Structure-Activity Relationships of the Fentanyl Scaffold: Identification of Antagonists as Potential Opioid Overdose Reversal Agents.

Opioid-related overdoses account for almost half of all drug overdose deaths in the United States and cause more preventable deaths every year than car crashes. Fentanyl, a highly potent mu opioid receptor (MOR) agonist and its analogues (fentalogues) are increasingly found in illicit drug samples, both where the primary drug of abuse is an opioid and where it is not. The prevalence of fentalogues in the illicit drug market is thought to be the primary driver of the increased number of opioid-related overdose deaths since 2016. In fact, fentanyl and its analogues are involved in more than 70% of opioid-related overdoses. The standard opioid overdose rescue therapy naloxone is often insufficient to reverse opioid overdoses caused by fentalogue agonists under current treatment paradigms. However, the pharmacology of many fentalogues is unknown. Moreover, within the fentalogue series of compounds, it is possible that antagonists could be identified that might be superior to naloxone as opioid overdose reversal agents. In this report, we explore the pharmacology of 70 fentalogues and identify compounds that behave as MOR antagonists in vitro and demonstrate with one of these reversals of fentanyl-induced respiratory depression in the mouse. Such compounds could provide leads for the development of effective agents for the reversal of opioid overdose.

Priorities, opportunities, and challenges for integrating microorganisms into Earth system models for climate change prediction.

Climate change jeopardizes human health, global biodiversity, and sustainability of the biosphere. To make reliable predictions about climate change, scientists use Earth system models (ESMs) that integrate physical, chemical, and biological processes occurring on land, the oceans, and the atmosphere. Although critical for catalyzing coupled biogeochemical processes, microorganisms have traditionally been left out of ESMs. Here, we generate a "top 10" list of priorities, opportunities, and challenges for the explicit integration of microorganisms into ESMs. We discuss the need for coarse-graining microbial information into functionally relevant categories, as well as the capacity for microorganisms to rapidly evolve in response to climate-change drivers. Microbiologists are uniquely positioned to collect novel and valuable information necessary for next-generation ESMs, but this requires data harmonization and transdisciplinary collaboration to effectively guide adaptation strategies and mitigation policy.

The language of marketing hyperbole and consumer perception-The case of Glasgow.

The aim of the study was to explore the potential of a timed word association task to generate detailed insights into the perception of Glasgow city and its people which could inform destination and brand marketing. Destination marketers have a challenge to convey the tourist destination image to attract and satisfy the expectations of its visitors. Yet destination perceptions are often the result of multiple tourist visitor experiences at a location, neglecting the voice of the resident. The extent to which word associations varied by participants' relationship to Glasgow was identified in terms of Aaker's brand personality scale, an extension of personality research on brands and destinations. Surveying of 1,219 UK participants generated a total of 5,993 terms (city; 1,144 unique) and 5,034 terms (people; 944 unique). The value of capturing the perceptions of a destination by its residents is identified. The results showed that the city of Glasgow was often described as cold and busy, while the people were primarily described as friendly and funny. Evidence was found in support of dual-processing theory suggesting word associations based on lived experiences of a city may be generated later (in terms of the order in which the terms were generated) in a word association task, while common linguistic associations (e.g. synonyms, antonyms, hierarchies etc.) tend to be generated earlier in the task. As hypothesised, analyses revealed a significant relationship between several of the Aaker-dimensions of brand personality, and the consumers' relationship to Glasgow, extending marketing research with an empirical approach to identifying differences in the perceived personality of a destination. The study offers a practical, fast, and replicable method for destination marketers to study consumer perception at scale, which is currently not widely utilised in this field. In particular, the use of semantic distance and word embeddings provides a readily available approach to automatically categorise content derived from word associations studies, or indeed, any text-based content. In contrast, financial investment in non-validated branding and destination marketing campaigns appear to be increasingly problematic. Advances were made in testing an approach to interpreting word associations through the lens of linguistic and situated simulation (LASS) theory to provide deeper analysis to both categorise and interpret consumers' perception. Traditional approaches to tourism marketing and destination branding rarely provide such a level of analytical appraisal. The analysis presented in this paper challenges the orthodoxy and validity of investment in brand and destination marketing at a city level and the potential for word association tasks to be used as a valuable alternative method to create more effective destination marketing and branding.

