BACKGROUND: The molecular tweezer CLR01 is a broad-spectrum inhibitor of abnormal protein self-assembly, which acts by binding selectively to Lys residues. CLR01 has been tested in several in vitro and in vivo models of amyloidoses all without signs of toxicity. With the goal of developing CLR01 as a therapeutic drug for Alzheimers disease and other amyloidoses, here we studied its safety and pharmacokinetics. METHODS: Toxicity studies were performed in 2-m old wild-type mice. Toxicity was evaluated by serum chemical analysis, histopathology analysis, and qualitative behavioral analysis. Brain penetration studies were performed using radiolabeled CLR01 in both wild-type mice and a transgenic mouse model of Alzheimers disease at 2-m, 12-m, and 22-m of age. Brain levels were measured from 0.5 - 72 h post administration. RESULTS: Examination of CLR01s effect on tubulin polymerization, representing normal protein assembly, showed disruption of the process only when 55-fold excess CLR01 was used, supporting the compounds putative process-specific mechanism of action.A single-injection of 100 mg/kg CLR01 in mice - 2,500-fold higher than the efficacious dose reported previously, induced temporary distress and liver injury, but no mortality. Daily injection of doses up to 10 mg/kg did not produce any signs of toxicity, suggesting a high safety margin.The brain penetration of CLR01 was found to be 1 - 3% of blood levels depending on age. Though CLR01 was almost completely removed from the blood by 8 h, unexpectedly, brain levels of CLR01 remained steady over 72 h. CONCLUSION: Estimation of brain levels compared to amyloid β-protein concentrations reported previously suggest that the stoichiometry obtained in vitro and in vivo is similar, supporting the mechanism of action of CLR01.The favorable safety margin of CLR01, together with efficacy shown in multiple animal models, support further development of CLR01 as a disease-modifying agent for amyloidoses.

Objectives

Limited data are available on how the closure of pediatric dental clinics because of the COVID-19 pandemic affected hospital pediatric emergency department (ED) visits in the United States. We evaluated changes in dental-related visits at a pediatric ED and associated urgent care centers (UCCs) after the shutdown of a large pediatric dental clinic because of the COVID-19 pandemic.

Methods

We conducted a single-center retrospective medical record review of 811 patients aged 0 to 17 years who presented to a pediatric ED or associated UCC at Rady Children's Hospital-San Diego for dental-related concerns from March 19, 2019, through January 17, 2021. Patients were classified into 3 periods: before shutdown, during shutdown, and after shutdown. We collected data on demographic characteristics; International Classification of Diseases, Tenth Revision codes; dental diagnosis; treatment; and COVID-19 test results. We compared the frequency and proportion of patients seen for dental-related concerns, dental diagnosis, and treatment during the 3 periods.

Results

The proportion of dental-related concerns in the ED doubled during the shutdown (0.7%) and was 1.5 times higher after the shutdown (0.6%) compared with before the shutdown (0.4%; P < .001). Significantly more patients were seen in EDs than in UCCs during and after the shutdown than before the shutdown (P = .005). During and after the shutdown, admission to the hospital for antibiotic treatment increased significantly to 6.5% and 7.9%, respectively, compared with before the shutdown (2.8%; P = .022), and nonaerosolized procedures and ED/UCC discharge increased to 13.4% and 9.3%, respectively, compared with before the shutdown (6.2%; P = .015).

Conclusions

Mitigating future closures of dental offices is important given the shifted burden of dental care to the ED.

Background

The "molecular tweezer" CLR01 is a broad-spectrum inhibitor of abnormal protein self-assembly, which acts by binding selectively to Lys residues. CLR01 has been tested in several in vitro and in vivo models of amyloidoses all without signs of toxicity. With the goal of developing CLR01 as a therapeutic drug for Alzheimer's disease and other amyloidoses, here we studied its safety and pharmacokinetics.

Methods

Toxicity studies were performed in 2-m old wild-type mice. Toxicity was evaluated by serum chemical analysis, histopathology analysis, and qualitative behavioral analysis. Brain penetration studies were performed using radiolabeled CLR01 in both wild-type mice and a transgenic mouse model of Alzheimer's disease at 2-m, 12-m, and 22-m of age. Brain levels were measured from 0.5 - 72 h post administration.

