Targeted Hybridization Capture of SARS-CoV-2 and Metagenomics Enables Genetic Variant Discovery and Nasal Microbiome Insights.

The emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic variants that may alter viral fitness highlights the urgency of widespread next-generation sequencing (NGS) surveillance. To profile genetic variants of the entire SARS-CoV-2 genome, we developed and clinically validated a hybridization capture SARS-CoV-2 NGS assay, integrating novel methods for panel design using double-stranded DNA (dsDNA) biotin-labeled probes, and built accompanying software. This test is the first hybrid capture-based NGS assay given Food and Drug Administration (FDA) emergency use authorization for detection of the SARS-CoV-2 virus. The positive and negative percent agreement (PPA and NPA, respectively) were defined in comparison to the results for an orthogonal real-time reverse transcription polymerase chain reaction (RT-PCR) assay (PPA and NPA, 96.7 and 100%, respectively). The limit of detection was established to be 800 copies/ml with an average fold enrichment of 46,791. Furthermore, utilizing the research-use-only analysis to profile the variants, we identified 55 novel mutations, including 11 in the functionally important spike protein. Finally, we profiled the full nasopharyngeal microbiome using metagenomics and found overrepresentation of 7 taxa and evidence of macrolide resistance in SARS-CoV-2-positive patients. This hybrid capture NGS assay, coupled with optimized software, is a powerful approach to detect and comprehensively map SARS-CoV-2 genetic variants for tracking viral evolution and guiding vaccine updates. IMPORTANCE This is the first FDA emergency-use-authorized hybridization capture-based next-generation sequencing (NGS) assay to detect the SARS-CoV-2 genome. Viral metagenomics and the novel hybrid capture NGS-based assay, along with its research-use-only analysis, can provide important genetic insights into SARS-CoV-2 and other emerging pathogens and improve surveillance and early detection, potentially preventing or mitigating new outbreaks. Better understanding of the continuously evolving SARS-CoV-2 viral genome and the impact of genetic variants may provide individual risk stratification, precision therapeutic options, improved molecular diagnostics, and population-based therapeutic solutions.

Understanding recent trends in childhood obesity in the United States.

The prevalence of childhood obesity in the United States has more than tripled over the last four decades from 5 percent in 1978 to 18.5 percent in 2016. There is evidence for a break in trend in recent years: after growing from 0.4 to 0.7 percentage point per year between 1978 and 2004, the rate of increase has slowed to 0.1 percentage point per year from 2004 to 2016. To better understand these trends, in this paper we analyze a range of datasets that collect information on childhood obesity. We analyze the data overall, across the age distribution, across birth cohorts, and for subgroups of interest. We find steady increases in cohort-level obesity prevalence through approximately age 10, with levels unchanged thereafter, suggesting a need for additional interventions at early ages. We find that the prevalence of obesity has diverged by race and gender in recent years, especially among children entering kindergarten. Compared with 5-year-olds in 1997, 5-year-olds in 2010 were 2 percentage points more likely to be obese overall. Black and Hispanic 5-year-olds were 5 and 3 percentage points more likely to be obese, respectively, while whites had a 1 percentage point increase in obesity. However, overall and among all subgroups the rate of growth in obesity from kindergarten through 3rd grade has declined in recent years. Together, these findings can inform a future research literature that aims to target obesity interventions where they will be most impactful.

Neural crest-mediated bone resorption is a determinant of species-specific jaw length.

