Vole genomics links determinate and indeterminate growth of teeth.

Continuously growing teeth are an important innovation in mammalian evolution, yet genetic regulation of continuous growth by stem cells remains incompletely understood. Dental stem cells responsible for tooth crown growth are lost at the onset of tooth root formation. Genetic signaling that initiates this loss is difficult to study with the ever-growing incisor and rooted molars of mice, the most common mammalian dental model species, because signals for root formation overlap with signals that pattern tooth size and shape (i.e., cusp patterns). Different species of voles (Cricetidae, Rodentia, Glires) have evolved rooted and unrooted molars that have similar size and shape, providing alternative models for studying roots. We assembled a de novo genome of Myodes glareolus, a vole with high-crowned, rooted molars, and performed genomic and transcriptomic analyses in a broad phylogenetic context of Glires (rodents and lagomorphs) to assess differential selection and evolution in tooth forming genes. We identified 15 dental genes with changing synteny relationships and six dental genes undergoing positive selection across Glires, two of which were undergoing positive selection in species with unrooted molars, Dspp and Aqp1. Decreased expression of both genes in prairie voles with unrooted molars compared to bank voles supports the presence of positive selection and may underlie differences in root formation. Bulk transcriptomics analyses of embryonic molar development in bank voles also demonstrated conserved patterns of dental gene expression compared to mice, with species-specific variation likely related to developmental timing and morphological differences between mouse and vole molars. Our results support ongoing evolution of dental genes across Glires, revealing the complex evolutionary background of convergent evolution for ever-growing molars.

Transcriptomic landscape of early hair follicle and epidermal development.

Morphogenesis of ectodermal organs, such as hair, tooth, and mammary gland, starts with the formation of local epithelial thickenings, or placodes, but it remains to be determined how distinct cell types and differentiation programs are established during ontogeny. Here, we use bulk and single-cell transcriptomics and pseudotime modeling to address these questions in developing hair follicles and epidermis and produce a comprehensive transcriptomic profile of cellular populations in the hair placode and interplacodal epithelium. We report previously unknown cell populations and marker genes, including early suprabasal and genuine interfollicular basal markers, and propose the identity of suprabasal progenitors. By uncovering four different hair placode cell populations organized in three spatially distinct areas, with fine gene expression gradients between them, we posit early biases in cell fate establishment. This work is accompanied by a readily accessible online tool to stimulate further research on skin appendages and their progenitors.

The developmental basis for scaling of mammalian tooth size.

When evolution leads to differences in body size, organs generally scale along. A well-known example of the tight relationship between organ and body size is the scaling of mammalian molar teeth. To investigate how teeth scale during development and evolution, we compared molar development from initiation through final size in the mouse and the rat. Whereas the linear dimensions of the rat molars are twice that of the mouse molars, their shapes are largely the same. Here, we focus on the first lower molars that are considered the most reliable dental proxy for size-related patterns due to their low within-species variability. We found that scaling of the molars starts early, and that the rat molar is patterned equally as fast but in a larger size than the mouse molar. Using transcriptomics, we discovered that a known regulator of body size, insulin-like growth factor 1 (Igf1), is more highly expressed in the rat molars compared to the mouse molars. Ex vivo and in vivo mouse models demonstrated that modulation of the IGF pathway reproduces several aspects of the observed scaling process. Furthermore, analysis of IGF1-treated mouse molars and computational modeling indicate that IGF signaling scales teeth by simultaneously enhancing growth and by inhibiting the cusp-patterning program, thereby providing a relatively simple mechanism for scaling teeth during development and evolution. Finally, comparative data from shrews to elephants suggest that this scaling mechanism regulates the minimum tooth size possible, as well as the patterning potential of large teeth.

Genome-wide mRNA profiling in urinary extracellular vesicles reveals stress gene signature for diabetic kidney disease.

Urinary extracellular vesicles (uEV) are a largely unexplored source of kidney-derived mRNAs with potential to serve as a liquid kidney biopsy. We assessed ∼200 uEV mRNA samples from clinical studies by genome-wide sequencing to discover mechanisms and candidate biomarkers of diabetic kidney disease (DKD) in Type 1 diabetes (T1D) with replication in Type 1 and 2 diabetes. Sequencing reproducibly showed >10,000 mRNAs with similarity to kidney transcriptome. T1D DKD groups showed 13 upregulated genes prevalently expressed in proximal tubules, correlated with hyperglycemia and involved in cellular/oxidative stress homeostasis. We used six of them (GPX3, NOX4, MSRB, MSRA, HRSP12, and CRYAB) to construct a transcriptional "stress score" that reflected long-term decline of kidney function and could even identify normoalbuminuric individuals showing early decline. We thus provide workflow and web resource for studying uEV transcriptomes in clinical urine samples and stress-linked DKD markers as potential early non-invasive biomarkers or drug targets.

