An integrated strategy for analyzing the unique developmental programs of different myoblast subtypes

An important but largely unmet challenge in understanding the mechanisms that govern the formation of specific organs is to decipher the complex and dynamic genetic programs exhibited by the diversity of cell types within the tissue of interest. Here, we use an integrated genetic, genomic, and computational strategy to comprehensively determine the molecular identities of distinct myoblast subpopulations within the Drosophila embryonic mesoderm at the time that cell fates are initially specified. A compendium of gene expression profiles was generated for primary mesodermal cells purified by flow cytometry from appropriately staged wild-type embryos and from 12 genotypes in which myogenesis was selectively and predictably perturbed. A statistical meta-analysis of these pooled datasets—based on expected trends in gene expression and on the relative contribution of each genotype to the detection of known muscle genes—provisionally assigned hundreds of differentially expressed genes to particular myoblast subtypes. Whole embryo in situ hybridizations were then used to validate the majority of these predictions, thereby enabling true-positive detection rates to be estimated for the microarray data. This combined analysis reveals that myoblasts exhibit much greater gene expression heterogeneity and overall complexity than was previously appreciated. Moreover, it implicates the involvement of large numbers of uncharacterized, differentially expressed genes in myogenic specification and subsequent morphogenesis. These findings also underscore a requirement for considerable regulatory specificity for generating diverse myoblast identities. Finally, to illustrate how the developmental functions of newly identified myoblast genes can be efficiently surveyed, a rapid RNA interference assay that can be scored in living embryos was developed and applied to selected genes. This integrated strategy for examining embryonic gene expression and function provides a substantially expanded framework for further studies of this model developmental system.     

Identification of Receptor-Tyrosine-Kinase-Signaling Target Genes Reveals Receptor-Specific Activities and Pathway Branchpoints During Drosophila Development

