Oxidase enzyme genes are differentially expressed during Acanthamoeba castellanii encystment.

Acanthamoeba castellanii, a ubiquitous protozoan, is responsible for significant diseases such as Acanthamoeba keratitis and granulomatous amoebic encephalitis. A crucial survival strategy of A. castellanii involves the formation of highly resistant cysts during adverse conditions. This study delves into the cellular processes underpinning encystment, focusing on gene expression changes related to reactive oxygen species (ROS) balance, with a particular emphasis on mitochondrial processes. Our findings reveal a dynamic response within the mitochondria during encystment, with the downregulation of key enzymes involved in oxidative phosphorylation (COX, AOX, and NADHalt) during the initial 48 h, followed by their overexpression at 72 h. This orchestrated response likely creates a pro-oxidative environment, facilitating encystment. Analysis of other ROS processing enzymes across the cell reveals differential expression patterns. Notably, antioxidant enzymes, such as catalases, glutaredoxins, glutathione S-transferases, peroxiredoxins, and thioredoxins, mirror the mitochondrial trend of downregulation followed by upregulation. Additionally, glycolysis and gluconeogenesis are downregulated during the early stages in order to potentially balance the metabolic requirement of the cyst. Our study underscores the importance of ROS regulation in Acanthamoeba encystment. Understanding these mechanisms offers insights into infection control and identifies potential therapeutic targets. This work contributes to unraveling the complex biology of A. castellanii and may aid in combatting Acanthamoeba-related infections. Further research into ROS and oxidase enzymes is warranted, given the organism's remarkable respiratory versatility.

A New Molecular Phylogeny of Salps (Tunicata: Thalicea: Salpida) and the Evolutionary History of Their Colonial Architecture.

Salps are marine pelagic tunicates with a complex life cycle, including a solitary and colonial stage composed of asexually budded individuals. These colonies develop into species-specific architectures with distinct zooid orientations, including transversal, oblique, linear, helical, and bipinnate chains, as well as whorls and clusters. The evolutionary history of salp colony architecture has remained obscured due to the lack of an ontology to characterize architectures, as well as a lack of phylogenetic taxon sampling and resolution of critical nodes. We (1) collected and sequenced eight species of salps that had never been sequenced before, (2) inferred the phylogenetic relationships among salps, and (3) reconstructed the evolutionary history of salp colony architecture. We collected salp specimens via offshore SCUBA diving, dissected tissue samples, extracted their DNA, amplified their 18S gene, and sequenced them using Sanger technology. We inferred the phylogeny of Salpida based on 18S using both Maximum Likelihood and Bayesian approaches. Using this phylogeny, we reconstructed the ancestral states of colony architecture using a Bayesian ordered Markov model informed by the presence and absence of specific developmental mechanisms that lead to each architecture. We find that the ancestral salp architecture is either oblique or linear, with every other state being derived. Moreover, linear chains have evolved independently at least three times. While transversal chains are developmentally basal and hypothesized to be ancestral, our phylogenetic topology and reconstructions strongly indicate that they are evolutionarily derived through the loss of zooid torsion. These traits are likely critical to multijet locomotory performance and evolving under natural selection. Our work showcases the need to study the broader diversity of salp species to gain a comprehensive understanding of their organismal biology, evolutionary history, and ecological roles in pelagic ecosystems.

