The First Hybrid International Educational Comprehensive Cleft Care Workshop.

OBJECTIVE: Describe the first hybrid global simulation-based comprehensive cleft care workshop, evaluate impact on participants, and compare experiences based on in-person versus virtual attendance. DESIGN: Cross-sectional survey-based evaluation. SETTING: International comprehensive cleft care workshop. PARTICIPANTS: Total of 489 participants. INTERVENTIONS: Three-day simulation-based hybrid comprehensive cleft care workshop. MAIN OUTCOME MEASURES: Participant demographic data, perceived barriers and interventions needed for global comprehensive cleft care delivery, participant workshop satisfaction, and perceived short-term impact on practice stratified by in-person versus virtual attendance. RESULTS: The workshop included 489 participants from 5 continents. The response rate was 39.9%. Participants perceived financial factors (30.3%) the most significant barrier and improvement in training (39.8%) as the most important intervention to overcome barriers facing cleft care delivery in low to middle-income countries. All participants reported a high level of satisfaction with the workshop and a strong positive perceived short-term impact on their practice. Importantly, while this was true for both in-person and virtual attendees, in-person attendees reported a significantly higher satisfaction with the workshop (28.63 ± 3.08 vs 27.63 ± 3.93; P = .04) and perceived impact on their clinical practice (22.37 ± 3.42 vs 21.02 ± 3.45 P = .01). CONCLUSION: Hybrid simulation-based educational comprehensive cleft care workshops are overall well received by participants and have a positive perceived impact on their clinical practices. In-person attendance is associated with significantly higher satisfaction and perceived impact on practice. Considering that financial and health constraints may limit live meeting attendance, future efforts will focus on making in-person and virtual attendance more comparable.

Reaction hijacking of tyrosine tRNA synthetase as a new whole-of-life-cycle antimalarial strategy.

Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5'-monophosphate-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5'-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum, namely tyrosine RS (PfYRS). ML901 exerts whole-life-cycle-killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.

A nitrite-oxidising bacterium constitutively consumes atmospheric hydrogen.

Chemolithoautotrophic nitrite-oxidising bacteria (NOB) of the genus Nitrospira contribute to nitrification in diverse natural environments and engineered systems. Nitrospira are thought to be well-adapted to substrate limitation owing to their high affinity for nitrite and capacity to use alternative energy sources. Here, we demonstrate that the canonical nitrite oxidiser Nitrospira moscoviensis oxidises hydrogen (H2) below atmospheric levels using a high-affinity group 2a nickel-iron hydrogenase [Km(app) = 32 nM]. Atmospheric H2 oxidation occurred under both nitrite-replete and nitrite-deplete conditions, suggesting low-potential electrons derived from H2 oxidation promote nitrite-dependent growth and enable survival during nitrite limitation. Proteomic analyses confirmed the hydrogenase was abundant under both conditions and indicated extensive metabolic changes occur to reduce energy expenditure and growth under nitrite-deplete conditions. Thermodynamic modelling revealed that H2 oxidation theoretically generates higher power yield than nitrite oxidation at low substrate concentrations and significantly contributes to growth at elevated nitrite concentrations. Collectively, this study suggests atmospheric H2 oxidation enhances the growth and survival of NOB amid variability of nitrite supply, extends the phenomenon of atmospheric H2 oxidation to an eighth phylum (Nitrospirota), and reveals unexpected new links between the global hydrogen and nitrogen cycles. Long classified as obligate nitrite oxidisers, our findings suggest H2 may primarily support growth and survival of certain NOB in natural environments.

A Clinical Report Utilizing the VITOM 3D® Microvideoscope for Cleft Palate Repair.

Cleft palate surgery has traditionally presented numerous problems for cleft surgeons including ergonomics, limited visual fields restricting the opportunity for demonstration and teaching. Additionally, the move toward online teaching means the ability to record or livestream video is paramount. The following report of eight cleft palate repairs highlights the novel use of the Vitom 3D® microvideoscope as an innovative technique for cleft palate repair with our early experience demonstrating significant ergonomic and teaching benefits.