Precise control of jaw length during development is crucial for proper form and function. Previously we have shown that in birds, neural crest mesenchyme (NCM) confers species-specific size and shape to the beak by regulating molecular and histological programs for the induction and deposition of cartilage and bone. Here we reveal that a hitherto unrecognized but similarly essential mechanism for establishing jaw length is the ability of NCM to mediate bone resorption. Osteoclasts are considered the predominant cells that resorb bone, although osteocytes have also been shown to participate in this process. In adults, bone resorption is tightly coupled to bone deposition as a means to maintain skeletal homeostasis. Yet, the role and regulation of bone resorption during growth of the embryonic skeleton have remained relatively unexplored. We compare jaw development in short-beaked quail versus long-billed duck and find that quail have substantially higher levels of enzymes expressed by bone-resorbing cells including tartrate-resistant acid phosphatase (TRAP), Matrix metalloproteinase 13 (Mmp13), and Mmp9. Then, we transplant NCM destined to form the jaw skeleton from quail to duck and generate chimeras in which osteocytes arise from quail donor NCM and osteoclasts come exclusively from the duck host. Chimeras develop quail-like jaw skeletons coincident with dramatically elevated expression of TRAP, Mmp13, and Mmp9. To test for a link between bone resorption and jaw length, we block resorption using a bisphosphonate, osteoprotegerin protein, or an MMP13 inhibitor, and this significantly lengthens the jaw. Conversely, activating resorption with RANKL protein shortens the jaw. Finally, we find that higher resorption in quail presages their relatively lower adult jaw bone mineral density (BMD) and that BMD is also NCM-mediated. Thus, our experiments suggest that NCM not only controls bone resorption by its own derivatives but also modulates the activity of mesoderm-derived osteoclasts, and in so doing enlists bone resorption as a key patterning mechanism underlying the functional morphology and evolution of the jaw.

Costs and benefits of mutational robustness in RNA viruses.

The accumulation of mutations in RNA viruses is thought to facilitate rapid adaptation to changes in the environment. However, most mutations have deleterious effects on fitness, especially for viruses. Thus, tolerance to mutations should determine the nature and extent of genetic diversity that can be maintained in the population. Here, we combine population genetics theory, computer simulation, and experimental evolution to examine the advantages and disadvantages of tolerance to mutations, also known as mutational robustness. We find that mutational robustness increases neutral diversity and, as expected, can facilitate adaptation to a new environment. Surprisingly, under certain conditions, robustness may also be an impediment for viral adaptation, if a highly diverse population contains a large proportion of previously neutral mutations that are deleterious in the new environment. These findings may inform therapeutic strategies that cause extinction of otherwise robust viral populations.

Evolution of a developmental mechanism: Species-specific regulation of the cell cycle and the timing of events during craniofacial osteogenesis.

Neural crest mesenchyme (NCM) controls species-specific pattern in the craniofacial skeleton but how this cell population accomplishes such a complex task remains unclear. To elucidate mechanisms through which NCM directs skeletal development and evolution, we made chimeras from quail and duck embryos, which differ markedly in their craniofacial morphology and maturation rates. We show that quail NCM, when transplanted into duck, maintains its faster timetable for development and autonomously executes molecular and cellular programs for the induction, differentiation, and mineralization of bone, including premature expression of osteogenic genes such as Runx2 and Col1a1. In contrast, the duck host systemic environment appears to be relatively permissive and supports osteogenesis independently by providing circulating minerals and a vascular network. Further experiments reveal that NCM establishes the timing of osteogenesis by regulating cell cycle progression in a stage- and species-specific manner. Altering the time-course of D-type cyclin expression mimics chimeras by accelerating expression of Runx2 and Col1a1. We also discover higher endogenous expression of Runx2 in quail coincident with their smaller craniofacial skeletons, and by prematurely over-expressing Runx2 in chick embryos we reduce the overall size of the craniofacial skeleton. Thus, our work indicates that NCM establishes species-specific size in the craniofacial skeleton by controlling cell cycle, Runx2 expression, and the timing of key events during osteogenesis.

Smad3 binds Scleraxis and Mohawk and regulates tendon matrix organization.