Genetic maps provide a means to estimate the probability of the co-inheritance of linked loci as they are transmitted across generations in both experimental and natural populations. However, in the age of whole-genome sequences, physical distances measured in base pairs of DNA provide the standard coordinates for navigating the myriad features of genomes. Although genetic and physical maps are colinear, there are well-characterized and sometimes dramatic heterogeneities in the average frequency of meiotic recombination events that occur along the physical extent of chromosomes. There also are documented differences in the recombination landscape between the two sexes. We have revisited high-resolution genetic map data from a large heterogeneous mouse population and have constructed a revised genetic map of the mouse genome, incorporating 10,195 single nucleotide polymorphisms using a set of 47 families comprising 3546 meioses. The revised map provides a different picture of recombination in the mouse from that reported previously. We have further integrated the genetic and physical maps of the genome and incorporated SSLP markers from other genetic maps into this new framework. We demonstrate that utilization of the revised genetic map improves QTL mapping, partially due to the resolution of previously undetected errors in marker ordering along the chromosome.GENETIC maps exist for hundreds of different species, and genetic map construction continues to play an important role in the characterization of genomes (Tanksley et al. 1992; Kong et al. 2002; Chowdhary and Raudsepp 2006; Stapley et al. 2008). A genetic map defines the linear order and relative distances among a set of marker loci in units that correspond to the frequency of meiotic recombination between the loci. Until recently mouse genetic maps based on simple sequence length polymorphism (SSLP) markers (Lyon 1976) have been sufficient for most experimental purposes since, unlike the hundreds of thousands of markers required in human genetic association studies, a relatively small number of markers is needed to map crosses between inbred mouse strains. However, recent developments in whole-genome high-resolution mapping in the mouse (Churchill et al. 2004; Valdar et al. 2006) and interest in examining recombination rates at an ultra-fine scale (Myers et al. 2005) have reawakened the need to develop a high-resolution genetic map in the mouse.The current standard genetic map of the mouse has been compiled from a substantial body of historical data and maintained by the Mouse Genome Informatics (MGI) project at The Jackson Laboratory (Bult et al. 2008). We will refer to it as the MGI map. The primary sources of data used to construct the MGI map were two mapping panels, described here. However, the current map is based on a consensus developed by the 2000 Chromosome Committee using all available published data. The map has continued to be maintained by MGI with the addition of new genetic markers and data but, because the map is based on consensus, published errors may have been perpetuated.The Jackson Laboratory developed a genetic map based on two sets of 94 progeny obtained from reciprocal backcrosses (BSB and BSS) between the inbred strains C57BL/6J and SPRET/EiJ (Rowe et al. 1994). These strains represent two different species of mouse (Mus musculus and Mus spretus). The map provides a wealth of genetic information, but problems with male fertility restrict breeding options and thus the map is female specific. The problems with male fertility may have resulted in some multi-locus distortion in the mapping panel (Montagutelli et al. 1996). Currently, 1372 and 4913 markers have been typed on the BSB and BSS backcross panels, respectively (Broman et al. 2002). Researchers at The Whitehead Institute and the Massachusetts Institute of Technology (MIT) developed a map of 4006 SSLP markers using an intercross population of 46 mice derived from strains OB (C57BL/6J-Lep^ob/ob) and CAST (CAST/EiJ) (Dietrich et al. 1994). Both parental strains OB and CAST are derived from M. musculus, but CAST is from a distinct subspecies, M. m. castaneus. The intercross mating strategy produces observable recombination from both male and female parents, but the two cannot be distinguished. Thus the map is sex averaged and based on 92 meioses. The Whitehead/MIT map was expanded to include 7377 SSLP markers (Dietrich et al. 1996). These are denoted as, e.g., D7Mit54, where “7” indicates the chromosome to which the marker is mapped and “54” is an arbitrary index. They are commonly referred to as “Mit” markers.Map resolution is limited by the number of observable recombination events in each of these panels. With 94 meioses (in each backcross) and an average of 14 recombination events/haploid genome transmitted, limiting resolution is on the order of 1 cM. A much larger panel would be needed to achieve subcentimorgan resolution and to accurately position high-density sets of SNP markers.Here we propose a new standard genetic map of the laboratory mouse based on data from a large heterogeneous stock (HS) mouse population descended from eight inbred strains (DBA/2J, C3H/HeJ, AKR/J, A/J, BALB/cJ, CBA/J, C57BL/6J, and LP/J) representing a diverse sample of the classical inbred strains (Petkov et al. 2004). Shifman et al. (2006) calculated genetic maps based on 11,247 informative SNP markers in 2293 HS individuals. The marker set is dense with 99% of the SNP intervals <500 kb, 81.2% <250 kb, and an estimated allele inheritance-based accuracy of 99.98%. Map positions were calculated separately for male and female meioses using CRIMAP software (Green et al. 1990), and the total length of the sex-averaged map is 1630 cM, as defined by the most distal SNP markers in their panel. The MGI map is 1783 cM on the basis of the most distal available marker position for each chromosome. However, on the basis of the most distal shared markers, the original Shifman map at 1612 cM is substantially longer than the MGI map at 1445 cM. It was not immediately clear if this discrepancy was due to the nature of recombination in the HS population or to their method of map estimation.There are at least two methodological problems with the HS map reported in Shifman et al. (2006). First, the map was constructed using a sliding window of 5–15 SNPs to handle eight multi-generation families within the CRIMAP software. Ideally, one considers all markers on a chromosome simultaneously in constructing a genetic map, and we found that this could be accomplished by splitting the complex pedigrees into sibships. Although splitting the pedigree results in slightly less efficient estimates of intermarker distances, this approach should incur no bias. Maps based on the full set of markers but with the complex pedigrees split into sibships are thus arguably better than maps based on the full pedigrees but with a sliding window of 5–15 markers. Second, analysis of families with incomplete parental genotypes may have contributed to an inflated map size. Sixteen of the 72 families lack parental genotypes or have genotypes for just one parent (15 of the 72), and many of them are small (26 have six or fewer siblings). Sibships with no parental genotype data of their own can give no information about sex-specific recombination rates. In conjunction with other sibships for which parental genotypes are available, they can provide some information, but the CRIMAP software (last modified in 1990) makes some approximations that result in a large bias even in the sex-averaged genetic maps for small sibships lacking parental genotype data.For these reasons, we recomputed the mouse genetic map on the basis of the original data reported and discuss the differences between the original Shifman map and the revised Shifman map below. The revised Shifman map provides a markedly different picture of recombination in the mouse: the estimated sex-averaged chromosome lengths correspond more closely to those in the original MGI map; the sex difference in the overall recombination rate is greatly reduced; and numerous narrow regions of high recombination rate, apparent in the original Shifman map, have disappeared.We propose the revised Shifman map as a new standard genetic map for the mouse. The new genetic map represents a substantial improvement over the existing MGI map due to the large number of meioses and to the genetic diversity of strains in the HS population. We have generated male, female, and sex-averaged genetic maps with physical positions and updated locus identifiers. We have established the correspondence between physical and genetic positions of 7080 Mit markers and corrected inconsistencies in the MGI map. We provide a web-based tool for the interpolation of new marker loci into the genetic map and for converting genetic map positions to NCBI mouse build 37 coordinates. Finally, we examine the effect of changing to this revised genetic map on QTL mapping in five previously published data sets (Beamer et al. 1999, 2001; Ishimori et al. 2004, 2008; Wergedal et al. 2006).     