Spanish Abstract Las salpas son tunicados pelágicos marinos con un ciclo vital complejo que incluye una fase solitaria y una colonial, compuesta de individuos gemados asexualmente. Al desarrollarse, estas colonias forman arquitecturas diferentes según la especie con orientaciones distintivas de los zooides. Estas arquitecturas incluyen las cadenas transversales, las oblícuas, las lineales, las solenoides, y también las formas de rueda y conglómero. La historia evolutiva de la arquitectura colonial de las salpas ha permanecido en la sombras debido a la falta de un ontología para caracterizar estas arquitecturas, además de por falta de representación taxonómica y resolución de nodos críticos en la filogenia. Hemos (1) muestreado y secuenciado ocho especies de salpa que nunca antes habían sido secuenciadas, (2) hemos inferido las relaciones filogenéticas entre salpas, y (3) reconstruido la historia evolutiva de la arquitectura colonial en salpas. Hemos obtenido especímenes de salpas mediante buceo con escafandra en alta mar, diseccionado muestras de tejido, extraído el ADN, amplificado y secuenciado el gen ribosomal 18S mediante tecnología Sanger. Hemos inferido la filogenia del orden Salpida en base al gen 18S utilizando tanto Máxima Verosimilitud como métodos Bayesianos. Utilizando esta filogenia, hemos reconstruido los estados ancestrales de la arquitectura colonial mediante un modelo Bayesiano Markov ordinal informado por la presencia y ausencia de mecanismos ontogenéticos que derivan en cada arquitectura. Hallamos que la arquitectura ancestral en las salpas fue o bien oblícua o lineal, con todos los demás estados derivados. Además, las cadenas lineales han evolucionado de forma independiente por lo menos tres veces. Las cadenas transversales son el estado basal en el desarrollo y han sido hipotetizadas como el estado ancestral. Sin embargo, nuestra topología filogenética y reconstrucciones indican firmemente que son en realidad derivadas a través de la pérdida del mecanismo de torsion de los zooides. Es probable que estos rasgos morfológicos sean críticos para la eficiencia locomotora de la propulsión multi-chorro y por tanto hayan evolucionado bajo el efecto de la selección natural. Nuestro trabajo demuestra la necesidad de estudiar la amplia diversidad de especies en las salpas para obtener una comprensión integral de su biología organísmica, historia evolutiva, y roles ecológicos en los ecosistemas pelágicos.

Gut microbiome of Crocodylus porosus and cellular stress: inhibition of nitric oxide, interleukin 1-beta, tumor necrosis factor-alpha, and prostaglandin E2 in cerebrovascular endothelial cells.

Crocodiles are renowned for their resilience and capacity to withstand environmental stressors, likely influenced by their unique gut microbiome. In this study, we determined whether selected gut bacteria of Crocodylus porosus exhibit anti-inflammatory effects in response to stress, by measuring nitric oxide release, interleukin 1-beta, tumor necrosis factor-alpha, and prostaglandin E2 in cerebrovascular endothelial cells. Using the Griess assay, the findings revealed that among several C. porosus gut bacterial isolates, the conditioned media containing the metabolites of two bacterial strains (CP27 and CP36) inhibited nitric oxide production significantly, in response to the positive control, i.e., taxol-treatment. Notably, CP27 and CP36 were more potent at reducing nitric oxide production than senloytic compounds (fisetin, quercetin). Using enzyme linked immunosorbent assays, the production of pro-inflammatory cytokines (IL-1ß, TNF-α, PGE2), was markedly reduced by treatment with CP27 and CP36, in response to stress. Both CP27 and CP36 contain a plethora of metabolites to exact their effects [(3,4-dihydroxyphenylglycol, 5-methoxytryptophan, nifedipine, 4-chlorotestosterone-17-acetate, 3-phenoxypropionic acid, lactic acid, f-Honaucin A, l,l-Cyclo(leucylprolyl), 3-hydroxy-decanoic acid etc.], indicative of their potential in providing protection against cellular stress. Further high-throughput bioassay-guided testing of gut microbial metabolites from crocodiles, individually as well as in combination, together with the underlying molecular mechanisms, in vitro and in vivo will elucidate their value in the rational development of innovative therapies against cellular stress/gut dysbiosis.

Dogs' health and demographics in wildlife-populated and tsetse-infested villages of Mambwe district, eastern Zambia.