TGFß plays a critical role in tendon formation and healing. While its downstream effector Smad3 has been implicated in the healing process, little is known about the role of Smad3 in normal tendon development or tenocyte gene expression. Using mice deficient in Smad3 (Smad3(-/-) ), we show that Smad3 ablation disrupts tendon architecture and has a dramatic impact on normal gene and protein expression during development as well as in mature tendon. In developing and adult tendon, loss of Smad3 results in reduced protein expression of the matrix components Collagen 1 and Tenascin-C. Additionally, when compared to wild type, tendon from adult Smad3(-/-) mice shows a down regulation of key tendon marker genes. Finally, we have established that Smad3 has the ability to physically interact with the critical transcriptional regulators Scleraxis and Mohawk. Together these results indicate a central role for Smad3 in normal tendon formation and in the maintenance of mature tendon.

Structured coculture of mesenchymal stem cells and disc cells enhances differentiation and proliferation.

PURPOSE: During in vivo stem cell differentiation, mature cells often induce the differentiation of nearby stem cells. Accordingly, prior studies indicate that a randomly mixed coculture can help transform mesenchymal stem cells (MSC) into nucleus pulposus cells (NPC). However, because in vivo signaling typically occurs heterotopically between adjacent cell layers, we hypothesized that a structurally organized coculture between MSC and NPC will result in greater cell differentiation and proliferation over single cell-type controls and cocultures with random organization. METHODS: We developed a novel bilaminar cell pellet (BCP) system where a sphere of MSC is enclosed in a shell of NPC by successive centrifugation. Controls were made using single cell-type pellets and coculture pellets with random organization. The pellets were evaluated for DNA content, gene expression, and histology. RESULTS: A bilaminar 3D organization enhanced cell proliferation and differentiation. BCP showed significantly more cell proliferation than pellets with one cell type and those with random organization. Enhanced differentiation of MSC within the BCP pellet relative to single cell-type pellets was demonstrated by quantitative RT-PCR, histology, and in situ hybridization. CONCLUSIONS: The BCP culture system increases MSC proliferation and differentiation as compared to single cell type or randomly mixed coculture controls.

Structured bilaminar coculture outperforms stem cells and disc cells in a simulated degenerate disc environment.

STUDY DESIGN: This study explores the use of bilaminar coculture pellets of mesenchymal stem cells (MSCs) and nucleus pulposus cells (NPCs) as a cell-based therapy for intervertebral disc regeneration. The pellets were tested under conditions that mimic the degenerative disc. OBJECTIVE: Our goal was to optimize our cell-based therapy in vitro under conditions representative of the eventual diseased tissue. SUMMARY OF BACKGROUND DATA: Harnessing the potential of stem cells is an important strategy for regenerative medicine. Our approach directed the behavior of stem cells by mimicking embryonic processes underlying cartilage and intervertebral disc development. Prior experiments have shown that bilaminar coculture can help differentiate MSC and substantially improve new matrix deposition. METHODS: We have designed a novel spherical bilaminar cell pellet (BCP) where MSCs are enclosed in a shell of NPC. There were 3 groups: MSC, NPC, and BCP. The pellets were tested under 3 different culture conditions: 1) in a bioreactor that provides pressure and hypoxia (mimicking normal disc conditions): 2) with inflammatory cytokines (IL-1b and TNF-a); and 3) a bioreactor with inflammation (mimicking painful disc conditions). RESULTS: When cultured in the bioreactor, the NPC pellets produced significantly more glycosaminoglycans (GAGs) per cell than the other groups: 70% to 80% more than the BCP and the MSC alone. When cultured in an inflammatory environment, the MSC and BCP groups produced 30% to 34% more GAGs per cell than NPC (P < 0.05). When the pellets were cultured in a bioreactor with inflammation, the BCP made 25% more GAGs per cell than the MSC and 57% more than the NPC (P < 0.05). CONCLUSION: This study shows that BCPs outperform controls in a simulated degenerated disc environment. Adapting inductive mechanisms from development to trigger differentiation and restore diseased tissue has many advantages. As opposed to strategies that require growth factor supplements or genetic manipulations, our method is self-sustaining, targeted, and minimally invasive injection.