Evolution of a minimal cell.

Possessing only essential genes, a minimal cell can reveal mechanisms and processes that are critical for the persistence and stability of life1,2. Here we report on how an engineered minimal cell3,4 contends with the forces of evolution compared with the Mycoplasma mycoides non-minimal cell from which it was synthetically derived. Mutation rates were the highest among all reported bacteria, but were not affected by genome minimization. Genome streamlining was costly, leading to a decrease in fitness of greater than 50%, but this deficit was regained during 2,000 generations of evolution. Despite selection acting on distinct genetic targets, increases in the maximum growth rate of the synthetic cells were comparable. Moreover, when performance was assessed by relative fitness, the minimal cell evolved 39% faster than the non-minimal cell. The only apparent constraint involved the evolution of cell size. The size of the non-minimal cell increased by 80%, whereas the minimal cell remained the same. This pattern reflected epistatic effects of mutations in ftsZ, which encodes a tubulin-homologue protein that regulates cell division and morphology5,6. Our findings demonstrate that natural selection can rapidly increase the fitness of one of the simplest autonomously growing organisms. Understanding how species with small genomes overcome evolutionary challenges provides critical insights into the persistence of host-associated endosymbionts, the stability of streamlined chassis for biotechnology and the targeted refinement of synthetically engineered cells2,7-9.

Microbiology and Climate Change: a Transdisciplinary Imperative.

Climate change is a complex problem involving nonlinearities and feedback that operate across scales. No single discipline or way of thinking can effectively address the climate crisis. Teams of natural scientists, social scientists, engineers, economists, and policymakers must work together to understand, predict, and mitigate the rapidly accelerating impacts of climate change. Transdisciplinary approaches are urgently needed to address the role that microorganisms play in climate change. Here, we demonstrate with case studies how diverse teams and perspectives provide climate-change insight related to the range expansion of emerging fungal pathogens, technological solutions for harmful cyanobacterial blooms, and the prediction of disease-causing microorganisms and their vector populations using massive networks of monitoring stations. To serve as valuable members of a transdisciplinary climate research team, microbiologists must reach beyond the boundaries of their immediate areas of scientific expertise and engage in efforts to build open-minded teams aimed at scalable technologies and adoptable policies.

Genome Sequence of Gordonia rubripertincta Phage Survivors, a Cluster CT Siphovirus.

Bacteriophage Survivors is a siphovirus isolated from Gordonia rubripertincta NRRL B-16540. Survivors has a 45,436-bp genome encoding 69 predicted protein-coding genes, of which 32 have assigned functions. Based on gene content similarity to sequenced actinobacteriophages, Survivors is assigned to phage cluster CT.

Phage-Encoded Sigma Factors Alter Bacterial Dormancy.

By entering a reversible state of reduced metabolic activity, dormant microorganisms are able to tolerate suboptimal conditions that would otherwise reduce their fitness. Dormancy may also benefit bacteria by serving as a refuge from parasitic infections. Here, we focus on dormancy in the Bacillota, where endospore development is transcriptionally regulated by the expression of sigma factors. A disruption of this process could influence the survivorship or reproduction of phages that infect spore-forming hosts with implications for coevolutionary dynamics. We characterized the distribution of sigma factors in over 4,000 genomes of diverse phages capable of infecting hosts that span the bacterial domain. From this, we identified homologs of sporulation-specific sigma factors in phages that infect spore-forming hosts. Unlike sigma factors required for phage reproduction, we provide evidence that sporulation-like sigma factors are nonessential for lytic infection. However, when expressed in the spore-forming Bacillus subtilis, some of these phage-derived sigma factors can activate the bacterial sporulation gene network and lead to a reduction in spore yield. Our findings suggest that the acquisition of host-like transcriptional regulators may allow phages to manipulate a complex and ancient trait in one of the most abundant cell types on Earth. IMPORTANCE As obligate parasites, phages exert strong top-down pressure on host populations with eco-evolutionary implications for community dynamics and ecosystem functioning. The process of phage infection, however, is constrained by bottom-up processes that influence the energetic and nutritional status of susceptible hosts. Many phages have acquired auxiliary genes from bacteria, which can be used to exploit host metabolism with consequences for phage fitness. In this study, we demonstrate that phages infecting spore-forming bacteria carry homologs of sigma factors, which their hosts use to orchestrate gene expression during spore development. By tapping into regulatory gene networks, phages may manipulate the physiology and survival strategies of nongrowing bacteria in ways that influence host-parasite coevolution.

