Studies of Key Enzymes Involved in the Biosynthesis of the Enediyne Antitumor Antibiotics Neocarzinostatin and C-1027

Thesis advisor: Steven D. BrunerThe enediyne antitumor antibiotics are produced by complex biosynthetic machinery in acetomycetes. This dissertation will focus on the study of three enzymes involved in key steps in the biosynthesis of two enediynes, neocarzinostatin and C-1027. Neocarzinostatin is biosynthesized by a number of enzymes that synthesize and decorate the enediyne core and the peripheral moieties. NcsB1 is one enzyme involved in functionalizing the naphthoic acid portion of neocarzinostatin, a key group involved in binding to target DNA duplexes. The enzyme has been shown to be a promiscuous (S)-adenosylmethionine-dependent O-methyltransferase responsible for methylating a variety of hydroxynaphthoic acids. Multiple crystal structures of NcsB1 cocomplexed to substrate and/or cofactor have been solved. These structures revealed a displacement of the C-terminal domain when not bound to substrate, a movement that likely opens up the active site for naphthoate binding. Additionally, the ternary complex structure of 1,4-dihydroxynaphthoic acid, (S)-adenosylhomocysteine, and NcsB1 was solved and showed a rotation of this alternate substrate in the binding pocket, allowing for methylation. These results led us to probe NcsB1 activity using active site mutants, demonstrating altered substrate specificity and revealing key residues in substrate binding. The final step of neocarzinostatin biosynthesis involves multiple enzymes that convergently assemble the multiple biosynthetic intermediates to form the chromophore. NcsB2, originally proposed to catalyze the attachment of the naphthoic acid moiety to the enediyne core, has been characterized in vitro. Studies into its substrate specificity as an adenylation domain led to a revised biosynthetic pathway of 2-hydroxy-7-methoxy-5-methyl naphthoic acid. Instead of catalyzing the attachment of an enzyme bound naphthoic acid to the enediyne core, NcsB2 was found to act as a CoA-ligase, activating a variety of naphthoic acids and forming a naphthoyl-CoA intermediate. The results of these studies present an outstanding opportunity to produce novel analogs of neocarzinostatin by manipulating its biosynthesis. C-1027 is an architecturally similar enediyne that is also biosynthesized in a convergent route. C-1027 is a member of a class of enediynes that contains a functionalized β-tyrosine derived from L-tyrosine. The first catalytic step towards this beta-tyrosine moiety is achieved by SgTAM, a tyrosine aminomutase that catalyzes a 2,3-amino shift on L-tyrosine to form (S)-β-tyrosine. The first X-ray crystal structure of SgTAM was recently solved by our group, revealing structural homology to ammonia lyases. Through site-directed mutagenesis, X-ray crystallography, and biochemical analysis, residues that influence the mechanism by which SgTAM catalyzes this difficult transformation were explored. From these studies, the enzymatic base and other pertinent residues involved in catalysis have been identified. In addition, residues that close the tunnel leading to the active site, thought to play a key role in mutase activity, were probed. Further study of rational mutants of SgTAM will allow us to engineer its activity to alter its substrate specificity and the type of product it produces.Thesis (PhD) — Boston College, 2009.Submitted to: Boston College. Graduate School of Arts and Sciences.Discipline: Chemistry

High-Flow Vascular Malformations in Children.

Children can have a variety of intracranial vascular anomalies ranging from small and incidental with no clinical consequences to complex lesions that can cause substantial neurologic deficits, heart failure, or profoundly affect development. In contrast to high-flow lesions with direct arterial-to-venous shunts, low-flow lesions such as cavernous malformations are associated with a lower likelihood of substantial hemorrhage, and a more benign course. Management of vascular anomalies in children has to incorporate an understanding of how treatment strategies may affect the normal development of the central nervous system. In this review, we discuss the etiologies, epidemiology, natural history, and genetic risk factors of three high-flow vascular malformations seen in children: brain arteriovenous malformations, intracranial dural arteriovenous fistulas, and vein of Galen malformations

The golden ticket: gaining in-person access to relatives in long-term care homes during the Covid-19 pandemic

