Mesenchymal Wnts are required for morphogenetic movements of calvarial osteoblasts during apical expansion.

Apical expansion of calvarial osteoblast progenitors from the cranial mesenchyme (CM) above the eye is integral to calvarial growth and enclosure of the brain. The cellular behaviors and signals underlying the morphogenetic process of calvarial expansion are unknown. Time-lapse light-sheet imaging of mouse embryos revealed calvarial progenitors intercalate in 3D in the CM above the eye, and exhibit protrusive and crawling activity more apically. CM cells express non-canonical Wnt/planar cell polarity (PCP) core components and calvarial osteoblasts are bidirectionally polarized. We found non-canonical ligand Wnt5a-/- mutants have less dynamic cell rearrangements and protrusive activity. Loss of CM-restricted Wntless (CM-Wls), a gene required for secretion of all Wnt ligands, led to diminished apical expansion of Osx+ calvarial osteoblasts in the frontal bone primordia in a non-cell autonomous manner without perturbing proliferation or survival. Calvarial osteoblast polarization, progressive cell elongation and enrichment for actin along the baso-apical axis were dependent on CM-Wnts. Thus, CM-Wnts regulate cellular behaviors during calvarial morphogenesis for efficient apical expansion of calvarial osteoblasts. These findings also offer potential insights into the etiologies of calvarial dysplasias.

What Do Animal Models Teach Us About Congenital Craniofacial Defects?

The formation of the head and face is a complex process which involves many different signaling cues regulating the migration, differentiation, and proliferation of the neural crest. This highly complex process is very error-prone, resulting in craniofacial defects in nearly 10,000 births in the United States annually. Due to the highly conserved mechanisms of craniofacial development, animal models are widely used to understand the pathogenesis of various human diseases and assist in the diagnosis and generation of preventative therapies and treatments. Here, we provide a brief background of craniofacial development and discuss several rare diseases affecting craniofacial bone development. We focus on rare congenital diseases of the cranial bone, facial jaw bones, and two classes of diseases, ciliopathies and RASopathies. Studying the animal models of these rare diseases sheds light not only on the etiology and pathology of each disease, but also provides meaningful insights towards the mechanisms which regulate normal development of the head and face.

The 14-3-3 Protein Homolog ArtA Regulates Development and Secondary Metabolism in the Opportunistic Plant Pathogen Aspergillus flavus.

The opportunistic plant-pathogenic fungus Aspergillus flavus produces carcinogenic mycotoxins termed aflatoxins (AF). Aflatoxin contamination of agriculturally important crops, such as maize, peanut, sorghum, and tree nuts, is responsible for serious adverse health and economic impacts worldwide. In order to identify possible genetic targets to reduce AF contamination, we have characterized the artA gene, encoding a putative 14-3-3 homolog in A. flavus The artA deletion mutant presents a slight decrease in vegetative growth and alterations in morphological development and secondary metabolism. Specifically, artA affects conidiation, and this effect is influenced by the type of substrate and culture condition. In addition, normal levels of artA are required for sclerotial development. Importantly, artA negatively regulates AF production as well as the concomitant expression of genes in the AF gene cluster. An increase in AF is also observed in seeds infected with the A. flavus strain lacking artA Furthermore, the expression of other secondary metabolite genes is also artA dependent, including genes in the cyclopiazonic acid (CPA) and ustiloxin gene clusters, in this agriculturally important fungus.IMPORTANCE In the current study, artA, which encodes a 14-3-3 homolog, was characterized in the agriculturally and medically important fungus Aspergillus flavus, specifically, its possible role governing sporulation, formation of resistant structures, and secondary metabolism. The highly conserved artA is necessary for normal fungal morphogenesis in an environment-dependent manner, affecting the balance between production of conidiophores and the formation of resistant structures that are necessary for the dissemination and survival of this opportunistic pathogen. This study reports a 14-3-3 protein affecting secondary metabolism in filamentous fungi. Importantly, artA regulates the biosynthesis of the potent carcinogenic compound aflatoxin B1 (AFB1) as well as the production of other secondary metabolites.

The effect of corrosion inhibitors on microbial communities associated with corrosion in a model flow cell system.

