Regulation of Endochondral Cartilage Growth in the Developing Avian Limb: Cooperative Involvement of Perichondrium and Periosteum

The perichondrium and periosteum have recently been suggested to be involved in the regulation of limb growth, serving as potential sources of signaling molecules that are involved in chondrocyte proliferation, maturation, and hypertrophy. Previously, we observed that removal of the perichondrium and periosteum from tibiotarsi in organ culture resulted in an overall increase in longitudinal cartilage growth, suggesting negative regulation originating from these tissues. To determine if the perichondrium and periosteum regulate growth through the production of diffusible factors, we have tested various conditioned media from these tissues for the ability to modify cartilage growth in tibiotarsal organ cultures from which these tissues have been removed. Both negative and positive regulatory activities were detected. Negative regulation was observed with conditioned medium from (1) cell cultures of the region bordering both the perichondrium and the periosteum, (2) co-cultures of perichondrial and periosteal cells, and (3) a mixture of conditioned media from perichondrial cell cultures and periosteal cell cultures. The requirement for regulatory factors from both the perichondrium and periosteum suggests a novel mechanism of regulation. Positive regulation was observed with conditioned media from several cell types, with the most potent activity being from articular perichondrial cells and hypertrophic chondrocytes.     

The Spindle Checkpoint of Budding Yeast Depends on a Tight Complex between the Mad1 and Mad2 Proteins

The spindle checkpoint arrests the cell cycle at metaphase in the presence of defects in the mitotic spindle or in the attachment of chromosomes to the spindle. When spindle assembly is disrupted, the budding yeast mad and bub mutants fail to arrest and rapidly lose viability. We have cloned the MAD2 gene, which encodes a protein of 196 amino acids that remains at a constant level during the cell cycle. Gel filtration and co-immunoprecipitation analyses reveal that Mad2p tightly associates with another spindle checkpoint component, Mad1p. This association is independent of cell cycle stage and the presence or absence of other known checkpoint proteins. In addition, Mad2p binds to all of the different phosphorylated isoforms of Mad1p that can be resolved on SDS-PAGE. Deletion and mutational analysis of both proteins indicate that association of Mad2p with Mad1p is critical for checkpoint function and for hyperphosphorylation of Mad1p.     

Differential pathogenesis of closely related 2018 Nigerian outbreak clade III Lassa virus isolates

Nigeria continues to experience ever increasing annual outbreaks of Lassa fever (LF). The World Health Organization has recently declared Lassa virus (LASV) as a priority pathogen for accelerated research leading to a renewed international effort to develop relevant animal models of disease and effective countermeasures to reduce LF morbidity and mortality in endemic West African countries. A limiting factor in evaluating medical countermeasures against LF is a lack of well characterized animal models outside of those based on infection with LASV strain Josiah originating form Sierra Leone, circa 1976. Here we genetically characterize five recent LASV isolates collected from the 2018 outbreak in Nigeria. Three isolates were further evaluated in vivo and despite being closely related and from the same spatial / geographic region of Nigeria, only one of the three isolates proved lethal in strain 13 guinea pigs and non-human primates (NHP). Additionally, this isolate exhibited atypical pathogenesis characteristics in the NHP model, most notably respiratory failure, not commonly described in hemorrhagic cases of LF. These results suggest that there is considerable phenotypic heterogeneity in LASV infections in Nigeria, which leads to a multitude of pathogenesis characteristics that could account for differences between subclinical and lethal LF infections. Most importantly, the development of disease models using currently circulating LASV strains in West Africa are critical for the evaluation of potential vaccines and medical countermeasures.     

Enhanced deoxyribozyme‐catalyzed RNA ligation in the presence of organic cosolvents

Deoxyribozyme and aptamer selections are typically conducted in aqueous buffer solutions. Using nonaqueous cosolvents in selection experiments will help expand the activity of deoxyribozymes with non‐oligonucleotide substrates and will allow identification of new aptamers for nonprotein targets. We undertook in vitro selections utilizing a small amount of methanol in the reaction to keep the herbicides alachlor and atrazine in solution with the goal of identifying deoxyribozymes that require these herbicides for activity. The resulting deoxyribozymes successfully catalyze RNA ligation, but do not require alachlor or atrazine. Surprisingly, some of these deoxyribozymes displayed better catalytic activity in the presence of methanol over just aqueous buffer. We investigated several organic cosolvents to see if this enhancement was limited to methanol and found that other cosolvents, including ethanol, DMSO, and DMF, supported activity; in some cases, greater enhancement was observed. On the basis of these results, we tested two other previously identified RNA‐ligating deoxyribozymes to assess their tolerance of cosolvents and determined that different deoxyribozymes showed different responses to the cosolvents. Our results demonstrate that deoxyribozymes can tolerate and, in some cases, display enhanced activity in alternative solvent conditions. These findings will facilitate the development of responsive deoxyribozyme systems utilizing components with limited water solubility. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 382–391, 2013.     