Is being in school better? The impact of school on children's BMI when starting age is endogenous.

In this paper, we investigate the impact of attending school on body weight and obesity using a regression-discontinuity design. As is the case with academic outcomes, school exposure is related to unobserved determinants of weight outcomes because some families choose to have their child start school late (or early). If one does not account for this endogeneity, it appears that an additional year of school exposure results in a greater BMI and a higher probability of being overweight or obese. When we compare the weight outcomes of similar age children with one versus two years of school exposure due to regulations on school starting age, the significant positive effects disappear, and most point estimates become negative, but insignificant. However, additional school exposure appears to improve weight outcomes of children for whom the transition to elementary school represents a more dramatic change in environment (those who spent less time in childcare prior to kindergarten).

Tissue-specific calibration of extracellular matrix material properties by transforming growth factor-ß and Runx2 in bone is required for hearing.

Physical cues, such as extracellular matrix stiffness, direct cell differentiation and support tissue-specific function. Perturbation of these cues underlies diverse pathologies, including osteoarthritis, cardiovascular disease and cancer. However, the molecular mechanisms that establish tissue-specific material properties and link them to healthy tissue function are unknown. We show that Runx2, a key lineage-specific transcription factor, regulates the material properties of bone matrix through the same transforming growth factor-ß (TGFß)-responsive pathway that controls osteoblast differentiation. Deregulated TGFß or Runx2 function compromises the distinctly hard cochlear bone matrix and causes hearing loss, as seen in human cleidocranial dysplasia. In Runx2+/⁻ mice, inhibition of TGFß signalling rescues both the material properties of the defective matrix, and hearing. This study elucidates the unknown cause of hearing loss in cleidocranial dysplasia, and demonstrates that a molecular pathway controlling cell differentiation also defines material properties of extracellular matrix. Furthermore, our results suggest that the careful regulation of these properties is essential for healthy tissue function.

Ameloblast differentiation in the human developing tooth: effects of extracellular matrices.

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin alpha2beta1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.

Childhood obesity: trends and potential causes.

The increase in childhood obesity over the past several decades, together with the associated health problems and costs, is raising grave concern among health care professionals, policy experts, children's advocates, and parents. Patricia Anderson and Kristin Butcher document trends in children's obesity and examine the possible underlying causes of the obesity epidemic. They begin by reviewing research on energy intake, energy expenditure, and "energy balance," noting that children who eat more "empty calories" and expend fewer calories through physical activity are more likely to be obese than other children. Next they ask what has changed in children's environment over the past three decades to upset this energy balance equation. In particular, they examine changes in the food market, in the built environment, in schools and child care settings, and in the role of parents-paying attention to the timing of these changes. Among the changes that affect children's energy intake are the increasing availability of energy-dense, high-calorie foods and drinks through schools. Changes in the family, particularly an increase in dual-career or single-parent working families, may also have increased demand for food away from home or pre-prepared foods. A host of factors have also contributed to reductions in energy expenditure. In particular, children today seem less likely to walk to school and to be traveling more in cars than they were during the early 1970s, perhaps because of changes in the built environment. Finally, children spend more time viewing television and using computers. Anderson and Butcher find no one factor that has led to increases in children's obesity. Rather, many complementary changes have simultaneously increased children's energy intake and decreased their energy expenditure. The challenge in formulating policies to address children's obesity is to learn how best to change the environment that affects children's energy balance.

Maternal employment and overweight children.

This paper seeks to determine whether a causal relationship exists between maternal employment and childhood weight problems. We use matched mother-child data from the National Longitudinal Survey of Youth (NLSY) and employ econometric techniques to control for observable and unobservable differences across individuals and families that may influence both children's weight and their mothers' work patterns. Our results indicate that a child is more likely to be overweight if his/her mother worked more hours per week over the child's life. Analyses by subgroups show that it is higher socioeconomic status mothers whose work intensity is particularly deleterious for their children's overweight status.