Context: Governments made emergency declarations to restrict the presence of family carers in long-term care homes (LTCHs) as part of infection control measures during the pandemic. Within Canada, two visitor statuses were created: ‘essential’ to the health of the resident and ‘non-essential’ or ‘social visitor’, who were subject to additional restrictions. Objective: This study explored family carers’ experiences navigating in-person access to their relatives in LTCH during the pandemic. Methods: Using interpretive description, a sample of 14 family carers (nine daughters, five spouses) living in British Columbia, Canada, participated in in-depth interviews via video call about their experiences between March 2020 and June 2021. Findings: Analyses illustrated variability in carers’ visitor status across families and over time. Two key themes were identified: 1) “Fighting a Losing Battle” describes how reductionist attitudes and policies minimized the role of caregiving and resulted in traumatic disruptions in familial relationships; 2) “Who’s In and Who’s Out” captures inequities in how visitor status policies were applied. Limitations: Restrictions on conducting research during the pandemic resulted in a smaller sample of family carer participants. Implications: Findings highlight the patchwork implementation of visitor policies over the initial 17 months of the pandemic and the precarious space family carers continue to occupy within the LTC sector. Future research should focus on formalising support for family presence during public health emergencies

Bringing high-grade arteriovenous malformations under control: clinical outcomes following multimodality treatment in children.

OBJECTIVE:Brain arteriovenous malformations (AVMs) consist of dysplastic blood vessels with direct arteriovenous shunts that can hemorrhage spontaneously. In children, a higher lifetime hemorrhage risk must be balanced with treatment-related morbidity. The authors describe a collaborative, multimodal strategy resulting in effective and safe treatment of pediatric AVMs. METHODS:A retrospective analysis of a prospectively maintained database was performed in children with treated and nontreated pediatric AVMs at the University of California, San Francisco, from 1998 to 2017. Inclusion criteria were age ≤ 18 years at time of diagnosis and an AVM confirmed by a catheter angiogram. RESULTS:The authors evaluated 189 pediatric patients with AVMs over the study period, including 119 ruptured (63%) and 70 unruptured (37%) AVMs. The mean age at diagnosis was 11.6 ± 4.3 years. With respect to Spetzler-Martin (SM) grade, there were 38 (20.1%) grade I, 40 (21.2%) grade II, 62 (32.8%) grade III, 40 (21.2%) grade IV, and 9 (4.8%) grade V lesions. Six patients were managed conservatively, and 183 patients underwent treatment, including 120 resections, 82 stereotactic radiosurgery (SRS), and 37 endovascular embolizations. Forty-four of 49 (89.8%) high-grade AVMs (SM grade IV or V) were treated. Multiple treatment modalities were used in 29.5% of low-grade and 27.3% of high-grade AVMs. Complete angiographic obliteration was obtained in 73.4% of low-grade lesions (SM grade I-III) and in 45.2% of high-grade lesions. A periprocedural stroke occurred in a single patient (0.5%), and there was 1 treatment-related death. The mean clinical follow-up for the cohort was 4.1 ± 4.6 years, and 96.6% and 84.3% of patients neurologically improved or remained unchanged in the ruptured and unruptured AVM groups following treatment, respectively. There were 16 bleeding events following initiation of AVM treatment (annual rate: 0.02 events per person-year). CONCLUSIONS:Coordinated multidisciplinary evaluation and individualized planning can result in safe and effective treatment of children with AVMs. In particular, it is possible to treat the majority of high-grade AVMs with an acceptable safety profile. Judicious use of multimodality therapy should be limited to appropriately selected patients after thorough team-based discussions to avoid additive morbidity. Future multicenter studies are required to better design predictive models to aid with patient selection for multimodal pediatric care, especially with high-grade AVMs

Synthesis of methylphosphonic acid by marine microbes: a source for methane in the aerobic ocean

Relative to the atmosphere, much of the aerobic ocean is supersaturated with methane; however, the source of this important greenhouse gas remains enigmatic. Catabolism of methylphosphonic acid by phosphorus-starved marine microbes, with concomitant release of methane, has been suggested to explain this phenomenon, yet methylphosphonate is not a known natural product, nor has it been detected in natural systems. Further, its synthesis from known natural products would require unknown biochemistry. Here we show that the marine archaeon Nitrosopumilus maritimus encodes a pathway for methylphosphonate biosynthesis and that it produces cell-associated methylphosphonate esters. The abundance of a key gene in this pathway in metagenomic data sets suggests that methylphosphonate biosynthesis is relatively common in marine microbes, providing a plausible explanation for the methane paradox

