Reconstructed human intestinal comet assay, a possible alternative in vitro model for genotoxicity assessment.

The aim of the present study was to evaluate the compatibility of reconstructed 3D human small intestinal microtissues to perform the in vitro comet assay. The comet assay is a common follow-up genotoxicity test to confirm or supplement other genotoxicity data. Technically, it can be performed utilizing a range of in vitro and in vivo assay systems. Here, we have developed a new reconstructed human intestinal comet (RICom) assay protocol for the assessment of orally ingested materials. The human intestine is a major site of food digestion and adsorption, first-pass metabolism as well as an early site of toxicant first contact and thus is a key site for evaluation. Reconstructed intestinal tissues were dosed with eight test chemicals: ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU), phenformin hydrochloride (Phen HCl), benzo[a]pyrene (BaP), 1,2-dimethylhydrazine hydrochloride (DMH), potassium bromate (KBr), glycidamide (GA), and etoposide (Etop) over a span of 48 h. The RICom assay correctly identified the genotoxicity of EMS, ENU, KBr, and GA. Phen HCl, a known non-genotoxin, did not induce DNA damage in the 3D reconstructed intestinal tissues whilst showing high cytotoxicity as assessed by the assay. The 3D reconstructed intestinal tissues possess sufficient metabolic competency for the successful detection of genotoxicity elicited by BaP, without the use of an exogenous metabolic system. In contrast, DMH, a chemical that requires liver metabolism to exert genotoxicity, did not induce detectable DNA damage in the 3D reconstructed intestinal tissue system. The genotoxicity of Etop, which is dependent on cellular proliferation, was also undetectable. These results suggest the RICom assay protocol is a promising tool for further investigation and safety assessment of novel ingested materials. We recommend that further work will broaden the scope of the 3D reconstructed intestinal tissue comet assay and facilitate broader analyses of genotoxic compounds having more varied modes of actions.

A combined bioinformatics and LC-MS-based approach for the development and benchmarking of a comprehensive database of Lymnaea CNS proteins.

Applications of key technologies in biomedical research, such as qRT-PCR or LC-MS-based proteomics, are generating large biological (-omics) datasets which are useful for the identification and quantification of biomarkers in any research area of interest. Genome, transcriptome and proteome databases are already available for a number of model organisms including vertebrates and invertebrates. However, there is insufficient information available for protein sequences of certain invertebrates, such as the great pond snail Lymnaea stagnalis, a model organism that has been used highly successfully in elucidating evolutionarily conserved mechanisms of memory function and dysfunction. Here, we used a bioinformatics approach to designing and benchmarking a comprehensive central nervous system (CNS) proteomics database (LymCNS-PDB) for the identification of proteins from the CNS of Lymnaea by LC-MS-based proteomics. LymCNS-PDB was created by using the Trinity TransDecoder bioinformatics tool to translate amino acid sequences from mRNA transcript assemblies obtained from a published Lymnaea transcriptomics database. The blast-style MMSeq2 software was used to match all translated sequences to UniProtKB sequences for molluscan proteins, including those from Lymnaea and other molluscs. LymCNS-PDB contains 9628 identified matched proteins that were benchmarked by performing LC-MS-based proteomics analysis with proteins isolated from the Lymnaea CNS. MS/MS analysis using the LymCNS-PDB database led to the identification of 3810 proteins. Only 982 proteins were identified by using a non-specific molluscan database. LymCNS-PDB provides a valuable tool that will enable us to perform quantitative proteomics analysis of protein interactomes involved in several CNS functions in Lymnaea, including learning and memory and age-related memory decline.

Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances.