Single visit versus multiple visit root canal

Single visit endodontics offers many advantages over multi visit treatment. Therefore, it is of interest to assess the preference of single visit over multiple visit root canals. We used 86,000 patient records and selected 9017 records matching the inclusion criteria for the analysis using statistical tools (Chi square test at p value <0.05). Data shows that people between 26 to 45 years are often affected with dental caries. Available data is biased towards multi visits rather than single visit regardless number of canals.     

Influence of glutamate and GABA transport on brain excitatory/inhibitory balance

An optimally functional brain requires both excitatory and inhibitory inputs that are regulated and balanced. A perturbation in the excitatory/inhibitory balance—as is the case in some neurological disorders/diseases (e.g. traumatic brain injury Alzheimer’s disease, stroke, epilepsy and substance abuse) and disorders of development (e.g. schizophrenia, Rhett syndrome and autism spectrum disorder)—leads to dysfunctional signaling, which can result in impaired cognitive and motor function, if not frank neuronal injury. At the cellular level, transmission of glutamate and GABA, the principle excitatory and inhibitory neurotransmitters in the central nervous system control excitatory/inhibitory balance. Herein, we review the synthesis, release, and signaling of GABA and glutamate followed by a focused discussion on the importance of their transport systems to the maintenance of excitatory/inhibitory balance.     

A microRNA network regulates proliferative timing and extracellular matrix synthesis during cellular quiescence in fibroblasts

Background

Although quiescence (reversible cell cycle arrest) is a key part in the life history and fate of many mammalian cell types, the mechanisms of gene regulation in quiescent cells are poorly understood. We sought to clarify the role of microRNAs as regulators of the cellular functions of quiescent human fibroblasts.

Results

Using microarrays, we discovered that the expression of the majority of profiled microRNAs differed between proliferating and quiescent fibroblasts. Fibroblasts induced into quiescence by contact inhibition or serum starvation had similar microRNA profiles, indicating common changes induced by distinct quiescence signals. By analyzing the gene expression patterns of microRNA target genes with quiescence, we discovered a strong regulatory function for miR-29, which is downregulated with quiescence. Using microarrays and immunoblotting, we confirmed that miR-29 targets genes encoding collagen and other extracellular matrix proteins and that those target genes are induced in quiescence. In addition, overexpression of miR-29 resulted in more rapid cell cycle re-entry from quiescence. We also found that let-7 and miR-125 were upregulated in quiescent cells. Overexpression of either one alone resulted in slower cell cycle re-entry from quiescence, while the combination of both together slowed cell cycle re-entry even further.

Conclusions

microRNAs regulate key aspects of fibroblast quiescence including the proliferative state of the cells as well as their gene expression profiles, in particular, the induction of extracellular matrix proteins in quiescent fibroblasts.     

Detection of IL28B SNP DNA from buccal epithelial cells, small amounts of serum, and dried blood spots

Background & Aims

Point mutations in the coding region of the interleukin 28 gene (rs12979860) have recently been identified for predicting the outcome of treatment of hepatitis C virus infection. This polymorphism detection was based on whole blood DNA extraction. Alternatively, DNA for genetic diagnosis has been derived from buccal epithelial cells (BEC), dried blood spots (DBS), and genomic DNA from serum. The aim of the study was to investigate the reliability and accuracy of alternative routes of testing for single nucleotide polymorphism allele rs12979860CC.

Methods

Blood, plasma, and sera samples from 200 patients were extracted (400 µL). Buccal smears were tested using an FTA card. To simulate postal delay, we tested the influence of storage at ambient temperature on the different sources of DNA at five time points (baseline, 48 h, 6 days, 9 days, and 12 days)

Results

There was 100% concordance between blood, plasma, sera, and BEC, validating the use of DNA extracted from BEC collected on cytology brushes for genetic testing. Genetic variations in HPTR1 gene were detected using smear technique in blood smear (3620 copies) as well as in buccal smears (5870 copies). These results are similar to those for whole blood diluted at 1/10. A minimum of 0.04 µL, 4 µL, and 40 µL was necessary to obtain exploitable results respectively for whole blood, sera, and plasma. No significant variation between each time point was observed for the different sources of DNA. IL28B SNPs analysis at these different time points showed the same results using the four sources of DNA.

Conclusion

We demonstrated that genomic DNA extraction from buccal cells, small amounts of serum, and dried blood spots is an alternative to DNA extracted from peripheral blood cells and is helpful in retrospective and prospective studies for multiple genetic markers, specifically in hard-to-reach individuals.     