Design of proteasome inhibitors with oral efficacy in vivo against Plasmodium falciparum and selectivity over the human proteasome.

The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) ß5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.

High Throughput Screening to Identify Selective and Nonpeptidomimetic Proteasome Inhibitors As Antimalarials.

The Ubiquitin Proteasome System is the main proteolytic pathway in eukaryotic cells, playing a role in key cellular processes. The essentiality of the Plasmodium falciparum proteasome is well validated, underlying its potential as an antimalarial target, but selective compounds are required to avoid cytotoxic effects in humans. Almost 550000 compounds were tested for the inhibition of the chymotrypsin-like activity of the P. falciparum proteasome using a Proteasome-GLO luminescence assay. Hits were confirmed in an orthogonal enzyme assay using Rho110-labeled peptides, and selectivity was assessed against the human proteasome. Four nonpeptidomimetic chemical families with some selectivity for the P. falciparum proteasome were identified and characterized in assays of proteasome trypsin and caspase activities and in parasite growth inhibition assays. Target engagement studies were performed, validating our approach. Hits identified are good starting points for the development of new antimalarial drugs and as tools to better understand proteasome function in P. falciparum.

Benthic habitat condition of the continental shelf surrounding oil and gas platforms in the Santa Barbara Channel, Southern California.

The continental shelf of southern California is an important location for the extraction of petroleum and natural gas. Many platforms in the region have been operating for more than four decades and are being targeted for decommissioning. Information on the condition of surrounding habitats to the platforms will be important for regulators. The condition of sediments near (250 m-2 km) four active oil/gas platforms was evaluated with measures of macrobenthic infauna, toxicity, and chemical composition using standardized assessment indices and compared to that of equivalent locations across the region without platforms. Assessment scores indicated that the sediments surrounding the oil platforms were in a relatively good state, with reference-condition infauna, minimal levels of chemical exposure, and five instances (25% of samples) of low-level toxicity. Samples from around the oil platforms were in overall similar condition to the region, with slightly better condition infauna, nearly identical chemistry, and slightly worse toxicity.

The importance of open science for biological assessment of aquatic environments.

Open science principles that seek to improve science can effectively bridge the gap between researchers and environmental managers. However, widespread adoption has yet to gain traction for the development and application of bioassessment products. At the core of this philosophy is the concept that research should be reproducible and transparent, in addition to having long-term value through effective data preservation and sharing. In this article, we review core open science concepts that have recently been adopted in the ecological sciences and emphasize how adoption can benefit the field of bioassessment for both prescriptive condition assessments and proactive applications that inform environmental management. An example from the state of California demonstrates effective adoption of open science principles through data stewardship, reproducible research, and engagement of stakeholders with multimedia applications. We also discuss technical, sociocultural, and institutional challenges for adopting open science, including practical approaches for overcoming these hurdles in bioassessment applications.

Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F420-0 in Mycobacteria.

F420 is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F420 in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F420 biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-phospholactate stimulates F420 biosynthesis. Analysis of F420 biosynthesis intermediates present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F420-0. To determine the structural basis of dehydro-F420-0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo-, substrate-, and product-bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic precomplex of this enzyme and demonstrate that dehydro-F420-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F420 in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance.IMPORTANCE Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F420, and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F420 is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F420 in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F420, determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F420.

A Review of 30 Years of Alveolar Bone Grafting in the Mixed Dentition Using a Standardized Protocol in Western Australia.