Good dog-keeping practices and access to veterinary care are essential for the well-being of dogs. As the main causes of morbidity and mortality in the rural canine population in Zambia are poorly understood, we followed a cohort of 162 indigenous dogs for six months in wildlife-populated and tsetse-infested villages of Mambwe district, eastern Zambia to gain deeper insights. Dogs lacked basic home and veterinary care, they were often starved and burdened with ticks, and some passed live adult worms in their stool. The frequent exposure of dogs to tsetse bites and consumption of fresh raw game meat and bones puts them at greater risk of acquiring African trypanosomiasis. Nearly 20 % of dogs were lost to follow-up, with the main causes being poor health (58.1 %), predation by wild carnivores (29 %), and owner culling or euthanasia (12.9 %). We observed that indigenous dogs' general well-being and survival were largely influenced by their environment, infectious diseases, injuries sustained during interaction with conspecifics and wildlife, and community attitudes and practices associated with dog ownership.

Bacterial flora varies throughout the saltwater crocodile (Crocodylus porosus) gastrointestinal tract.

OBJECTIVE: The objective of this study was to determine bacterial flora throughout the gastrointestinal tract of a saltwater crocodile (Crocodylus porosus) using 16S rRNA gene analysis. ANIMALS: A convention on international trade in endangered species (CITES) of wild fauna and flora registered crocodile farm, provided a healthy male saltwater crocodile, Crocodylus porosus for this study. PROCEDURES: Three samples were taken from the oral cavity, 3 samples from the proximal region of the small intestine (jejunum), and 3 samples from the distal part of the large intestine of the gastrointestinal tract of C. porosus were obtained using sterile cotton swabs. Next, swabs were placed in 15 mL sterile centrifuge tubes, individually, and kept on ice for immediate transportation to the laboratory. This was followed by 16S rRNA gene analysis using specific primers (341F-CCTAYGGGRBGCASCAG, and 806R-GGACTACNNGGGTATCTAAT). Amplicons were sequenced on Illumina paired-end platform, and bacterial gastrointestinal communities, the relative abundance of taxa, and principal component and coordinate analysis were performed. RESULTS: The findings revealed that bacterial community structures from differing regions exhibited several differences. The number of observed bacterial operational taxonomic units (OTUs) was 153 in the oral cavity, 239 in the small intestine, and 119 in the large intestine of C. porosus. The small intestine reflects the highest richness. In contrast, the large intestine exhibited the least richness of microbial communities. Relative abundance of taxa showed that Proteobacteria, Bacteroidetes, and Firmicutes were dominant in all 3 sample sites. Pseudomonas differed in the oral cavity and the large intestine, with the latter exhibiting less distribution of Pseudomonas. Stenotrophomonas and Castellaniella were higher in the oral cavity, while the relative abundance of Comamonas and Salmonella was higher in the small intestine. Conversely, the relative abundance of Salmonella and Pannonibacter was augmented in the large intestine. CLINICAL RELEVANCE: For the first time, this study demonstrates the bacterial diversity along the segments of the gastrointestinal tract of C. porosus. Bacterial flora varies throughout the gastrointestinal tract. Although further studies using large cohorts are warranted; however, our findings suggest that microbiome composition may have the potential as a biomarker in determining the overall health and well-being of C. porosus.

Mass spectrometric analysis of bioactive conditioned media of bacteria isolated from reptilian gut.

Aim: To determine whether selected gut bacteria of crocodile exhibit antibacterial properties. Materials & methods: Two bacteria isolated from Crocodylus porosus gut were used, namely: Pseudomonas aeruginosa and Aeromonas dhakensis. Conditioned media were tested against pathogenic bacteria and metabolites were analyzed using liquid chromatography-mass spectrometry. Results & conclusion: Antibacterial assays revealed that conditioned media showed potent effects against pathogenic Gram-positive and Gram-negative bacteria. LC-MS revealed identity of 210 metabolites. The abundant metabolites were, N-Acetyl-L-tyrosine, Acetaminophen, Trans-Ferulic acid, N, N-Dimethylformamide, Pyrocatechol, Cyclohexanone, Diphenhydramine, Melatonin, Gamma-terpinene, Cysteamine, 3-phenoxypropionic acid, Indole-3-carbinol, Benzaldehyde, Benzocaine, 2-Aminobenzoic acid, 3-Methylindole. These findings suggest that crocodile gut bacteria are potential source of novel bioactive molecules that can be utilized as pre/post/antibiotics for the benefit of human health.