Dormancy dampens the microbial distance-decay relationship.

Much of Earth's biodiversity has the capacity to engage in dormancy, a reversible state of reduced metabolic activity. By increasing resilience to unfavourable conditions, dormancy leads to the accumulation of 'seed banks'. These reservoirs of genetic and phenotypic diversity should diminish the strength of environmental filtering and increase rates of dispersal. Although prevalent among single-celled organisms, evidence that dormancy influences patterns of microbial biogeography is lacking. We constructed geographical and environmental distance-decay relationships (DDRs) for the total (DNA) and active (RNA) portions of bacterial communities in a regional-scale 16S rRNA survey of forested ponds in Indiana, USA. As predicted, total communities harboured greater diversity and exhibited weaker DDRs than active communities. These observations were robust to random resampling and different community metrics. To evaluate the processes underlying the biogeographic patterns, we developed a platform of mechanistic models that used the geographical coordinates and environmental characteristics of our study system. Based on more than 106 simulations, our models approximated the empirical DDRs when there was strong environmental filtering along with the presence of long-lived seed banks. By contrast, the inclusion of dispersal generally decreased model performance. Together, our findings support recent theoretical predictions that seed banks can influence the biogeographic patterns of microbial communities. This article is part of the theme issue 'Conceptual challenges in microbial community ecology'.

Structure-Activity Relationships of 7-Substituted Dimethyltyrosine-Tetrahydroisoquinoline Opioid Peptidomimetics.

The opioid receptors modulate a variety of biological functions, including pain, mood, and reward. As a result, opioid ligands are being explored as potential therapeutics for a variety of indications. Multifunctional opioid ligands, which act simultaneously at more than one type of opioid receptor, show promise for use in the treatment of addiction, pain, and other conditions. Previously, we reported the creation of bifunctional kappa opioid receptor (KOR) agonist/mu opioid receptor (MOR) partial agonist ligands from the classically delta opioid receptor (DOR) antagonist selective dimethyltyrosine-tetrahydroisoquinoline (Dmt-Tiq) scaffold through the addition of a 7-benzyl pendant on the tetrahydroisoquinoline ring. This study further explores the structure-activity relationships surrounding 7-position pendants on the Dmt-Tiq scaffold. Some analogues maintain a KOR agonist/MOR partial agonist profile, which is being explored in the development of a treatment for cocaine addiction. Others display a MOR agonist/DOR antagonist profile, which has potential to be used in the creation of a less addictive pain medication. Ultimately, we report the synthesis and in vitro evaluation of novel opioid ligands with a variety of multifunctional profiles.

How, When, and Where Relic DNA Affects Microbial Diversity.

Extracellular or "relic" DNA is one of the largest pools of nucleic acids in the biosphere. Relic DNA can influence a number of important ecological and evolutionary processes, but it may also affect estimates of microbial abundance and diversity, which has implications for understanding environmental, engineered, and host-associated ecosystems. We developed models capturing the fundamental processes that regulate the size and composition of the relic DNA pools to identify scenarios leading to biased estimates of biodiversity. Our models predict that bias increases with relic DNA pool size, but only when the species abundance distributions (SADs) of relic and intact DNA are distinct from one another. We evaluated our model predictions by quantifying relic DNA and assessing its contribution to bacterial diversity using 16S rRNA gene sequences collected from different ecosystem types, including soil, sediment, water, and the mammalian gut. On average, relic DNA made up 33% of the total bacterial DNA pool but exceeded 80% in some samples. Despite its abundance, relic DNA had a minimal effect on estimates of taxonomic and phylogenetic diversity, even in ecosystems where processes such as the physical protection of relic DNA are common and predicted by our models to generate bias. Our findings are consistent with the expectation that relic DNA from different taxa degrades at a constant and equal rate, suggesting that it may not fundamentally alter estimates of microbial diversity.IMPORTANCE The ability to rapidly obtain millions of gene sequences and transcripts from a range of environments has greatly advanced understanding of the processes that regulate microbial communities. However, nucleic acids extracted from complex samples do not come only from viable microorganisms. Dead microorganisms can generate large pools of relic DNA that distort insight into the ecology and evolution of microbial systems. Here, we develop a conceptual and quantitative framework for understanding how relic DNA influences the structure of microbiomes. Our theoretical models and empirical results demonstrate that a large relic DNA pool does not automatically lead to biased estimates of microbial diversity. Rather, relic DNA effects emerge in combination with microscale processes that alter the commonness and rarity of sequences found in heterogeneous DNA pools.