BACKGROUND: Outcomes for a continuously applied alveolar bone grafting protocol, established in 1982, are reported and compared to previously published outcomes from the authors' unit and elsewhere. METHODS: A descriptive, retrospective cohort study of alveolar bone grafting outcomes at a tertiary referral cleft center was performed. Records of all alveolar bone grafts between 2002 and 2014 were reviewed (224 grafts). Three-year postoperative periapical radiographs were evaluated using the Bergland, Kindelan, and standardized way to assess graft scores by an external rater. Incomplete records, a syndromic diagnosis, or primary surgery performed elsewhere resulted in 123 grafts being excluded, leaving 101 grafts for assessment. The distribution of scores was compared to the authors' previous studies and international reports. The authors also tested for any impact on the outcome based on cleft type, laterality, timing for incisor or canine eruption, and surgeon experience. RESULTS: A total of 95.6 percent of applicable grafts (66 of 69) were considered "successful" by Bergland scores and 96 percent by Kindelan scores. Eighty-nine percent of grafts were "very good" based on standardized way to assess graft score. No significant differences were detected in outcomes based on timing, cleft type, or laterality. Surgeon experience had a significant impact (p < 0.05) on outcome for Bergland and Kindelan scores. Distribution of Bergland scores did not differ from the authors' earlier studies demonstrating consistent outcomes for over 30 years. CONCLUSIONS: The Western Australian alveolar bone grafting protocol has consistently achieved a very high success rate (96 percent) for over 30 years despite multiple staff changes. These results compared well with best-reported outcomes from worldwide cleft centers. Surgeon training and experience were significant in achieving these outcomes. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Decreased K13 Abundance Reduces Hemoglobin Catabolism and Proteotoxic Stress, Underpinning Artemisinin Resistance.

Increased tolerance of Plasmodium falciparum to front-line artemisinin antimalarials (ARTs) is associated with mutations in Kelch13 (K13), although the precise role of K13 remains unclear. Here, we show that K13 mutations result in decreased expression of this protein, while mislocalization of K13 mimics resistance-conferring mutations, pinpointing partial loss of function of K13 as the relevant molecular event. K13-GFP is associated with ∼170 nm diameter doughnut-shaped structures at the parasite periphery, consistent with the location and dimensions of cytostomes. Moreover, the hemoglobin-peptide profile of ring-stage parasites is reduced when K13 is mislocalized. We developed a pulse-SILAC approach to quantify protein turnover and observe less disruption to protein turnover following ART exposure when K13 is mislocalized. Our findings suggest that K13 regulates digestive vacuole biogenesis and the uptake/degradation of hemoglobin and that ART resistance is mediated by a decrease in heme-dependent drug activation, less proteotoxicity, and increased survival of parasite ring stages.

The structure of the PA28-20S proteasome complex from Plasmodium falciparum and implications for proteostasis.

The activity of the proteasome 20S catalytic core is regulated by protein complexes that bind to one or both ends. The PA28 regulator stimulates 20S proteasome peptidase activity in vitro, but its role in vivo remains unclear. Here, we show that genetic deletion of the PA28 regulator from Plasmodium falciparum (Pf) renders malaria parasites more sensitive to the antimalarial drug dihydroartemisinin, indicating that PA28 may play a role in protection against proteotoxic stress. The crystal structure of PfPA28 reveals a bell-shaped molecule with an inner pore that has a strong segregation of charges. Small-angle X-ray scattering shows that disordered loops, which are not resolved in the crystal structure, extend from the PfPA28 heptamer and surround the pore. Using single particle cryo-electron microscopy, we solved the structure of Pf20S in complex with one and two regulatory PfPA28 caps at resolutions of 3.9 and 3.8 Å, respectively. PfPA28 binds Pf20S asymmetrically, strongly engaging subunits on only one side of the core. PfPA28 undergoes rigid body motions relative to Pf20S. Molecular dynamics simulations support conformational flexibility and a leaky interface. We propose lateral transfer of short peptides through the dynamic interface as a mechanism facilitating the release of proteasome degradation products.

Organic contaminants as an ecological tool to explore niche partitioning: a case study using three pelagic shark species.