Crocodiles thrive in unsanitary conditions, feed on rotten meat, and endure conditions that are detrimental to human health. In addition to their immune system, we speculate that their microbial gut flora produce substances contributing to their "hardiness" and "longevity". Herein, we showed that selected bacteria isolated from crocodile gut produced potent antibacterial properties against multiple drug-resistant pathogenic Gram-negative and Gram-positive bacteria. LC­MS/MS revealed the identity of gut microbial metabolites. These findings suggest that analyses of crocodile gut bacteria may reveal potential drug leads that can be utilized as probiotics/pre/post/antibiotics for the benefit of human health, however intensive future research is needed to realize these expectations.

mRNA Sequencing Reveals Upregulation of Glutathione S-Transferase Genes during Acanthamoeba Encystation.

Some members of the genus Acanthamoeba are facultative pathogens typically with a biphasic lifestyle: trophozoites and cysts. Acanthamoeba is capable of infecting the cornea, resulting in Acanthamoeba keratitis. The cyst is one of the key components for the persistence of infection. Gene expression during Acanthamoeba encystation showed an upregulation of glutathione S-transferase (GST) genes and other closely related proteins. mRNA sequencing showed GST, and five genes with similar sequences were upregulated after 24 h of inducing encystation. GST overexpression was verified with qPCR using the HPRT and the cyst-specific protein 21 genes as controls. The GST inhibitor ethacrynic acid was found to decrease cell viability by 70%. These results indicate a role of GST in successful encystation, possibly by maintaining redox balance. GST and associated processes could be targets for potential treatments alongside regular therapies to reduce relapses of Acanthamoeba infection.

Acanthamoeba castellanii exhibits intron retention during encystment.

Intron retention (IR) refers to the mechanism of alternative splicing in which an intron is not excised from the mature transcript. IR in the cosmopolitan free-living amoeba Acanthamoeba castellanii has not been studied. We performed an analysis of RNA sequencing data during encystment to identify genes that presented differentially retained introns during this process. We show that IR increases during cyst formation, indicating a potential mechanism of gene regulation that could help downregulate metabolism. We identify 69 introns from 67 genes that are differentially retained comparing the trophozoite stage and encystment after 24 and 48 h. These genes include several hypothetical proteins. We show different patterns of IR during encystment taking as examples a lipase, a peroxin-3 protein, an Fbox domain containing protein, a proteasome subunit, a polynucleotide adenylyltransferase, and a tetratricopeptide domain containing protein. A better understanding of IR in Acanthamoeba, and even other protists, could help elucidate changes in life cycle and combat disease such as Acanthamoeba keratitis in which the cyst is key for its persistence.

Epigenetic-Mediated Antimicrobial Resistance: Host versus Pathogen Epigenetic Alterations.

Since the discovery of antibiotics, humans have been benefiting from them by decreasing the morbidity and mortality associated with bacterial infections. However, in the past few decades, misuse of antibiotics has led to the emergence of bacterial infections resistant to multiple drugs, a significant health concern. Bacteria exposed to inappropriate levels of antibiotics lead to several genetic changes, enabling them to survive in the host and become more resistant. Despite the understanding and targeting of genetic-based biochemical changes in the bacteria, the increasing levels of antibiotic resistance are not under control. Many reports hint at the role of epigenetic modifications in the bacterial genome and host epigenetic reprogramming due to interaction with resistant pathogens. Epigenetic changes, such as the DNA-methylation-based regulation of bacterial mutation rates or bacteria-induced histone modification in human epithelial cells, facilitate its long-term survival. In this review article, epigenetic changes leading to the development of antibiotic resistance in clinically relevant bacteria are discussed. Additionally, recent lines of evidence focusing on human host epigenetic changes due to the human-pathogen interactions are presented. As genetic mechanisms cannot explain the transient nature of antimicrobial resistance, we believe that epigenetics may provide new frontiers in antimicrobial discovery.

Crocodylus porosus Sera a Potential Source to Identify Novel Epigenetic Targets: In Silico Analysis.