A model flow cell system was designed to investigate pitting corrosion in pipelines associated with microbial communities. A microbial inoculum producing copious amounts of H2S was enriched from an oil pipeline biofilm sample. Reservoirs containing a nutrient solution and the microbial inoculum were pumped continuously through six flow cells containing mild steel corrosion coupons. Two cells received corrosion inhibitor "A", two received corrosion inhibitor "B", and two ("untreated") received no additional chemicals. Coupons were removed after 1 month and analyzed for corrosion profiles and biofilm microbial communities. Coupons from replicate cells showed a high degree of similarity in pitting parameters and in microbial community profiles, as determined by 16S rRNA gene sequence libraries but differed with treatment regimen, suggesting that the corrosion inhibitors differentially affected microbial species. Viable microbial biomass values were more than 10-fold higher for coupons from flow cells treated with corrosion inhibitors than for coupons from untreated flow cells. The total number of pits >10 mils diameter and maximum pitting rate were significantly correlated with each other and the total number of pits with the estimated abundance of sequences classified as Desulfomicrobium. The maximum pitting rate was significantly correlated with the sum of the estimated abundance of Desulfomicrobium plus Clostridiales, and with the sum of the estimated abundance of Desulfomicrobium plus Betaproteobacteria. The lack of significant correlation with the estimated abundance of Deltaproteobacteria suggests not all Deltaproteobacteria species contribute equally to microbiologically influenced corrosion (MIC) and that it is not sufficient to target one bacterial group when monitoring for MIC.

Mesenchymal Wnts are required for morphogenetic movements of calvarial osteoblasts during apical expansion.

Apical expansion of calvarial osteoblast progenitors from the cranial mesenchyme (CM) above the eye is integral for calvarial growth and enclosure of the brain. The cellular behaviors and signals underlying the morphogenetic process of calvarial expansion are unknown. During apical expansion, we found that mouse calvarial primordia have consistent cellular proliferation, density, and survival with complex tissue scale deformations, raising the possibility that morphogenetic movements underlie expansion. Time lapse light sheet imaging of mouse embryos revealed that calvarial progenitors intercalate in 3D to converge supraorbital arch mesenchyme mediolaterally and extend it apically. In contrast, progenitors located further apically exhibited protrusive and crawling activity. CM cells express non-canonical Wnt/Planar Cell Polarity (PCP) core components and calvarial osteoblasts are bidirectionally polarized. We found non-canonical ligand, Wnt5a-/- mutants have less dynamic cell rearrangements, protrusive activity, and a flattened head shape. Loss of cranial mesenchyme-restricted Wntless (CM-Wls), a gene required for secretion of all Wnt ligands, led to diminished apical expansion of OSX+ calvarial osteoblasts in the frontal bone primordia in a non-cell autonomous manner without perturbing proliferation or survival. Calvarial osteoblast polarization, progressive cell elongation and enrichment for actin cytoskeleton protein along the baso-apical axis were dependent on CM-Wnts. Thus, CM-Wnts regulate cellular behaviors during calvarial morphogenesis and provide tissue level cues for efficient apical expansion of calvarial osteoblasts. These findings also offer potential insights into the etiologies of calvarial dysplasias.

Molecular tools to track bacteria responsible for fuel deterioration and microbiologically influenced corrosion.

Investigating the susceptibility of various fuels to anaerobic biodegradation has become complicated with the recognition that the fuels themselves are not sterile. Bacterial DNA could be obtained when various fuels were filtered through a hydrophobic teflon (0.22 µm) membrane filter. Bacterial 16S rRNA genes from these preparations were PCR amplified, cloned, and the resulting libraries sequenced to identify the fuel-borne bacterial communities. The most common sequence, found in algal- and camelina-based biofuels as well as in ultra-low sulfur diesel (ULSD) and F76 diesel, was similar to that of a Tumebacillus. The next most common sequence was similar to Methylobacterium and was found in the biofuels and ULSD. Higher level phylogenetic groups included representatives of the Firmicutes (Bacillus, Lactobacillus and Streptococcus), several Actinobacteria, Deinococcus-Thermus, Chloroflexi, Cyanobacteria, Bacteroidetes, Alphaproteobacteria (Methylobacterium and Sphingomonadales), Betaproteobacteria (Oxalobacteraceae and Burkholderiales) and Deltaproteobacteria. All of the fuel-associated bacterial sequences, except those obtained from a few facultative microorganisms, were from aerobes and only remotely affiliated with sequences that resulted from anaerobic successional events evident when ULSD was incubated with a coastal seawater and sediment inoculum. Thus, both traditional and alternate fuel formulations harbor a characteristic microflora, but these microorganisms contributed little to the successional patterns that ultimately resulted in fuel decomposition, sulfide formation and metal biocorrosion. The findings illustrate the value of molecular approaches to track the fate of bacteria that might come in contact with fuels and potentially contribute to corrosion problems throughout the energy value chain.

Injection of human neuroblastoma cells into neural crest streams in live zebrafish embryos.

Cancer cell behavior is highly microenvironment dependent, but we have a limited understanding of malignant cell-microenvironment interactions in vivo. Here, we describe a protocol for xenotransplanting human neuroblastoma (NB) cells into streams of migrating neural crest stem cells in zebrafish embryos, followed by confocal time-lapse imaging and cell tracking. This high-resolution model system facilitates the quantitative spatiotemporal analysis of cancer cell-cell and cell-environment interactions. For complete details on the use and execution of this protocol, please refer to Treffy et al. (2021).

Wnt-Dependent Activation of ERK Mediates Repression of Chondrocyte Fate during Calvarial Development.