The micronucleus (MN) assay is widely used as part of a battery of tests applied to evaluate the genotoxic potential of chemicals, including new food additives and novel food ingredients. Micronucleus assays typically utilise homogenous in vitro cell lines which poorly recapitulate the physiology, biochemistry and genomic events in the gut, the site of first contact for ingested materials. Here we have adapted and validated the MN endpoint assay protocol for use with complex 3D reconstructed intestinal microtissues; we have named this new protocol the reconstructed intestine micronucleus cytome (RICyt) assay. Our data suggest the commercial 3D microtissues replicate the physiological, biochemical and genomic responses of native human small intestine to exogenous compounds. Tissues were shown to maintain log-phase proliferation throughout the period of exposure and expressed low background MN. Analysis using the RICyt assay protocol revealed the presence of diverse cell types and nuclear anomalies (cytome) in addition to MN, indicating evidence for comprehensive DNA damage and mode(s) of cell death reported by the assay. The assay correctly identified and discriminated direct-acting clastogen, aneugen and clastogen requiring exogenous metabolic activation, and a non-genotoxic chemical. We are confident that the genotoxic response in the 3D microtissues more closely resembles the native tissues due to the inherent tissue architecture, surface area, barrier effects and tissue matrix interactions. This proof-of-concept study highlights the RICyt MN cytome assay in 3D reconstructed intestinal microtissues is a promising tool for applications in predictive toxicology.

Complete overdenture fabrication for a 12-year-old child with dentinogenesis imperfecta type 2.

Dentinogenesis imperfecta type 2 (DI-2), also known as hereditary opalescent dentin, is a rare, genetically linked condition that affects both primary and permanent teeth. Severe attrition requiring full-mouth rehabilitation is a common finding associated with DI-2. Dental rehabilitation options include a variety of invasive and noninvasive restorative techniques dictated by the age of the patient. Growth and development must be considered and may result in a restorative challenge for the dental practitioner, particularly when the patient in question is a child. This case report describes the fabrication of an overdenture to reestablish function, esthetics, and self-esteem in a 12-year-old patient. A 2-stage restorative treatment was followed by a satisfactory 6-month recall examination, indicating that the prostheses provided a successful outcome until more definitive restorative treatment can be accomplished in adulthood.

Dentist and practice characteristics associated with restorative treatment of enamel caries in permanent teeth: multiple-regression modeling of observational clinical data from the National Dental PBRN.

PURPOSE: Current evidence in dentistry recommends non-surgical treatment to manage enamel caries lesions. However, surveyed practitioners report they would restore enamel lesions that are confined to the enamel. Actual clinical data were used to evaluate patient, dentist, and practice characteristics associated with restoration of enamel caries, while accounting for other factors. METHODS: Data from a National Dental Practice-Based Research Network observational study of consecutive restorations placed in previously unrestored permanent tooth surfaces and practice/demographic data from 229 participating network dentists were combined. ANOVA and logistic regression, using generalized estimating equations (GEE) and variable selection within blocks, were used to test the hypothesis that patient, dentist, and practice characteristics were associated with variations in enamel restorations of occlusal and proximal caries compared to dentin lesions, accounting for dentist and patient clustering. RESULTS: Network dentists from five regions placed 6,891 restorations involving occlusal and/or proximal caries lesions. Enamel restorations accounted for 16% of enrolled occlusal caries lesions and 6% of enrolled proximal caries lesions. Enamel occlusal restorations varied significantly (P < 0.05) by patient age and race/ethnicity, dentists' use of caries risk assessment, network region, and practice type. Enamel proximal restorations varied significantly (P < 0.05) by dentist race/ethnicity, network region, and practice type.

A circuit mechanism linking past and future learning through shifts in perception.

Long-term memory formation is energetically costly. Neural mechanisms that guide an animal to identify fruitful associations therefore have important survival benefits. Here, we elucidate a circuit mechanism in Lymnaea, which enables past memory to shape new memory formation through changes in perception. Specifically, strong classical conditioning drives a positive shift in perception that facilitates the robust learning of a subsequent and otherwise ineffective weak association. Circuit dissection approaches reveal the neural control network responsible, characterized by a mutual inhibition motif. This both sets perceptual state and acts as the master controller for gating new learning. Pharmacological circuit manipulation in vivo fully substitutes for strong paradigm learning, shifting the network into a more receptive state to enable subsequent weak paradigm learning. Thus, perceptual change provides a conduit to link past and future memory storage. We propose that this mechanism alerts animals to learning-rich periods, lowering the threshold for new memory acquisition.

Assessing the risk of a clinically significant infection from a Microneedle Array Patch (MAP) product.