We have previously found that sera from Crocodylus porosus contain anticancer agents and the treatment of MCF7 cells with this serum resulted in the differential expression of 51 genes. The purpose of this study was to use in silico analysis to identify genes that might be epigenetically modulated in cells treated with crocodile serum and to understand the role of potential genes as novel candidates with epigenetic therapeutic potential. The findings report five proto-oncogenes (TUBA1B, SLC2A1, PGK1, CCND1, and NCAPD2) and two tumor suppressor genes (RPLP2, RPL37) as novel therapeutic targets. Furthermore, we present a comprehensive overview of relevant studies on epigenetic regulation of these genes along with an insight into their clinical implications. Therefore, elucidating the molecules present in the serum and gut bacteria of reptiles such as crocodiles may offer insights into the role of these genes on longevity, health, disease, and life expectancy.

Statins Induce Actin Cytoskeleton Disassembly and an Apoptosis-Like Process in Acanthamoeba spp.

Acanthamoeba is a ubiquitous opportunistic protozoan pathogen that is known to cause blinding keratitis and rare, but usually fatal, granulomatous encephalitis. The difficulty in treating infections and the toxicity issues of the current treatments emphasize the need to use alternative agents with amoebicidal activity. The aim of this study was to evaluate the in vitro antiamoebic activity of three third-generation statins-cerivastatin, pitavastatin and rosuvastatin-against both cysts and trophozoites of the following four strains of Acanthamoeba: A. castellanii Neff, A. polyphaga, A. griffini and A. quina. Furthermore, programmed cell death (PCD) induction traits were evaluated by measuring chromatin condensation, damages at the mitochondrial level, production of reactive oxygen species (ROS) and the distribution of actin cytoskeleton fibers. Acanthamoeba castellanii Neff was the strain most sensitive to all the statins, where cerivastatin showed the lowest amoebicidal activity for both trophozoite and cyst forms (0.114 ± 0.050 and 0.704 ± 0.129 µM, respectively). All the statins were able to cause DNA condensation, collapse in the mitochondrial membrane potential and a reduction in ATP level production, and disorganization of the total actin fibers in the cytoskeleton of all the evaluated Acanthamoeba strains. Our results showed that the tested statins were able to induce PCD compatible events in the treated amoebae, including chromatin condensation, collapse in the mitochondrial potential and ATP levels, cytoskeleton disassembly and ROS generation.

Gut microbiome-immune system interaction in reptiles.

Reptiles are ectothermic amniotes in a world dominated by endotherms. Reptiles originated more than 300 million years ago and they often dwell in polluted environments which may expose them to pathogenic micro-organisms, radiation and/or heavy metals. Reptiles also possess greater longevity and may live much longer than similar-sized land mammals, for example, turtles, tortoises, crocodiles and tuatara are long-lived reptiles living up to 100 years or more. Many recent studies have emphasized the pivotal role of the gut microbiome on its host; thus, we postulated that reptilian gut microbiome and/or its metabolites and the interplay with their robust immune system may contribute to their longevity and overall hardiness. Herein, we discuss the composition of the reptilian gut microbiome, immune system-gut microbiome cross-talk, antimicrobial peptides, reptilian resistance to infectious diseases and cancer, ageing, as well the current knowledge of the genome and epigenome of these remarkable species. Preliminary studies have demonstrated that microbial gut flora of reptiles such as crocodiles, tortoises, water monitor lizard and python exhibit remarkable anticancer and antibacterial properties, as well as comprise novel gut bacterial metabolites and antimicrobial peptides. The underlying mechanisms between the gut microbiome and the immune system may hold clues to developing new therapies overall for health, and possible extrapolation to exploit the ancient defence systems of reptiles for Homo sapiens benefit.

Inter-domain horizontal gene transfer of nickel-binding superoxide dismutase.