Wnt signaling regulates cell fate decisions in diverse contexts during development, and loss of Wnt signaling in the cranial mesenchyme results in a robust and binary cell fate switch from cranial bone to ectopic cartilage. The Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) and Wnt signaling pathways are activated during calvarial osteoblast cell fate selection. Here, we test the hypothesis that ERK signaling is a mediator of Wnt-dependent cell fate decisions in the cranial mesenchyme. First, we show that loss of Erk1/2 in the cranial mesenchyme results in a diminished domain of osteoblast marker expression and increased expression of cartilage fate markers and ectopic cartilage formation in the frontal bone primordia. Second, we show that mesenchyme Wnt/ß-catenin signaling and Wntless are required for ERK activation in calvarial osteoblasts. Third, we demonstrate that Wnt and ERK signaling pathways function together to repress SOX9 expression in mouse cranial mesenchyme. Our results demonstrate an interaction between the Wnt and ERK signaling pathways in regulating lineage selection in a subset of calvarial cells and provide new insights into Wnt-dependent cell fate decisions.

Severe Hypoalbuminemia at Admission Is Strongly Associated with Worse Prognosis in Older Adults with SARS-CoV-2 Infection.

Serum albumin levels have been associated with prognosis in several conditions among older adults. The aim of this study is to assess the prognostic value in mortality of serum albumin in older adults with SARS-CoV-2 infection. METHODS: Cohort observational study with consecutive older-adults (≥65 years old), with confirmed SARS-CoV-2 infection admitted to a university hospital between March-May 2020. A logistic regression model was fitted to assess the impact of albumin levels on in-hospital mortality adjusted by potential confounders. RESULTS: Among a total of 840 patients admitted to the hospital, 405 (48%) were older adults with a total of 92 deaths (23%) among them. Those who died were older, had more comorbidities, higher inflammation status and lower levels of serum albumin at admission [3.10 g/dL (0.51) vs. 3.45 g/dL (0.45); p < 0.01. Serum albumin levels at admission were negatively correlated with inflammatory markers such as C-Reactive protein (Pearson Coeff -0.4634; p < 0.001) or IL-6 (Pearson's Coeff -0.244; p = 0.006) at admission but also to other clinical outcomes such time to clinical stability (Pearson's Coeff -0.259; p < 0.001). Severe hypoalbuminemia associated with increased risk of mortality was defined as ≤3 g/dL at admission according to the AUC/ROC analysis (0.72 95% CI 0.63-0.81) In a multivariate logistic regression model adjusting by age, inflammation, comorbidities and severity at admission severe hypoalbuminemia was a strong predictor of in-hospital mortality (OR 2.18 95% CI 1.03-4.62; p = 0.039). CONCLUSION: Severe hypoalbuminemia with ≤3 g/dL is an independent risk factor for mortality among older adults with SARS-CoV-2 infection. There is a consistent correlation between albumin levels and inflammatory biomarkers. Further studies are needed to determine whether the supplementation of albumin as coadjuvant treatment will have a positive impact on the prognosis of this infection.

Wnt/ß-catenin Signaling Pathway Regulates Specific lncRNAs That Impact Dermal Fibroblasts and Skin Fibrosis.

Wnt/ß-catenin signaling is required for embryonic dermal fibroblast cell fate, and dysregulation of this pathway is sufficient to promote fibrosis in adult tissue. The downstream modulators of Wnt/ß-catenin signaling required for controlling cell fate and dermal fibrosis remain poorly understood. The discovery of regulatory long non-coding RNAs (lncRNAs) and their pivotal roles as key modulators of gene expression downstream of signaling cascades in various contexts prompted us to investigate their roles in Wnt/ß-catenin signaling. Here, we have identified lncRNAs and protein-coding RNAs that are induced by ß-catenin activity in mouse dermal fibroblasts using next generation RNA-sequencing. The differentially expressed protein-coding mRNAs are enriched for extracellular matrix proteins, glycoproteins, and cell adhesion, and many are also dysregulated in human fibrotic tissues. We identified 111 lncRNAs that are differentially expressed in response to activation of Wnt/ß-catenin signaling. To further characterize the role of mouse lncRNAs in this pathway, we validated two novel Wnt signaling- Induced Non-Coding RNA (Wincr) transcripts referred to as Wincr1 and Wincr2. These two lncRNAs are highly expressed in mouse embryonic skin and perinatal dermal fibroblasts. Furthermore, we found that Wincr1 expression levels in perinatal dermal fibroblasts affects the expression of key markers of fibrosis (e.g., Col1a1 and Mmp10), enhances collagen contraction, and attenuates collective cell migration. Our results show that ß-catenin signaling-responsive lncRNAs may modulate dermal fibroblast behavior and collagen accumulation in dermal fibrosis, providing new mechanistic insights and nodes for therapeutic intervention.