Whole-Genome Sequence of the Soil Bacterium Micrococcus sp. KBS0714.

We present here a draft genome assembly of Micrococcus sp. KBS0714, which was isolated from agricultural soil. The genome provides insight into the strategies that Micrococcus spp. use to contend with environmental stressors such as desiccation and starvation in environmental and host-associated ecosystems.

Microbial contributions to subterranean methane sinks.

Sources and sinks of methane (CH4 ) are critical for understanding global biogeochemical cycles and their role in climate change. A growing number of studies have reported that CH4 concentrations in cave ecosystems are depleted, leading to the notion that these subterranean environments may act as sinks for atmospheric CH4 . Recently, it was hypothesized that this CH4 depletion may be caused by radiolysis, an abiotic process whereby CH4 is oxidized via interactions with ionizing radiation derived from radioactive decay. An alternate explanation is that the depletion of CH4 concentrations in caves could be due to biological processes, specifically oxidation by methanotrophic bacteria. We theoretically explored the radiolysis hypothesis and conclude that it is a kinetically constrained process that is unlikely to lead to the rapid loss of CH4 in subterranean environments. We present results from a controlled laboratory experiment to support this claim. We then tested the microbial oxidation hypothesis with a set of mesocosm experiments that were conducted in two Vietnamese caves. Our results reveal that methanotrophic bacteria associated with cave rocks consume CH4 at a rate of 1.3-2.7 mg CH4  · m-2  · d-1 . These CH4 oxidation rates equal or exceed what has been reported in other habitats, including agricultural systems, grasslands, deciduous forests, and Arctic tundra. Together, our results suggest that depleted concentrations of CH4 in caves are most likely due to microbial activity, not radiolysis as has been recently claimed. Microbial methanotrophy has the potential to oxidize CH4 not only in caves, but also in smaller-size open subterranean spaces, such as cracks, fissures, and other pores that are connected to and rapidly exchange with the atmosphere. Future studies are needed to understand how subterranean CH4 oxidation scales up to affect local, regional, and global CH4 cycling.

Microscale Insight into Microbial Seed Banks.

Microbial dormancy leads to the emergence of seed banks in environmental, engineered, and host-associated ecosystems. These seed banks act as reservoirs of diversity that allow microbes to persist under adverse conditions, including extreme limitation of resources. While microbial seed banks may be influenced by macroscale factors, such as the supply of resources, the importance of microscale encounters between organisms and resource particles is often overlooked. We hypothesized that dimensions of spatial, trophic, and resource complexity determine rates of encounter, which in turn, drive the abundance, productivity, and size of seed banks. We tested this using >10,000 stochastic individual based models (IBMs) that simulated energetic, physiological, and ecological processes across combinations of resource, spatial, and trophic complexity. These IBMs allowed realistic dynamics and the emergence of seed banks from ecological selection on random variation in species traits. Macroscale factors like the supply and concentration of resources had little effect on resource encounter rates. In contrast, encounter rates were strongly influenced by interactions between dispersal mode and spatial structure, and also by the recalcitrance of resources. In turn, encounter rates drove abundance, productivity, and seed bank dynamics. Time series revealed that energetically costly traits can lead to large seed banks and that recalcitrant resources can lead to greater stability through the formation of seed banks and the slow consumption of resources. Our findings suggest that microbial seed banks emerge from microscale dimensions of ecological complexity and their influence on resource limitation and energetic costs.