Results

Examination of CLR01's effect on tubulin polymerization, representing normal protein assembly, showed disruption of the process only when 55-fold excess CLR01 was used, supporting the compound's putative "process-specific" mechanism of action.A single-injection of 100 mg/kg CLR01 in mice - 2,500-fold higher than the efficacious dose reported previously, induced temporary distress and liver injury, but no mortality. Daily injection of doses up to 10 mg/kg did not produce any signs of toxicity, suggesting a high safety margin.The brain penetration of CLR01 was found to be 1 - 3% of blood levels depending on age. Though CLR01 was almost completely removed from the blood by 8 h, unexpectedly, brain levels of CLR01 remained steady over 72 h.

Conclusion

Estimation of brain levels compared to amyloid β-protein concentrations reported previously suggest that the stoichiometry obtained in vitro and in vivo is similar, supporting the mechanism of action of CLR01.The favorable safety margin of CLR01, together with efficacy shown in multiple animal models, support further development of CLR01 as a disease-modifying agent for amyloidoses.

Alzheimer's disease is a progressive neurodegenerative disease that manifests as memory loss, cognitive dysfunction, and dementia. Animal models of Alzheimer's disease have been instrumental in understanding the underlying pathological mechanism and in evaluation of potential therapies. The triple transgenic (3 × Tg) mouse model of AD is unique because it recapitulates both pathologic hallmarks of Alzheimer's disease--amyloid plaques and neurofibrillary tangles. The earliest cognitive deficits in this model have been shown at 6-m of age by most groups, necessitating aging of the mice to this age before initiating evaluation of the cognitive effects of therapies. To assess cognitive deficits in the 3 × Tg mice, originally we employed a typical Barnes maze protocol of 15 training trials, but found no significant deficits in aged mice. Therefore, we shortened the protocol to include only 5 training trials to increase difficulty. We found cognitive deficits using this protocol using mainly measures from the probe day, rather than the training trials. This also decreased the effort involved with data analysis. We compared 3 × Tg and wild-type mice at 4-m- and 15-m of age using both the original, long training, and the short training paradigms. We found that differences in learning between 3 × Tg and wild-type mice disappeared after the 4(th) training trial. Measures of learning and memory on the probe day showed significant differences between 3 × Tg and wild-type mice following the short, 5-training trial protocol but not the long, 15-training trial protocol. Importantly, we detected cognitive dysfunction already at 4-m of age in 3 × Tg mice using the short Barnes-maze protocol. The ability to test learning and memory in 4-m old 3 × Tg mice using a shortened Barnes maze protocol offers considerable time and cost savings and provides support for the utilization of this model at pre-pathology stages for therapeutic studies.

BACKGROUND: Clinical management of shrimp allergy is hampered by the lack of accurate tests. Molecular diagnosis has been shown to more accurately reflect the clinical reactivity but the full spectrum of shrimp allergens and their clinical relevance are yet to be established. We therefore sought to comprehend the allergen repertoire of shrimp, investigate and compare the sensitization pattern and diagnostic value of the allergens in allergic subjects of two distinct populations. METHODS: Sera were collected from 85 subjects with challenge-proven or doctor-diagnosed shrimp allergy in Hong Kong and Thailand. The IgE-binding proteins of Penaeus monodon were probed by Western blotting and identified by mass spectrometry. Recombinant shrimp allergens were synthesized and analyzed for IgE sensitization by ELISA. RESULTS: Ten IgE-binding proteins were identified, and a comprehensive panel of 11 recombinant shrimp allergens was generated. The major shrimp allergens among Hong Kong subjects were troponin C (Pen m 6) and glycogen phosphorylase (Pen m 14, 47.1%), tropomyosin (Pen m 1, 41.2%) and sarcoplasmic-calcium binding protein (Pen m 4, 35.3%), while those among Thai subjects were Pen m 1 (68.8%), Pen m 6 (50.0%) and fatty acid-binding protein (Pen m 13, 37.5%). Component-based tests yielded significantly higher area under curve values (0.77-0.96) than shrimp extract-IgE test (0.70-0.75). Yet the best component test differed between populations; Pen m 1-IgE test added diagnostic value only in the Thai cohort, whereas sensitizations to other components were better predictors of shrimp allergy in Hong Kong patients. CONCLUSION: Pen m 14 was identified as a novel shrimp allergen predictive of challenge outcome. Molecular diagnosis better predicts shrimp allergy than conventional tests, but the relevant component is population dependent.