Epidural analgesia and cesarean delivery in multiple sclerosis post-partum relapses: the Italian cohort study

Background

Few studies have systematically addressed the role of epidural analgesia and caesarean delivery in predicting the post-partum disease activity in women with Multiple Sclerosis (MS).The objective of this study was to assess the impact of epidural analgesia (EA) and caesarean delivery (CD) on the risk of post-partum relapses and disability in women with MS.

Methods

In the context of an Italian prospective study on the safety of immunomodulators in pregnancy, we included pregnancies occurred between 2002 and 2008 in women with MS regularly followed-up in 21 Italian MS centers. Data were gathered through a standardized, semi-structured interview, dealing with pregnancy outcomes, breastfeeding, type of delivery (vaginal or caesarean) and EA. The risk of post-partum relapses and disability progression (1 point on the Expanded Disability Status Sclae, EDSS, point, confirmed after six months) was assessed through a logistic multivariate regression analysis.

Results

We collected data on 423 pregnancies in 415 women. Among these, 349 pregnancies resulted in full term deliveries, with a post-partum follow-up of at least one year (mean follow-up period 5.5±3.1 years). One hundred and fifty-five patients (44.4%) underwent CD and 65 (18.5%) EA. In the multivariate analysis neither CD, nor EA were associated with a higher risk of post-partum relapses. Post-partum relapses were related to a higher EDSS score at conception (OR=1.42; 95% CI 1.11-1.82; p=0.005), a higher number of relapses in the year before pregnancy (OR=1.62; 95% CI 1.15-2.29; p=0.006) and during pregnancy (OR=3.07; 95% CI 1.40-6.72; p=0.005). Likewise, CD and EA were not associated with disability progression on the EDSS after delivery. The only significant predictor of disability progression was the occurrence of relapses in the year after delivery (disability progression in the year after delivery: OR= 4.00; 95% CI 2.0-8.2; p<0.001; disability progression over the whole follow-up period: OR= 2.0; 95% CI 1.2-3.3; p=0.005).

Conclusions

Our findings, show no correlation between EA, CD and postpartum relapses and disability. Therefore these procedures can safely be applied in MS patients. On the other hand, post-partum relapses are significantly associated with increased disability, which calls for the need of preventive therapies after delivery.

     

Direct cord implantation in brachial plexus avulsions: revised technique using a single stage combined anterior (first) posterior (second) approach and end-to-side side-to-side grafting neurorrhaphy

Background

The superiority of a single stage combined anterior (first) posterior (second) approach and end-to-side side-to-side grafting neurorrhaphy in direct cord implantation was investigated as to providing adequate exposure to both the cervical cord and the brachial plexus, as to causing less tissue damage and as to being more extensible than current surgical approaches.

Methods

The front and back of the neck, the front and back of the chest up to the midline and the whole affected upper limb were sterilized while the patient was in the lateral position; the patient was next turned into the supine position, the plexus explored anteriorly and the grafts were placed; the patient was then turned again into the lateral position, and a posterior cervical laminectomy was done. The grafts were retrieved posteriorly and side grafted to the anterior cord. Using this approach, 5 patients suffering from complete traumatic brachial plexus palsy, 4 adults and 1 obstetric case were operated upon and followed up for 2 years. 2 were C5,6 ruptures and C7,8T1 avulsions. 3 were C5,6,7,8T1 avulsions. C5,6 ruptures were grafted and all avulsions were cord implanted.

Results

Surgery in complete avulsions led to Grade 4 improvement in shoulder abduction/flexion and elbow flexion. Cocontractions occurred between the lateral deltoid and biceps on active shoulder abduction. No cocontractions occurred after surgery in C5,6 ruptures and C7,8T1 avulsions, muscle power improvement extended into the forearm and hand; pain disappeared.

Limitations include

spontaneous recovery despite MRI appearance of avulsions, fallacies in determining intraoperative avulsions (wrong diagnosis, wrong level); small sample size; no controls rule out superiority of this technique versus other direct cord reimplantation techniques or other neurotization procedures; intra- and interobserver variability in testing muscle power and cocontractions.

Conclusion

Through providing proper exposure to the brachial plexus and to the cervical cord, the single stage combined anterior (first) and posterior (second) approach might stimulate brachial plexus surgeons to go more for direct cord implantation. In this study, it allowed for placing side grafts along an extensive donor recipient area by end-to-side, side-to-side grafting neurorrhaphy and thus improved results.

Level of evidence

Level IV, prospective case series.