phobia of Vomiting (SPOV) compared with a wait list and to use assessment scales that are specific for a SPOV. METHOD: 24 participants (23 women and 1 man) were randomly allocated to either 12 sessions of CBT or a wait list. RESULTS: At the end of the treatment, CBT was significantly more efficacious than the wait list with a large effect size (Cohen's d=1.53) on the Specific Phobia of Vomiting Inventory between the two groups after 12 sessions. Six (50%) of the participants receiving CBT achieved clinically significant change compared to 2 (16%) participants in the wait list group. Eight (58.3%) participants receiving CBT achieved reliable improvement compared to 2 (16%) participants in the wait list group. CONCLUSIONS: A SPOV is a condition treatable by CBT but further developments are required to increase efficacy.

Physical, mental, and social catastrophic cognitions as prognostic factors in cognitive-behavioral and pharmacological treatments for panic disorder.

The authors explored the prognostic value of 3 different types of catastrophic cognitions in the treatment of panic disorder with and without mild-to-moderate agoraphobia using a sample of 143 participants who received either cognitive-behavioral therapy (CBT) or imipramine in a randomized controlled trial. Stronger fears of social catastrophes both prior to and following treatment with CBT or imipramine were associated with a poorer outcome. In contrast, cognitions involving physical or mental catastrophes were unrelated to outcome, regardless of whether these thoughts were measured prior to or following treatment. These findings are consistent with the notion that although the intensity of physical catastrophe cognitions may best discriminate between panic disorder and other anxiety disorders, it is the intensity of social catastrophe cognitions that is most closely tied to success in treating this disorder.