Microneedle Array Patches (MAPs) are an emerging dosage form that creates transient micron-sized disruptions in the outermost physical skin barrier, the stratum corneum, to facilitate delivery of active pharmaceutical ingredients to the underlying tissue. Numerous MAP products are proposed and there is significant clinical potential in priority areas such as vaccination. However, since their inception scientists have hypothesized about the risk of a clinically significant MAP-induced infection. Safety data from two major Phase 3 clinical trials involving hundreds of participants, who in total received tens of thousands of MAP applications, does not identify any clinically significant infections. However, the incumbent data set is not extensive enough to make definitive generalizable conclusions. A comprehensive assessment of the infection risk is therefore advised for MAP products, and this should be informed by clinical and pre-clinical data, theoretical analysis and informed opinions. In this article, a group of key stakeholders identify some of the key product- and patient-specific factors that may contribute to the risk of infection from a MAP product and provide expert opinions in the context of guidance from regulatory authorities. Considerations that are particularly pertinent to the MAP dosage form include the specifications of the finished product (e.g. microbial specification), it's design features, the setting for administration, the skill of the administrator, the anatomical application site, the target population and the clinical context. These factors, and others discussed in this article, provide a platform for the development of MAP risk assessments and a stimulus for early and open dialogue between developers, regulatory authorities and other key stakeholders, to expedite and promote development of safe and effective MAP products.

The role of non-coding RNAs in the formation of long-term associative memory after single-trial learning in Lymnaea.

Investigations of the molecular mechanisms of long-term associative memory have revealed key roles for a number of highly evolutionarily conserved molecular pathways in a variety of different vertebrate and invertebrate model systems. One such system is the pond snail Lymnaea stagnalis, in which, like in other systems, the transcription factors CREB1 and CREB2 and the enzyme NOS play essential roles in the consolidation of long-term associative memory. More recently, epigenetic control mechanisms, such as DNA methylation, histone modifications, and control of gene expression by non-coding RNAs also have been found to play important roles in all model systems. In this minireview, we will focus on how, in Lymnaea, even a single episode of associative learning can activate CREB and NO dependent cascades due to the training-induced up- or downregulation of the expression levels of recently identified short and long non-coding RNAs.

Persistent sodium current is a nonsynaptic substrate for long-term associative memory.

Although synaptic plasticity is widely regarded as the primary mechanism of memory [1], forms of nonsynaptic plasticity, such as increased somal or dendritic excitability or membrane potential depolarization, also have been implicated in learning in both vertebrate and invertebrate experimental systems [2-7]. Compared to synaptic plasticity, however, there is much less information available on the mechanisms of specific types of nonsynaptic plasticity involved in well-defined examples of behavioral memory. Recently, we have shown that learning-induced somal depolarization of an identified modulatory cell type (the cerebral giant cells, CGCs) of the snail Lymnaea stagnalis encodes information that enables the expression of long-term associative memory [8]. The Lymnaea CGCs therefore provide a highly suitable experimental system for investigating the ionic mechanisms of nonsynaptic plasticity that can be linked to behavioral learning. Based on a combined behavioral, electrophysiological, immunohistochemical, and computer simulation approach, here we show that an increase of a persistent sodium current of this neuron underlies its delayed and persistent depolarization after behavioral single-trial classical conditioning. Our findings provide new insights into how learning-induced membrane level changes are translated into a form of long-lasting neuronal plasticity already known to contribute to maintained adaptive modifications at the network and behavioral level [8].

Different circuit and monoamine mechanisms consolidate long-term memory in aversive and reward classical conditioning.