Gene duplication is a major evolutionary force driving adaptation and speciation, as it allows for the acquisition of new functions and can augment or diversify existing functions. Here, we report a gene duplication event that yielded another outcome--the generation of antagonistic functions. One product of this duplication event--UPF3B--is critical for the nonsense-mediated RNA decay (NMD) pathway, while its autosomal counterpart--UPF3A--encodes an enigmatic protein previously shown to have trace NMD activity. Using loss-of-function approaches in vitro and in vivo, we discovered that UPF3A acts primarily as a potent NMD inhibitor that stabilizes hundreds of transcripts. Evidence suggests that UPF3A acquired repressor activity through simple impairment of a critical domain, a rapid mechanism that may have been widely used in evolution. Mice conditionally lacking UPF3A exhibit "hyper" NMD and display defects in embryogenesis and gametogenesis. Our results support a model in which UPF3A serves as a molecular rheostat that directs developmental events.

[This corrects the article on p. 1154 in vol. 8, PMID: 28694797.].

Although the taxonomy of Burkholderia has been extensively scrutinized, significant uncertainty remains regarding the generic boundaries and composition of this large and heterogeneous taxon. Here we used the amino acid and nucleotide sequences of 106 conserved proteins from 92 species to infer robust maximum likelihood phylogenies with which to investigate the generic structure of Burkholderia sensu lato. These data unambiguously supported five distinct lineages, of which four correspond to Burkholderia sensu stricto and the newly introduced genera Paraburkholderia, Caballeronia, and Robbsia. The fifth lineage was represented by P. rhizoxinica. Based on these findings, we propose 13 new combinations for those species previously described as members of Burkholderia but that form part of Caballeronia. These findings also suggest revision of the taxonomic status of P. rhizoxinica as it is does not form part of any of the genera currently recognized in Burkholderia sensu lato. From a phylogenetic point of view, Burkholderia sensu stricto has a sister relationship with the Caballeronia+Paraburkholderia clade. Also, the lineages represented by P. rhizoxinica and R. andropogonis, respectively, emerged prior to the radiation of the Burkholderia sensu stricto+Caballeronia+Paraburkholderia clade. Our findings therefore constitute a solid framework, not only for supporting current and future taxonomic decisions, but also for studying the evolution of this assemblage of medically, industrially and agriculturally important species.

Unsolved Mendelian cases often lack obvious pathogenic coding variants, suggesting potential non-coding etiologies. Here, we present a single cell multi-omic framework integrating embryonic mouse chromatin accessibility, histone modification, and gene expression assays to discover cranial motor neuron (cMN) cis-regulatory elements and subsequently nominate candidate non-coding variants in the congenital cranial dysinnervation disorders (CCDDs), a set of Mendelian disorders altering cMN development. We generate single cell epigenomic profiles for ~86,000 cMNs and related cell types, identifying ~250,000 accessible regulatory elements with cognate gene predictions for ~145,000 putative enhancers. We evaluate enhancer activity for 59 elements using an in vivo transgenic assay and validate 44 (75%), demonstrating that single cell accessibility can be a strong predictor of enhancer activity. Applying our cMN atlas to 899 whole genome sequences from 270 genetically unsolved CCDD pedigrees, we achieve significant reduction in our variant search space and nominate candidate variants predicted to regulate known CCDD disease genes MAFB, PHOX2A, CHN1, and EBF3 - as well as candidates in recurrently mutated enhancers through peak- and gene-centric allelic aggregation. This work delivers non-coding variant discoveries of relevance to CCDDs and a generalizable framework for nominating non-coding variants of potentially high functional impact in other Mendelian disorders.