The ability of aerobic microorganisms to regulate internal and external concentrations of the reactive oxygen species (ROS) superoxide directly influences the health and viability of cells. Superoxide dismutases (SODs) are the primary regulatory enzymes that are used by microorganisms to degrade superoxide. SOD is not one, but three separate, non-homologous enzymes that perform the same function. Thus, the evolutionary history of genes encoding for different SOD enzymes is one of convergent evolution, which reflects environmental selection brought about by an oxygenated atmosphere, changes in metal availability, and opportunistic horizontal gene transfer (HGT). In this study, we examine the phylogenetic history of the protein sequence encoding for the nickel-binding metalloform of the SOD enzyme (SodN). The genomic potential to produce SodN is widespread among bacteria, including Actinobacteriota (Actinobacteria), Chloroflexota (Chloroflexi), Cyanobacteria, Proteobacteria, Patescibacteria, and others. The gene is also present in many archaea, with Thermoplasmatota and Nanoarchaeota representing the vast majority of archaeal sodN diversity. A comparison of organismal and SodN protein phylogenetic trees reveals several instances of HGT, including multiple inter-domain transfers of the sodN gene from the bacterial domain to the archaeal domain. Nearly half of the archaeal members with sodN live in the photic zone of the marine water column. The sodN gene is widespread and characterized by apparent vertical gene transfer in some sediment- or soil-associated lineages within the Actinobacteriota and Chloroflexota phyla, suggesting the ancestral sodN likely originated in one of these clades before expanding its taxonomic and biogeographic distribution to additional microbial groups in the surface ocean in response to decreasing iron availability. In addition to decreasing iron quotas, nickel-binding SOD has the added benefit of withstanding high reactant and product ROS concentrations without damaging the enzyme, making it particularly well suited for the modern surface ocean.

Development of anti-acanthamoebic approaches.

Acanthamoeba keratitis is a sight-endangering eye infection, and causative organism Acanthamoeba presents a significant concern to public health, given escalation of contact lens wearers. Contemporary therapy is burdensome, necessitating prompt diagnosis and aggressive treatment. None of the contact lens disinfectants (local and international) can eradicate Acanthamoeba effectively. Using a range of compounds targeting cellulose, ion channels, and biochemical pathways, we employed bioassay-guided testing to determine their anti-amoebic effects. The results indicated that acarbose, indaziflam, terbuthylazine, glimepiride, inositol, vildagliptin and repaglinide showed anti-amoebic effects. Compounds showed minimal toxicity on human cells. Therefore, effects of the evaluated compounds after conjugation with nanoparticles should certainly be the subject of future studies and will likely lead to promising leads for potential applications.

Capillary Electromigration Techniques Coupled to Mass Spectrometry: Applications to Food Analysis.

Analysis of food is essential for safety, quality control, government regulations, and recommendations to answer basic research questions. Capillary electrophoresis-mass spectrometry (CE-MS) is a powerful hyphenated technique in food, beverages, and foodomics for analytes ranging from small organic ions and biochemical compounds to macromolecules. Advantages of CE-MS for food analysis include high efficiency, high resolution, low cost of reagent consumption, fast and green approach in various food research areas. This review offers a comprehensive evaluation of CE-MS application for food analysis published in the open literature in the last decade (July 2010-October 2020). The principles of various CE-MS modes, CE-inductively coupled plasma mass spectrometry, ionization interfaces, and sample preparation methods for multiple types of liquid and solid food analysis are compiled. The latest advances and potential trends are outlined in several food analysis areas where CE-MS could be beneficial.

Ink Release and Swimming Behavior in the Oceanic Ctenophore Eurhamphaea vexilligera.

Of the more than 150 ctenophore species, the oceanic ctenophore Eurhamphaea vexilligera is notable for its bright orange-yellow ink, secreted from numerous small vesicles that line its substomodeal comb rows. To date, in situ observations by scuba divers have proved the most fruitful method of observing these animals' natural behavior. We present the results of one such contemporary scuba-based observation of E. vexilligera, conducted in the Gulf Stream waters off the coast of Florida, using high-resolution photography and video. Utilizing underwater camera systems purpose built for filming gelatinous zooplankton, we observed E. vexilligera ink release and swimming behavior in situ. From these data, we describe the timeline and mechanics of E. vexilligera ink release in detail, as well as the animal's different swimming behaviors and resulting ink dispersal patterns. We also describe a rolling swimming behavior, accompanied and possibly facilitated by a characteristic change in overall body shape. These observations provide further insight into the behavioral ecology of this distinctive ctenophore and may serve as the foundation for future kinematic studies.