Chromosomal neighbourhoods allow identification of organ specific changes in gene expression.

Although most genes share their chromosomal neighbourhood with other genes, distribution of genes has not been explored in the context of individual organ development; the common focus of developmental biology studies. Because developmental processes are often associated with initially subtle changes in gene expression, here we explored whether neighbouring genes are informative in the identification of differentially expressed genes. First, we quantified the chromosomal neighbourhood patterns of genes having related functional roles in the mammalian genome. Although the majority of protein coding genes have at least five neighbours within 1 Mb window around each gene, very few of these neighbours regulate development of the same organ. Analyses of transcriptomes of developing mouse molar teeth revealed that whereas expression of genes regulating tooth development changes, their neighbouring genes show no marked changes, irrespective of their level of expression. Finally, we test whether inclusion of gene neighbourhood in the analyses of differential expression could provide additional benefits. For the analyses, we developed an algorithm, called DELocal that identifies differentially expressed genes by comparing their expression changes to changes in adjacent genes in their chromosomal regions. Our results show that DELocal removes detection bias towards large changes in expression, thereby allowing identification of even subtle changes in development. Future studies, including the detection of differential expression, may benefit from, and further characterize the significance of gene-gene neighbour relationships.

System-level analyses of keystone genes required for mammalian tooth development.

When a null mutation of a gene causes a complete developmental arrest, the gene is typically considered essential for life. Yet, in most cases, null mutations have more subtle effects on the phenotype. Here we used the phenotypic severity of mutations as a tool to examine system-level dynamics of gene expression. We classify genes required for the normal development of the mouse molar into different categories that range from essential to subtle modification of the phenotype. Collectively, we call these the developmental keystone genes. Transcriptome profiling using microarray and RNAseq analyses of patterning stage mouse molars show highly elevated expression levels for genes essential for the progression of tooth development, a result reminiscent of essential genes in single-cell organisms. Elevated expression levels of progression genes were also detected in developing rat molars, suggesting evolutionary conservation of this system-level dynamics. Single-cell RNAseq analyses of developing mouse molars reveal that even though the size of the expression domain, measured in the number of cells, is the main driver of organ-level expression, progression genes show high cell-level transcript abundances. Progression genes are also upregulated within their pathways, which themselves are highly expressed. In contrast, a high proportion of the genes required for normal tooth patterning are secreted ligands that are expressed in fewer cells than their receptors and intracellular components. Overall, even though expression patterns of individual genes can be highly different, conserved system-level principles of gene expression can be detected using phenotypically defined gene categories.

Preoperative beta blockade is associated with increased rates of 30-day major adverse cardiac events in critical limb ischemia patients undergoing infrainguinal revascularization.

OBJECTIVE: The association between beta blockers and cardiovascular or limb-related outcomes after revascularization for critical limb ischemia (CLI) remains unclear. The objective of this study was to assess the impact of preoperative beta blockade on 30-day major adverse cardiac events (MACEs) and major adverse limb events (MALEs) in patients undergoing infrainguinal revascularization for CLI. We hypothesized that rates of MALEs and MACEs will be higher in patients not receiving preoperative beta blockade. METHODS: The National Surgical Quality Improvement Program vascular targeted file for 2011 to 2014 identified patients receiving beta blockade and undergoing infrainguinal endovascular intervention and open bypass for CLI. Primary outcomes including 30-day MACE (stroke, myocardial infarction [MI], or death) and MALE (untreated loss of patency, reintervention, or amputation) were compared between patients taking and not taking preoperative beta blockers. Multivariate logistic regression identified independent predictors of MACEs and MALEs. RESULTS: A total of 11,785 revascularizations were performed for CLI during the study period (7408 bypasses vs 4377 endovascular interventions). Preoperative beta blockers were used by 7365 patients, including 4541 (61.7%) in the open bypass cohort and 2824 (64.5%) in the endovascular group (P < .01). MACEs and MI were significantly higher in patients with preoperative beta blockers (MACEs, 5.8% vs 3.4% [P < .0001]; MI, 3.1% vs 1.8% [P < .0001]). After controlling for cardiac risk factors, beta blockers independently predicted MACEs (odds ratio [OR], 1.27; P = .03) and MI (OR, 1.36; P = .03) but not stroke (OR, 1.17; P = .58) or 30-day mortality (OR, 1.22; P = .19). Beta-blocker use did not have an effect on MALEs (OR, 0.99; P = .88). CONCLUSIONS: In patients with CLI, preoperative beta blockade was an independent predictor of 30-day MI and MACEs after controlling for other cardiovascular risk factors. Beta blockers did not have an impact on short-term limb-related outcomes. The association between beta blockade and revascularization for CLI deserves further investigation.