Longitudinal patterns of change in eye-hand coordination in children aged 8-16 years.

Enhanced eye-hand coordination (EHC) is associated with greater participation in physical activity. No longitudinal studies have examined the change in throw-catch EHC from childhood to mid-adolescence. We investigated the development of EHC with an object control test from childhood to mid-adolescence in boys and girls. Evaluated at age 8, 10, 12 and 16 years, EHC was measured as the aggregate success rate of a throw and wall-rebound catch test. The test involved 40 attempts of progressive increasing difficulty, as determined by increased distances from a wall and transitions from two-handed to one-handed catches. Outcomes were treated as quasi-binomial and modelled by generalised linear mixed logistic regression analysis. EHC improved with age from childhood to mid-adolescence, although boys were more adept at each age (p<0.001). The patterns of change in EHC with increasing age varied according to the degree of difficulty of the task (p<0.001); throw and two-handed catch proficiency developing earlier than throw and one-handed catch in both sexes. Boys' EHC was better than girls' as early as age 8 years and male proficiency was maintained through to mid-adolescence. The proficiency of throw and two-handed catch rates developed faster than throw and one-handed catch rates for both sexes.

Knowledge of the indications for endoscopic ultrasound (EUS) among gastroenterologists and non-gastroenterologists.

The level of awareness among Irish doctors of the appropriate indications for endoscopic ultrasound (EUS) is unknown. This study assessed knowledge of EUS indications among consultants and trainees in 3 Irish teaching hospitals. A questionnaire was designed to test knowledge of EUS indications in 4 organ systems: oesophagus, gastroduodenum, hepatopancreatobiliary system and colorectum. The questionnaire was distributed to consultants and trainees (both gastroenterology and non-gastroenterology) in 3 major Irish teaching hospitals. The survey was distributed to 86 doctors, all of whom replied: 18 consultants (11 gastroenterologists,) 40 registrars (28 gastroenterology) and 26 SHOs/interns. Knowledge of appropriate EUS indications was best among consultant gastroenterologists (82%) and GI registrars (79%), compared with non-GI consultants (74%), non-GI registrars (72%) and SHOs/interns (68%). Among gastroenterologists and GI registrars, knowledge levels of oesophageal (89%, 85%) and gastroduodenal applications (92%, 95%) was best while knowledge of colorectal applications (75%, 71%) was poorest. GI consultants and GI registrars display good knowledge of appropriate EUS indications although their knowledge of applications for colorectal disease is poorest. Future studies focusing on the education of non-gastroenterologists of the role of EUS would be helpful.

Outcome of accident and emergency room triaged patients with low risk non-variceal upper gastrointestinal haemorrhage.

An acute upper gastrointestinal haemorrhage (UGIH) is an expensive healthcare problem estimated to cost more than 2.5 billion dollars per annum in the United States. Recent British Society of Gastroenterology (BSG) guidelines (2002) advise that patients with low risk non variceal UGIH have a benign outcome and may be suitable for a relatively short hospital stay with attendant economic saving. We evaluated current clinical experience, endoscopy findings and length of hospital stay in our hospital. We carried out a two year retrospective data analysis of 395 Accident and Emergency Room triaged patients admitted with a low risk non variceal UGIH. Data variables included age, sex, presence of co morbid illness, endoscopic findings, Rockall Risk Score, mortality within the hospitalisation period and length and cost of hospital stay. Of the 395 haemodynamically stable patients identified, requiring 1,644 hospitalisation days, 320/395 (85.8%) had a Rockall Score < or =3. No endoscopic intervention was required in 366/395 (92.7%) of patients with a 2% overall mortality. The mean hospital length of stay (LOS) was 4.16, costing in excess of 1002,840 Euro. Following routine clinical practice for low risk non variceal UGIH, the subsequent duration of hospital stay was unnecessarily prolonged and costly. This highlights the need to initiate change, to monitor resource utilisation and implement early hospital discharge in appropriate patients.

Management of Barrett's oesophagus in 2001 in Ireland.