Effectiveness and Safety of Adalimumab Biosimilar SB5 in IBD:Outcomes in Originator to SB5 Switch, Double Biosimilar Switch and Bio-Naieve SB5 Observational Cohorts

BACKGROUND AND AIMS: Multiple adalimumab [ADA] biosimilars are now approved for use in inflammatory bowel disease [IBD]; however, effectiveness and safety data remain scarce. We aimed to investigate long-term outcomes of the ADA biosimilar SB5 in IBD patients following a switch from the ADA originator [SB5-switch cohort] or after start of SB5 [SB5-start cohort]. METHODS: We performed an observational cohort study in a tertiary IBD referral centre. All IBD patients treated with Humira underwent an elective switch to SB5. We identified all these patients in a biological prescription database that prospectively registered all ADA start and stop dates including brand names. Data on IBD phenotype, C-reactive protein [CRP], drug persistence, ADA drug and antibody levels, and faecal calprotectin were collected. RESULTS: In total, 481 patients were treated with SB5, 256 in the SB5-switch cohort (median follow-up: 13.7 months [IQR 8.6–15.2]) and 225 in the SB5-start cohort [median follow-up: 8.3 months [4.2–12.8]). Of the SB5-switch cohort, 70.8% remained on SB5 beyond 1 year; 90/256 discontinued SB5, mainly due to adverse events [46/90] or secondary loss of response [37/90]. In the SB5-start cohort, 81/225 discontinued SB5, resulting in SB5-drug persistence of 60.3% beyond 1 year. No differences in clinical remission [p = 0.53], CRP [p = 0.80], faecal calprotectin [p = 0.40] and ADA trough levels [p = 0.55] were found between baseline, week 26 and week 52 following switch. Injection site pain was the most frequently reported adverse event. CONCLUSION: Switching from ADA originator to SB5 appeared effective and safe in this study with over 12 months of follow-up

Humanity’s diverse predatory niche and its ecological consequences

Although humans have long been predators with enduring nutritive and cultural relationships with their prey, seldom have conservation ecologists considered the divergent predatory behavior of contemporary, industrialized humans. Recognizing that the number, strength and diversity of predator-prey relationships can profoundly influence biodiversity, here we analyze humanity’s modern day predatory interactions with vertebrates and estimate their ecological consequences. Analysing IUCN ‘use and trade’ data for ~47,000 species, we show that fishers, hunters and other animal collectors prey on more than a third (~15,000 species) of Earth’s vertebrates. Assessed over equivalent ranges, humans exploit up to 300 times more species than comparable non-human predators. Exploitation for the pet trade, medicine, and other uses now affects almost as many species as those targeted for food consumption, and almost 40% of exploited species are threatened by human use. Trait space analyses show that birds and mammals threatened by exploitation occupy a disproportionally large and unique region of ecological trait space, now at risk of loss. These patterns suggest far more species are subject to human-imposed ecological (e.g., landscapes of fear) and evolutionary (e.g., harvest selection) processes than previously considered. Moreover, continued overexploitation will likely bear profound consequences for biodiversity and ecosystem function

Effective health care for older people living and dying in care homes: A realist review