There has been considerable recent interest in comparing the circuit and monoamine-based mechanisms of aversive and reward-associative conditioning in a number of vertebrate and invertebrate model systems. The mollusc Lymnaea stagnalis provides a unique opportunity to explore changes in the neural and chemical pathways underlying these two different types of conditioning as its feeding circuitry has been thoroughly characterised. Animals can learn after a single trial to associate the same CS (amyl acetate) either with a punishment (quinine) or reward (sucrose), showing either a reduced or an elevated feeding response, respectively, to the CS. We previously showed that reward conditioning strengthened the direct excitatory pathway from the lips to the feeding central pattern generator in the buccal ganglia through the activation of feeding interneurons in the cerebral ganglia. Now we demonstrate that aversive conditioning enhances the strength of a different inhibitory pathway that suppresses feeding but has no effect on the excitatory pathway. Here we show that consolidation of long-term memory (LTM) in reward conditioning depends on dopamine but not octopamine. In contrast, aversive LTM depends on octopamine but not dopamine. Octopamine is the invertebrate equivalent of noradrenalin, so these results on the monoamine dependence of reward and aversive conditioning in Lymnaea resemble, at the transmitter receptor level, those in mammals but are the opposite of those in another invertebrate group, the insects.

Interneuronal mechanisms for learning-induced switch in a sensory response that anticipates changes in behavioral outcomes.

Sensory cues in the natural environment predict reward or punishment, important for survival. For example, the ability to detect attractive tastes indicating palatable food is essential for foraging while the recognition of inedible substrates prevents harm. While some of these sensory responses are innate, they can undergo fundamental changes due to prior experience associated with the stimulus. However, the mechanisms underlying such behavioral switching of an innate sensory response at the neuron and network levels require further investigation. We used the model learning system of Lymnaea stagnalis1-3 to address the question of how an anticipated aversive outcome reverses the behavioral response to a previously effective feeding stimulus, sucrose. Key to the switching mechanism is an extrinsic inhibitory interneuron of the feeding network, PlB (pleural buccal4,5), which is inhibited by sucrose to allow a feeding response. After multi-trial aversive associative conditioning, pairing sucrose with strong tactile stimuli to the head, PlB's firing rate increases in response to sucrose application to the lips and the feeding response is suppressed; this learned response is reversed by the photoinactivation of a single PlB. A learning-induced persistent change in the cellular properties of PlB that results in an increase rather than a decrease in its firing rate in response to sucrose provides a neurophysiological mechanism for this behavioral switch. A key interneuron, PeD12 (Pedal-Dorsal 12), of the defensive withdrawal network5,6 does not mediate the conditioned suppression of feeding, but its facilitated output contributes to the sensitization of the withdrawal response.

The growing cohort of seniors with HIV/AIDS: changing the scope of Medicare Part D.

HIV/AIDS medications are generally expensive and government assistance is often necessary to limit high out-of-pocket patient costs. Lowered patient out-of-pocket costs were the objective of government involvement in drug provision through legislation creating Medicare Part D. However, the Medicare program faces a surge in those beneficiaries living longer on more effective antiretroviral drugs. Higher prevalence of HIV/AIDS patients means more opportunity for transmission of the infection and recidivistic behavior such as non-adherence to medication regimens. Along with the resulting increased frequency of opportunistic infections in HIV/AIDS patients comes the requirement for aggressive pharmacological treatment. To meet this need, Medicare Part D provides drugs for the treatment of opportunistic infections occurring in HIV/AIDS patients. Problematically, though, Medicare Part D contains so many choices that it tends to overwhelm patients and sometimes even the providers and insurance companies as well. The multiplicity of choices in this highly complex program for the aged and infirm often leads to confusion and incorrect choices by beneficiaries. Furthermore, the advent of tiered cost-sharing or formulary management by Medicare Part D providers, besides controlling out-of-pocket costs, controls which medications are covered and limits the quantity that is dispensed. HIV/AIDS treatment in the present day requires a highly accessible medication provision program that is only now beginning to evolve as Medicare Part D.

A psychosocial approach to dentistry for the underserved: incorporating theory into practice.