Microdosing and standard-dosing take-home buprenorphine from the emergency department: A feasibility study.

OBJECTIVE: Emergency department (ED)-initiated buprenorphine may prevent overdose. Microdosing is a novel approach that does not require withdrawal, which can be a barrier to standard inductions. We aimed to evaluate the feasibility of an ED-initiated buprenorphine/naloxone program providing standard-dosing and microdosing take-home packages and of randomizing patients to either intervention. METHODS: We broadly screened patients ≥18 years old for opioid use disorder at a large, urban ED. In a first phase, we provided consecutive patients with 3-day standard-dosing packages, and then we provided a subsequent group with 6-day microdosing packages. In a second phase, we randomized patients to standard dosing or microdosing. We attempted 7-day telephone follow-ups and 30-day in-person community follow-ups. The primary feasibility outcome was number of patients enrolled and accepting randomization. Secondary outcomes were numbers screened, follow-up rates, and 30-day opioid agonist therapy retention. RESULTS: We screened 3954 ED patients and identified 94 with opioid use disorders. Of the patients, 26 (27.7%) declined participation: 10 identified a negative prior experience with buprenorphine/naloxone as the reason, 5 specifically cited precipitated withdrawal, and none cited randomization. We enrolled 68 patients. A total of 14 left the ED against medical advice, 8 were excluded post-enrollment, 21 received standard dosing, and 25 received microdosing. The 7-day and 30-day follow-up rates were 9/46 (19.6%) and 15/46 (32.6%), respectively. At least 5/21 (23.8%) provided standard dosing and 8/25 (32.0%) provided microdosing remained on opioid agonist therapy at 30 days. CONCLUSIONS: ED-initiated take-home standard-dosing and microdosing buprenorphine/naloxone programs are feasible, and a randomized controlled trial would be acceptable to our target population.

Is Naegleria fowleri an Emerging Parasite?

Naegleria fowleri causes an uncommon but deadly disease called primary amoebic meningoencephalitis (PAM). There has been an increase of reported PAM cases, particularly since 2000. Although water is the dominant route of transmission of PAM, infection through soil/dust is a possible alternative route. We have observed differences in epidemiology between the southern states in the USA and the Indian subcontinent (ISC). The patient age range is greater in the ISC than in the USA, and there are more infections in the ISC which are not water-associated. We show that PAM is under-reported and argue that climate change will increase the incidence of PAM, and that the geographic range of N. fowleri will spread polewards.

Critical Issues in Dental and Medical Management of Obstructive Sleep Apnea.

This critical review focuses on obstructive sleep apnea (OSA) and its management from a dental medicine perspective. OSA is characterized by ≥10-s cessation of breathing (apnea) or reduction in airflow (hypopnea) ≥5 times per hour with a drop in oxygen and/or rise in carbon dioxide. It can be associated with sleepiness and fatigue, impaired mood and cognition, cardiometabolic complications, and risk for transportation and work accidents. Although sleep apnea is diagnosed by a sleep physician, its management is interdisciplinary. The dentist's role includes 1) screening patients for OSA risk factors (e.g., retrognathia, high arched palate, enlarged tonsils or tongue, enlarged tori, high Mallampati score, poor sleep, supine sleep position, obesity, hypertension, morning headache or orofacial pain, bruxism); 2) referring to an appropriate health professional as indicated; and 3) providing oral appliance therapy followed by regular dental and sleep medical follow-up. In addition to the device features and provider expertise, anatomic, behavioral, demographic, and neurophysiologic characteristics can influence oral appliance effectiveness in managing OSA. Therefore, OSA treatment should be tailored to each patient individually. This review highlights some of the putative action mechanisms related to oral appliance effectiveness and proposes future research directions.