Increased warm ischemia time during vessel harvest decreases the primary patency of cryopreserved conduits in patients undergoing lower extremity bypass.

OBJECTIVE: Autologous vein is the preferred conduit for lower extremity bypass. However, it is often unavailable because of prior harvest or inadequate for bypass owing to insufficient caliber. Cryopreserved cadaveric vessels can be used as conduits for lower extremity revascularization when autogenous vein is not available and the use of prosthetic grafts is not appropriate. Many studies have shown that donor characteristics influence clinical outcomes in solid organ transplantation, but little is known regarding their impact in vascular surgery. The purpose of this study was to examine the effects donor variables have on patients undergoing lower extremity bypass with cryopreserved vessels. METHODS: The tissue processing organization was queried for donor blood type, warm ischemia times (WITs), and serial numbers of cryopreserved vessels implanted at a single center from 2010 to 2016. The serial numbers were then matched with their respective patients using the institutional Clinical Data Repository and patient data were obtained from the Clinical Data Repository and chart review. Primary outcomes were primary patency of the bypass conduits and limb salvage. Time to loss of patency was evaluated using Kaplan-Meier methods and a Cox proportional hazards model determined risk-adjusted predictors of patency and limb salvage. RESULTS: Sixty patients underwent lower extremity bypass with 65 cryopreserved vessels (23 superficial femoral arteries, 41 saphenous veins, 1 femoral vein). Thirty-eight procedures were reoperations. There were 21 inflow, 44 outflow, and 44 infrainguinal procedures. Preexisting comorbidities did not differ significantly between those who lost patency and those who did not. The mean WIT among the entire cohort was 892.3 ± 389.1 minutes (range, 158.0-1434.0 minutes). The median follow-up was 394 days. Kaplan-Meier analysis demonstrated an overall 1-year primary patency rate of 51%. Primary patency at 1 year was 67% and 41% for inflow and outflow procedures, respectively, and did not differ significantly between the two groups (P = .15). Donor-to-recipient ABO incompatibility was not associated with loss of primary patency. The 1-year amputation-free survival was 74%. Primary patency significantly decreased with each hourly increase in WIT on risk-adjusted analysis (hazard ratio, 1.1; P = .02). CONCLUSIONS: Higher cryopreserved vessel WIT was associated with increased risk-adjusted loss of primary patency in this cohort. At 1 year, the overall primary patency was 51% and amputation-free survival was 74%. Vascular surgeons should be aware that WIT may affect outcomes for lower extremity bypass.

Hair follicle dermal condensation forms via Fgf20 primed cell cycle exit, cell motility, and aggregation.

Mesenchymal condensation is a critical step in organogenesis, yet the underlying molecular and cellular mechanisms remain poorly understood. The hair follicle dermal condensate is the precursor to the permanent mesenchymal unit of the hair follicle, the dermal papilla, which regulates hair cycling throughout life and bears hair inductive potential. Dermal condensate morphogenesis depends on epithelial Fibroblast Growth Factor 20 (Fgf20). Here, we combine mouse models with 3D and 4D microscopy to demonstrate that dermal condensates form de novo and via directional migration. We identify cell cycle exit and cell shape changes as early hallmarks of dermal condensate morphogenesis and find that Fgf20 primes these cellular behaviors and enhances cell motility and condensation. RNAseq profiling of immediate Fgf20 targets revealed induction of a subset of dermal condensate marker genes. Collectively, these data indicate that dermal condensation occurs via directed cell movement and that Fgf20 orchestrates the early cellular and molecular events.

How do eubacterial organisms manage aggregation-prone proteome?