BACKGROUND: Endoscopic surveillance of patients with Barrett's oesophagus is recommended to detect early carcinoma. The practice patterns of endoscopists since the publication of more recent management guidelines remain unknown. METHODS: All endoscopists (n=68) in the Irish Medical Directory and their trainees were sent a postal questionnaire on Barrett's surveillance. RESULTS: Fifty-five per cent (30/54) perform surveillance on all patients with Barrett's oesophagus and 38% on selected patients. In patients with no dysplasia, repeat endoscopy was more commonly practiced annually (28/54) than every two to three years (23/54). Surgeons were more likely to perform surveillance annually than gastroenterologists (75% vs 40%). Only 26% of endoscopists took four-quadrant biopsies every 2 cm. Intervention was recommended by a majority (28/54) of endoscopists in a patient with high grade dysplasia. A majority of respondents (47/54) would have surveillance if they were found to have Barrett's oesophagus. CONCLUSION: Most endoscopists in Ireland do not adhere to recent guidelines in their management of Barrett's oesophagus. Surgical endoscopists perform surveillance more frequently than their medical colleagues.

Cholesterol biosynthesis regulation and protein changes in rat liver following treatment with fluvastatin.

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is a key regulator in cholesterol biosynthesis and HMG CoA reductase inhibitors (statins) have become a widely prescribed family of lipid lowering agents. Cholesterol synthesis occurs predominantly in liver which is the target organ of statins. We studied the effects of fluvastatin (Lescol), a member of the statin family, on hepatic protein regulation. Male F344 rats treated with 0.8 mg/kg per day fluvastatin or 24 mg/kg per day fluvastatin for 7 days showed treatment-related changes in 58 liver proteins (P<0.005). Major effects were evident in the cholesterol biosynthesis pathway including the induction of enzymes upstream and downstream of the target enzyme HMG CoA reductase. Treatment also triggered alterations in key enzymes of carbohydrate metabolism and was associated with changes in a heterogeneous set of cellular stress proteins involved in cytoskeletal structure, calcium homeostasis and protease activity. The latter set of protein alterations indicates that hepatotoxicity is associated with high-dose treatment. Based on the results it is suggested that HMG-CoA synthase and isopentenyl-diphosphate delta-isomerase may be explored as alternative drug targets and that the induction levels of these enzymes may serve as a measure of potency of individual statin drugs. It is proposed that efficacy and cellular stress markers discovered in this study may be used in a high throughput screen (HTS) assay format to compare efficiently and accurately the therapeutic windows of different members of the statin family.

Long-term treatment with subantimicrobial dose doxycycline exerts no antibacterial effect on the subgingival microflora associated with adult periodontitis.

BACKGROUND: The purpose of this study was to determine whether treatment with subantimicrobial dose doxycycline (SDD), 20 mg bid, exerted an antimicrobial effect on the microflora associated with adult periodontitis. METHODS: Following the approval of the protocol and informed consent forms by the respective IRBs at the University of Florida and West Virginia University, 76 subjects with adult periodontitis were entered and randomly assigned to receive SDD or placebo. A split-mouth design was utilized, with each subject receiving subgingival scaling and root planing (SRP) in two quadrants immediately following baseline data collection, while the remaining two quadrants were left unscaled (non-SRP). Microbial samples were collected prior to treatment, after 3, 6, and 9 months of treatment, and after 3 months of no treatment. The samples were examined by microscopy and by enumeration on selective and non-selective media. RESULTS: All treatments resulted in statistically significant decreases in the proportions of spirochetes and motile rods (P <0.05) and in an increase in the proportion of coccoid forms (P <0.0001) relative to baseline. No between-treatment differences were detected between the SDD and placebo treatments in either the SRP or non-SRP design, with the exception of the small and large spirochetal groups. The spirochetal proportions present in the SDD group were significantly lower (P<0.05) than the paired placebo group during the 9-month treatment and was preceded by a significant decrease (P<0.01) in the proportion of microbiologic sample sites that bled on probing. No between- treatment differences were detected in any of the other microbial parameters. CONCLUSION: The microbial differences observed were attributed to the anticollagenase and anti-inflammatory properties of SDD and not to an antimicrobial effect.