CACNA1H mutations in autism spectrum disorders

Autism spectrum disorders (ASD) are neurodevelopmental conditions characterized by impaired social interaction, communication skills, and restricted and repetitive behavior. The genetic causes for autism are largely unknown. Previous studies implicate CACNA1C (L-type Ca(V)1.2) calcium channel mutations in a disorder associated with autism (Timothy syndrome). Here, we identify missense mutations in the calcium channel gene CACNA1H (T-type Ca(V)3.2) in 6 of 461 individuals with ASD. These mutations are located in conserved and functionally relevant domains and are absent in 480 ethnically matched controls (p = 0.014, Fisher's exact test). Non-segregation within the pedigrees between the mutations and the ASD phenotype clearly suggest that the mutations alone are not responsible for the condition. However, functional analysis shows that all these mutations significantly reduce Ca(V)3.2 channel activity and thus could affect neuronal function and potentially brain development. We conclude that the identified mutations could contribute to the development of the ASD phenotype.     

Measuring size and composition of species pools: a comparison of dark diversity estimates

Ecological theory and biodiversity conservation have traditionally relied on the number of species recorded at a site, but it is agreed that site richness represents only a portion of the species that can inhabit particular ecological conditions, that is, the habitat‐specific species pool. Knowledge of the species pool at different sites enables meaningful comparisons of biodiversity and provides insights into processes of biodiversity formation. Empirical studies, however, are limited due to conceptual and methodological difficulties in determining both the size and composition of the absent part of species pools, the so‐called dark diversity. We used >50,000 vegetation plots from 18 types of habitats throughout the Czech Republic, most of which served as a training dataset and 1083 as a subset of test sites. These data were used to compare predicted results from three quantitative methods with those of previously published expert estimates based on species habitat preferences: (1) species co‐occurrence based on Beals' smoothing approach; (2) species ecological requirements, with envelopes around community mean Ellenberg values; and (3) species distribution models, using species environmental niches modeled by Biomod software. Dark diversity estimates were compared at both plot and habitat levels, and each method was applied in different configurations. While there were some differences in the results obtained by different methods, particularly at the plot level, there was a clear convergence, especially at the habitat level. The better convergence at the habitat level reflects less variation in local environmental conditions, whereas variation at the plot level is an effect of each particular method. The co‐occurrence agreed closest the expert estimate, followed by the method based on species ecological requirements. We conclude that several analytical methods can estimate species pools of given habitats. However, the strengths and weaknesses of different methods need attention, especially when dark diversity is estimated at the plot level.     

Zinc finger protein ZFP57 requires its co-factor to recruit DNA methyltransferases and maintains DNA methylation imprint in embryonic stem cells via its transcriptional repression domain

Previously, we discovered that ZFP57 is a maternal-zygotic effect gene, and it maintains DNA methylation genomic imprint at multiple imprinted regions in mouse embryos. Despite these findings, it remains elusive how DNA methyltransferases are targeted to the imprinting control regions to initiate and maintain DNA methylation imprint. To gain insights into these essential processes in genomic imprinting, we examined how ZFP57 maintains genomic DNA methylation imprint in mouse embryonic stem (ES) cells. Here we demonstrate that the loss of ZFP57 in mouse ES cells led to a complete loss of genomic DNA methylation imprint at multiple imprinted regions, similar to its role in mouse embryos. However, reintroduction of ZFP57 into Zfp57-null ES cells did not result in reacquisition of DNA methylation imprint, suggesting that the memory for genomic imprinting had been lost or altered in Zfp57-null ES cells in culture. Interestingly, ZFP57 and DNA methyltransferases could form complexes in the presence of KAP1/TRIM28/TIF1β when co-expressed in COS cells. We also found that the wild-type exogenous ZFP57 but not the mutant ZFP57 lacking the KRAB box that interacts with its co-factor KAP1/TRIM28/TIF1β could substitute for the endogenous ZFP57 in maintaining the DNA methylation imprint in ES cells. These results suggest that ZFP57 may recruit DNA methyltransferases to its target regions to maintain DNA methylation imprint, and this interaction is likely facilitated by KAP1/TRIM28/TIF1β.