Background: Care home residents in England have variable access to health care services. There is currently no coherent policy or consensus about the best arrangements to meet these needs. The purpose of this review was to explore the evidence for how different service delivery models for care home residents support and/or improve wellbeing and health-related outcomes in older people living and dying in care homes. Methods: We conceptualised models of health care provision to care homes as complex interventions. We used a realist review approach to develop a preliminary understanding of what supported good health care provision to care homes. We completed a scoping of the literature and interviewed National Health Service and Local Authority commissioners, providers of services to care homes, representatives from the Regulator, care home managers, residents and their families. We used these data to develop theoretical propositions to be tested in the literature to explain why an intervention may be effective in some situations and not others. We searched electronic databases and related grey literature. Finally the findings were reviewed with an external advisory group. Results: Strategies that support and sustain relational working between care home staff and visiting health care professionals explained the observed differences in how health care interventions were accepted and embedded into care home practice. Actions that encouraged visiting health care professionals and care home staff jointly to identify, plan and implement care home appropriate protocols for care, when supported by ongoing facilitation from visiting clinicians, were important. Contextual factors such as financial incentives or sanctions, agreed protocols, clinical expertise and structured approaches to assessment and care planning could support relational working to occur, but of themselves appeared insufficient to achieve change. Conclusion: How relational working is structured between health and care home staff is key to whether health service interventions achieve health related outcomes for residents and their respective organisations. The belief that either paying clinicians to do more in care homes and/or investing in training of care home staff is sufficient for better outcomes was not supported.This research was funded by National Institute of Health Research Health Service Delivery and Research programme (HSDR 11/021/02)

Increasing vegetable intakes: rationale and systematic review of published interventions

Purpose While the health benefits of a high fruit and vegetable consumption are well known and considerable work has attempted to improve intakes, increasing evidence also recognises a distinction between fruit and vegetables, both in their impacts on health and in consumption patterns. Increasing work suggests health benefits from a high consumption specifically of vegetables, yet intakes remain low, and barriers to increasing intakes are prevalent making intervention difficult. A systematic review was undertaken to identify from the published literature all studies reporting an intervention to increase intakes of vegetables as a distinct food group. Methods Databases—PubMed, PsychInfo and Medline—were searched over all years of records until April 2015 using pre-specified terms. Results Our searches identified 77 studies, detailing 140 interventions, of which 133 (81 %) interventions were conducted in children. Interventions aimed to use or change hedonic factors, such as taste, liking and familiarity (n = 72), use or change environmental factors (n = 39), use or change cognitive factors (n = 19), or a combination of strategies (n = 10). Increased vegetable acceptance, selection and/or consumption were reported to some degree in 116 (83 %) interventions, but the majority of effects seem small and inconsistent. Conclusions Greater percent success is currently found from environmental, educational and multi-component interventions, but publication bias is likely, and long-term effects and cost-effectiveness are rarely considered. A focus on long-term benefits and sustained behaviour change is required. Certain population groups are also noticeably absent from the current list of tried interventions

Multilocus Sequence Typing as a Replacement for Serotyping in Salmonella enterica

Salmonella enterica subspecies enterica is traditionally subdivided into serovars by serological and nutritional characteristics. We used Multilocus Sequence Typing (MLST) to assign 4,257 isolates from 554 serovars to 1092 sequence types (STs). The majority of the isolates and many STs were grouped into 138 genetically closely related clusters called eBurstGroups (eBGs). Many eBGs correspond to a serovar, for example most Typhimurium are in eBG1 and most Enteritidis are in eBG4, but many eBGs contained more than one serovar. Furthermore, most serovars were polyphyletic and are distributed across multiple unrelated eBGs. Thus, serovar designations confounded genetically unrelated isolates and failed to recognize natural evolutionary groupings. An inability of serotyping to correctly group isolates was most apparent for Paratyphi B and its variant Java. Most Paratyphi B were included within a sub-cluster of STs belonging to eBG5, which also encompasses a separate sub-cluster of Java STs. However, diphasic Java variants were also found in two other eBGs and monophasic Java variants were in four other eBGs or STs, one of which is in subspecies salamae and a second of which includes isolates assigned to Enteritidis, Dublin and monophasic Paratyphi B. Similarly, Choleraesuis was found in eBG6 and is closely related to Paratyphi C, which is in eBG20. However, Choleraesuis var. Decatur consists of isolates from seven other, unrelated eBGs or STs. The serological assignment of these Decatur isolates to Choleraesuis likely reflects lateral gene transfer of flagellar genes between unrelated bacteria plus purifying selection. By confounding multiple evolutionary groups, serotyping can be misleading about the disease potential of S. enterica. Unlike serotyping, MLST recognizes evolutionary groupings and we recommend that Salmonella classification by serotyping should be replaced by MLST or its equivalents