BACKGROUND: Dentistry for the underserved is more than an egalitarian social issue--it is a key factor in the health and social progress of our nation. The first signs or manifestations of several diseases such as varicella (i.e., chicken pox and shingles), STDs, and influenza become apparent in the oral cavity. The value of access to quality dentistry is an immeasurable factor in maintaining general medical health of people and fulfilling their psychosocial needs of pain reduction and enhanced cosmetics. In the United States, for the most part, only the middle and upper classes receive non-extraction, restorative, and prosthetic dentistry that is economically within their ability to pay. In addition, uninsured and poverty-level individuals often must face overwhelming long waiting lists, unnecessary referrals, lack of choice, and bureaucratic hurdles when seeking primary dental care. Therefore, it seems pertinent to put forth the question: What are the critical values and beliefs of psychosocial theory that can underscore the practice of dentistry for underserved populations in the United States? METHODS: The widely employed public health theory, the health belief model (HBM), is applied to evaluate psychosocial factors in dental care for the underserved. The HBM is used to predict and explain behavioral changes in dental health and associated belief patterns. RESULTS: The HBM as applied to dentistry for the underserved predicts self-perceptions of susceptibility and seriousness of dental disease, health status, cues to action, and self-efficacy. Furthermore, patients can make judgments about benefits, costs, and risks of dental treatment. CONCLUSIONS: A theoretical approach to dentistry employing the HBM, mediated by values and culture, can provide significant insights into patient thinking, beliefs, and perceptions. These insights can mediate access to and use of primary care dental services by underserved populations. PRACTICE IMPLICATIONS: Evidence-based practice (i.e., based on research using the scientific method) has been put forth as the future of modern dentistry. However, the practice of dentistry need not just be evidence-based, but have its roots clearly grounded in theory.

Integrating child dental care at Community Smiles: the director's goals fulfilled....

Community Smiles/Dade County Dental Research Clinic provides dental care to the indigent population of Miami-Dade County. A local board of directors governs the organization, with dental procedures performed by volunteer professionals from the community. The research clinic partners with community organizations to obtain sustained funding from diverse sources. The clinic has a long-term commitment to the growth and development of children in the community. Certainly, changing the structure and focus of the clinic toward children's dental care and seeking community funding and resources to institute this program was an experiment. In his four years as clinic director and chief executive officer (CEO) at Community Smiles, the late Dr. Robert M. Wolf brought increased clinic productivity and organizational change that expanded community involvement. Dr. Wolf's tenure at Community Smiles brought general increases in patients care in terms of patients visits, new patients and number of procedures performed. However, the key to his administration as clinic director and CEO was the production and integration of a children's dentistry program into the mainstream activities of the clinic. Furthermore, he oversaw the successful corporate reorganization of Community Smiles as the clinic emerged under a non-profit corporate structure employing multi-faceted community resources. Emphasizing new dental programs for children in the community is culturally and socially competent--positively impacting the public health. Community Smiles became a venue where disparities were largely eliminated and access to dental treatment increased. Health care was promoted as Community Smiles became a place that helped build a healthier community.

The unlimited potential of the great pond snail, Lymnaea stagnalis.

Only a limited number of animal species lend themselves to becoming model organisms in multiple biological disciplines: one of these is the great pond snail, Lymnaea stagnalis. Extensively used since the 1970s to study fundamental mechanisms in neurobiology, the value of this freshwater snail has been also recognised in fields as diverse as host-parasite interactions, ecotoxicology, evolution, genome editing and 'omics', and human disease modelling. While there is knowledge about the natural history of this species, what is currently lacking is an integration of findings from the laboratory and the field. With this in mind, this article aims to summarise the applicability of L. stagnalis and points out that this multipurpose model organism is an excellent, contemporary choice for addressing a large range of different biological questions, problems and phenomena.

The Prolonged Use of VV ECMO Support in COVID-19: A Case Report.

COVID-19 has resulted in unprecedented global health and economic challenges. The reported mortality in patients with COVID-19 requiring mechanical ventilation is high. VV ECMO may serve as a lifesaving rescue therapy for a minority of patients with COVID-19; however, its impact on overall survival of these patients is unknown. To date, few reports describe successful discharge from ECMO in COVID-19 after a prolonged ECMO run. The only Australian case of a COVID-19 patient, supported by prolonged VV ECMO in conjunction with prone ventilation, complicated by significant airway bleeding, and successfully decannulated after forty-two days, is described. VV ECMO is a resource-intense form of respiratory support. Providing complex therapies such as VV ECMO during a pandemic has its unique challenges. This case report provides a unique insight into the potential clinical sequelae of COVID-19, supported in an intensive care environment which was not resource-limited at the time, and adds to the evolving experience of prolonged VV ECMO support for ARDS with a goal to lung recovery.