Chemical contaminant profiles are linked to an animal's niche, providing a potential tool by which to assess resource partitioning in pelagic species. As proof of concept, we examined contaminant signatures in three species of sharks (Isurus oxyrinchus, Prionace glauca, and Alopias vulpinus) known to overlap in both space and time. Since these sharks comprise a predatory guild within the Southern California Bight (SCB), we predicted that species may partition spatial and dietary resources to limit the extent of competitive exclusion. Indeed, species were distinguishable by both total contaminant loads and their contaminant fingerprint, as random forest analysis found that species could be correctly classified 96% of the time. Our results demonstrate the utility of chemical analyses for ecological studies, and how contaminant tracers can be used in combination with traditional methods to elucidate how species may undergo niche partitioning to reduce competition for overlapping resources within predatory guilds.

A Low-Cost Continuous Turbidity Monitor.

Turbidity describes the cloudiness, or clarity, of a liquid. It is a principal indicator of water quality, sensitive to any suspended solids present. Prior work has identified the lack of low-cost turbidity monitoring as a significant hurdle to overcome to improve water quality in many domains, especially in the developing world. Low-cost hand-held benchtop meters have been proposed. This work adapts and verifies the technology for continuous monitoring. Lab tests show the low-cost continuous monitor can achieve 1 nephelometric turbidity unit (NTU) accuracy in the range 0-100 NTU and costs approximately 64 USD in components to construct. This level of accuracy yields useful and actionable data about water quality and may be sufficient in certain applications where cost is a primary constraint. A 38-day continuous monitoring trial, including a step change in turbidity, showed promising results with a median error of 0.45 and 1.40 NTU for two different monitors. However, some noise was present in the readings resulting in a standard deviation of 1.90 and 6.55 NTU, respectively. The cause was primarily attributed to ambient light and bubbles in the piping. By controlling these noise sources, we believe the low-cost continuous turbidity monitor could be a useful tool in multiple domains.

Species-Specific Characteristics Influence Contaminant Accumulation Trajectories and Signatures Across Ontogeny in Three Pelagic Shark Species.

Factors influencing organic contaminant accumulation in sharks, especially across ontogeny, are not well-known. Contaminant concentrations were measured in three species of sharks (Blue, Shortfin Mako, and Common Thresher) across a range of size classes (neonatal to adult) that vary in their ecological and physiological characteristics. Empirical data was compared to a theoretical framework that predicted the shape of lifetime accumulation curves. We found that a one-size-fits-all accumulation model was not appropriate as species-specific characteristics had a significant effect on contaminant accumulation trajectories. Maternal offloading likely has an important effect on determining neonatal shark contaminant starting points, and trophic ecology and physiology may interact to affect the shape of species' contaminant accumulation curves. Makos were found to have the highest accumulation potential and Blues the lowest, with Threshers being intermediate in accumulation potential. Changes in species' ecology and/or physiology were also reflected in contaminant signature changes over ontogeny. If contaminant concentrations are to be used as a proxy for risk, species-specific characteristics need to be taken into account when estimating contaminant exposure and its potential negative effects on shark health and human consumption safety.

Selecting Comparator Sites for Ecological Causal Assessment Based on Expected Biological Similarity.

Sites in poor ecological condition often require causal assessment to determine appropriate follow-up actions. Site-specific causal assessments can be time consuming. To streamline the process, we describe a quantitative method that expedites a key component of causal assessment: identifying a group of ecologically similar (comparator) sites that are used to compare and contrast biological condition and stressor exposure at the site of interest. A good set of comparator sites should: 1. Be capable of supporting similar biota to the impaired site in the absence of disturbance; 2. Comprise a gradient of biotic condition; and 3. Contain enough sites to assess variability. We used expected biological similarity to select good sets of comparator sites from a large pool of potential sites. Expected biological similarity was measured as Bray-Curtis dissimilarity values (BC) calculated from the expected benthic macroinvertebrate taxa lists produced by a predictive biotic index of stream health. Sets of comparator sites were created for 15 demonstration sites across Southern California in poor condition. We examined the stressor and biological data collected at the 15 sites and their comparators to assess the likelihood that four example stressors - total nitrogen, ammonia, specific conductivity, and bifenthrin - contribute to the poor biotic conditions that were observed. We were able to select more than 100 comparator sites for all but 1 of the 15 demonstration sites at a BC <0.1. These sets of comparator sites were then used to evaluate the four example stressors using two commonly used causal assessment types of evidence. Elevated conductivity was the most frequently supported likely cause among the demonstration sites, though total nitrogen and bifenthrin were also indicated at some sites. Though our specific approach was tailored for application in California's stream bioassessment framework, the concepts could be adapted for any bioassessment program with a large amount of sample data and an associated predictive index of biotic condition. Furthermore, this approach lays the groundwork for developing a novel approach to causal assessment that begins with a rapid, screening-level evaluation of stressors common in a region using these data-rich groups of comparator sites, which then informs follow-up management actions.