Eubacterial genomes vary considerably in their nucleotide composition. The percentage of genetic material constituted by guanosine and cytosine (GC) nucleotides ranges from 20% to 70%.  It has been posited that GC-poor organisms are more dependent on protein folding machinery. Previous studies have ascribed this to the accumulation of mildly deleterious mutations in these organisms due to population bottlenecks. This phenomenon has been supported by protein folding simulations, which showed that proteins encoded by GC-poor organisms are more prone to aggregation than proteins encoded by GC-rich organisms. To test this proposition using a genome-wide approach, we classified different eubacterial proteomes in terms of their aggregation propensity and chaperone-dependence using multiple machine learning models. In contrast to the expected decrease in protein aggregation with an increase in GC richness, we found that the aggregation propensity of proteomes increases with GC content. A similar and even more significant correlation was obtained with the GroEL-dependence of proteomes: GC-poor proteomes have evolved to be less dependent on GroEL than GC-rich proteomes. We thus propose that a decrease in eubacterial GC content may have been selected in organisms facing proteostasis problems.

Selection of relevant features from amino acids enables development of robust classifiers.

Machine learning (ML) has been extensively applied to develop models and to understand high-throughput data of biological processes. However, new ML models, trained with novel experimental results, are required to build regularly for more precise predictions. ML methods can build models from numeric data, whereas biological data are generally textual (DNA, protein sequences) or images and needs feature calculation algorithms to generate quantitative features. Programming skills along with domain knowledge are required to develop these algorithms. Therefore, the process of knowledge discovery through ML is decelerated due to lack of generic tools to construct features and to build models directly from the data. Hence, we developed a schema that calculates about 5,000 features, selects relevant features and develops protein classifiers from the training data. To demonstrate the general applicability and robustness of our method, fungal adhesins and nuclear receptor proteins were used for building classifiers which outperformed existing classifiers when tested on independent data. Next, we built a classifier for mitochondrial proteins of Plasmodium falciparum which causes human malaria because the latest corresponding classifiers are not publically accessible. Our classifier attained 98.18 % accuracy and 0.95 Matthews correlation coefficient by fivefold cross-validation and outperformed existing classifiers on independent test set. We implemented this schema as user-friendly and open source application Pro-Gyan ( http://code.google.com/p/pro-gyan/ ), to build and share executable classifiers without programming knowledge.

Esophageal diverticulum arising from a prolonged retained esophageal foreign body.

Esophageal foreign body impaction (EFBI) is a rare condition in childhood which needs urgent removal. However, if left untreated, its chronic impaction may lead to serious secondary complications. Symptoms associated with chronic EFBI are attributed to airway disease or gastroesophageal reflux, which further delays definitive diagnosis and management. We present a girl with ingestion of a bottle cap diaphragm that was embedded in her esophagus resulting in esophageal narrowing and a secondary diverticulum formation. As the disk was unable to be removed using standard grasping forceps, the authors used an injector needle to impale the disk and successfully removed it without any complications.

IGVBrowser--a genomic variation resource from diverse Indian populations.

The Indian Genome Variation Consortium (IGVC) project, an initiative of the Council for Scientific and Industrial Research, has been the first large-scale comprehensive study of the Indian population. One of the major aims of the project is to study and catalog the variations in nearly thousand candidate genes related to diseases and drug response for predictive marker discovery, founder identification and also to address questions related to ethnic diversity, migrations, extent and relatedness with other world population. The Phase I of the project aimed at providing a set of reference populations that would represent the entire genetic spectrum of India in terms of language, ethnicity and geography and Phase II in providing variation data on candidate genes and genome wide neutral markers on these reference set of populations. We report here development of the IGVBrowser that provides allele and genotype frequency data generated in the IGVC project. The database harbors 4229 SNPs from more than 900 candidate genes in contrasting Indian populations. Analysis shows that most of the markers are from genic regions. Further, a large fraction of genes are implicated in cardiovascular, metabolic, cancer and immune system-related diseases. Thus, the IGVC data provide a basal level variation data in Indian population to study genetic diseases and pharmacology. Additionally, it also houses data on ∼50,000 (Affy 50 K array) genome wide neutral markers in these reference populations. In IGVBrowser one can analyze and compare genomic variations in Indian population with those reported in HapMap along with annotation information from various primary data sources. Database URL: http://igvbrowser.igib.res.in.