Role of delayed nonsynaptic neuronal plasticity in long-term associative memory.

BACKGROUND: It is now well established that persistent nonsynaptic neuronal plasticity occurs after learning and, like synaptic plasticity, it can be the substrate for long-term memory. What still remains unclear, though, is how nonsynaptic plasticity contributes to the altered neural network properties on which memory depends. Understanding how nonsynaptic plasticity is translated into modified network and behavioral output therefore represents an important objective of current learning and memory research. RESULTS: By using behavioral single-trial classical conditioning together with electrophysiological analysis and calcium imaging, we have explored the cellular mechanisms by which experience-induced nonsynaptic electrical changes in a neuronal soma remote from the synaptic region are translated into synaptic and circuit level effects. We show that after single-trial food-reward conditioning in the snail Lymnaea stagnalis, identified modulatory neurons that are extrinsic to the feeding network become persistently depolarized between 16 and 24 hr after training. This is delayed with respect to early memory formation but concomitant with the establishment and duration of long-term memory. The persistent nonsynaptic change is extrinsic to and maintained independently of synaptic effects occurring within the network directly responsible for the generation of feeding. Artificial membrane potential manipulation and calcium-imaging experiments suggest a novel mechanism whereby the somal depolarization of an extrinsic neuron recruits command-like intrinsic neurons of the circuit underlying the learned behavior. CONCLUSIONS: We show that nonsynaptic plasticity in an extrinsic modulatory neuron encodes information that enables the expression of long-term associative memory, and we describe how this information can be translated into modified network and behavioral output.

Practices participating in a dental PBRN have substantial and advantageous diversity even though as a group they have much in common with dentists at large.

BACKGROUND: Practice-based research networks offer important opportunities to move recent advances into routine clinical practice. If their findings are not only generalizable to dental practices at large, but can also elucidate how practice characteristics are related to treatment outcome, their importance is even further elevated. Our objective was to determine whether we met a key objective for The Dental Practice-Based Research Network (DPBRN): to recruit a diverse range of practitioner-investigators interested in doing DPBRN studies. METHODS: DPBRN participants completed an enrollment questionnaire about their practices and themselves. To date, more than 1100 practitioners from the five participating regions have completed the questionnaire. The regions consist of: Alabama/Mississippi, Florida/Georgia, Minnesota, Permanente Dental Associates, and Scandinavia (Denmark, Norway, and Sweden). We tested the hypothesis that there are statistically significant differences in key characteristics among DPBRN practices, based on responses from dentists who participated in DPBRN's first network-wide study (n = 546). RESULTS: There were statistically significant, substantive regional differences among DPBRN-participating dentists, their practices, and their patient populations. CONCLUSION: Although as a group, participants have much in common with practices at large; their substantial diversity offers important advantages, such as being able to evaluate how practice differences may affect treatment outcomes, while simultaneously offering generalizability to dentists at large. This should help foster knowledge transfer in both the research-to-practice and practice-to-research directions.

Dentists in practice-based research networks have much in common with dentists at large: evidence from the Dental Practice-Based Research Network.

Practice-based research networks (PBRNs) aim to improve clinical practice by engaging dental practitioners in studies that are directly relevant to daily clinical practice. The Dental Practice-Based Research Network (DPBRN) consists of dentists from seven U.S. states and three Scandinavian countries. All DPBRN dentists complete an enrollment questionnaire about their practices and themselves; as of this writing, 1,086 have done so. To quantify the similarities between DPBRN dentists and U.S. dentists at large, this article compared DPBRN practice characteristics to those of dentists who responded to the 2004 ADA Survey of dental practice, which is not limited to ADA members. DPBRN dentists were similar to U.S. dentists in terms of gender, race, ethnicity, number of offices, percentage of patients with insurance coverage, number of operatories, patient visits per week, days for a new appointment, and waiting room time. DPBRN dentists were statistically more likely to be recent graduates. The commonalities should increase the likelihood that DPBRN studies will be applicable to U.S. practices, thereby fostering knowledge transfer in both research-to-practice and practice-to-research.