Target Validation and Identification of Novel Boronate Inhibitors of the Plasmodium falciparum Proteasome.

The Plasmodium proteasome represents a potential antimalarial drug target for compounds with activity against multiple life cycle stages. We screened a library of human proteasome inhibitors (peptidyl boronic acids) and compared activities against purified P. falciparum and human 20S proteasomes. We chose four hits that potently inhibit parasite growth and show a range of selectivities for inhibition of the growth of P. falciparum compared with human cell lines. P. falciparum was selected for resistance in vitro to the clinically used proteasome inhibitor, bortezomib, and whole genome sequencing was applied to identify mutations in the proteasome ß5 subunit. Active site profiling revealed inhibitor features that enable retention of potent activity against the bortezomib-resistant line. Substrate profiling reveals P. falciparum 20S proteasome active site preferences that will inform attempts to design more selective inhibitors. This work provides a starting point for the identification of antimalarial drug leads that selectively target the P. falciparum proteasome.

Artemisinin kills malaria parasites by damaging proteins and inhibiting the proteasome.

Artemisinin and its derivatives (collectively referred to as ARTs) rapidly reduce the parasite burden in Plasmodium falciparum infections, and antimalarial control is highly dependent on ART combination therapies (ACTs). Decreased sensitivity to ARTs is emerging, making it critically important to understand the mechanism of action of ARTs. Here we demonstrate that dihydroartemisinin (DHA), the clinically relevant ART, kills parasites via a two-pronged mechanism, causing protein damage, and compromising parasite proteasome function. The consequent accumulation of proteasome substrates, i.e., unfolded/damaged and polyubiquitinated proteins, activates the ER stress response and underpins DHA-mediated killing. Specific inhibitors of the proteasome cause a similar build-up of polyubiquitinated proteins, leading to parasite killing. Blocking protein synthesis with a translation inhibitor or inhibiting the ubiquitin-activating enzyme, E1, reduces the level of damaged, polyubiquitinated proteins, alleviates the stress response, and dramatically antagonizes DHA activity.

Adaptation and application of multivariate AMBI (M-AMBI) in US coastal waters.

The multivariate AMBI (M-AMBI) is an extension of the AZTI Marine Biotic Index (AMBI) that has been used extensively in Europe, but not in the United States. In a previous study, we adapted AMBI for use in US coastal waters (US AMBI), but saw biases in salinity and score distribution when compared to locally calibrated indices. In this study we modified M-AMBI for US waters and compared its performance to that of US AMBI. Index performance was evaluated in three ways: 1) concordance with local indices presently being used as management tools in three geographic regions of US coastal waters, 2) classification accuracy for sites defined a priori as good or bad and 3) insensitivity to natural environmental gradients. US M-AMBI was highly correlated with all three local indices and removed the compression in response seen in moderately disturbed sites with US AMBI. US M-AMBI and US AMBI did a similar job correctly classifying sites as good or bad in local validation datasets (83 to 100% accuracy vs. 84 to 95%, respectively). US M-AMBI also removed the salinity bias of US AMBI so that lower salinity sites were not more likely to be incorrectly classified as impaired. The US M-AMBI appears to be an acceptable index for comparing condition across broad-scales such as estuarine and coastal waters surveyed by the US EPA's National Coastal Condition Assessment, and may be applicable to areas of the US coast that do not have a locally derived benthic index.