Identifying environmental barriers to participation: Usability of a health-literacy informed problem-identification approach for parents of young children with developmental disabilities.

BACKGROUND: Parents of very young children recently diagnosed with developmental disabilities (DD) need to identify environmental barriers to their children's participation and adopt an adaptive orientation to solving these problems. Given the health service disparities for diverse families, parents may benefit from easy to use problem-identification approaches that address environmental barriers stemming from community and policy contexts. This feasibility study evaluated the usability of a health literacy-informed, structured, environment-focused problem-identification approach for parents of young children with DD. METHODS: We used purposeful, convenience sampling to enrol 9 mothers of children ages 1-3 with DD (4 racial/ethnic minorities, 3 high school education, 4 annual household income <$20,000). We developed a structured problem-identification approach guided by a social ecological model featuring home, community, and policy contexts. The approach was applied to 3 short stories during a narrative elicitation interview. Two researchers independently coded parent responses for the type of barrier and solution identified with and without the approach. RESULTS: Parents identified 121 environmental barriers without the approach. When using the approach and prompted to consider home, community, and policy barriers, parents identified an additional 222 environmental barriers; the greatest number of barriers were aligned with International Classification of Functioning, Disability, and Health-Children and Youth environment Chapter 5 "Services, systems, and policies." Using the approach, parents with a postgraduate education and annual household income >$80,000 identified the most environmental barriers, and parents reporting the lowest annual household incomes identified the fewest environmental barriers. When parents attributed participation challenges to an environmental barrier, ~57% of solutions required parents to interact with individuals at the community or policy level. CONCLUSIONS: This study suggests that parents with a range of background characteristics can use a structured, environment-focused problem-identification approach. With the approach, parents are more likely to attribute participation challenges to environmental barriers and adopt a problem-solving orientation focused on changes to the community and policy context.

Saliva-microbe interactions and salivary gland dysfunction.

Adequate salivary secretion is crucial to both oral and general health, since it provides a complex milieu for support of the microbial populations of the mouth, while at the same time containing antimicrobial products that help control these microbial populations. This paper summarizes several aspects of salivary component function, gland secretion mechanisms, and immunopathogenesis as related to oral health and disease. Salivary components mediate microbial attachment to oral surfaces, and also interact with planktonic microbial surfaces to facilitate agglutination and elimination of pathogens from the oral cavity. Adhesive interactions are often mediated by lectin-like bacterial proteins that bind to glycan motifs on salivary glycoproteins. An important salivary antimicrobial protein is histatin 5 (Hst 5), which shows potent and selective antifungal activity and also susceptibility to proteolytic degradation. Coupling of Hst 5 with the carrier molecule spermidine significantly enhanced killing of C. albicans and resistance to proteolytic degradation, compared with the parent peptide. Loss of salivary secretion may be caused by disorders such as Sjögren's syndrome (SS) or ectodermal dysplasia, or may be a side-effect of radiation therapy. Two new approaches to the treatment of salivary gland dysfunction include the use of resolvins and the creation of differentiated acinar structures to construct an artificial salivary gland. B-cells contribute to the pathogenesis of SS by releasing cytokines and autoantibodies and by influencing T-cell differentiation. CXCL13, a potent B-cell chemokine associated with autoimmune diseases, is elevated locally and systemically in SS and may represent a novel biomarker or therapeutic target in the management and treatment of SS.

High-Resolution Transcriptomic Landscape of the Human Submandibular Gland.

Saliva-secreting and transporting cells are part of the complex cellular milieu of the human salivary gland, where they play important roles in normal glandular physiology and diseased states. However, comprehensive molecular characterization, particularly at single-cell resolution, is still incomplete, in part due to difficulty in procuring normal human tissues. Here, we perform an in-depth analysis of male and female adult human submandibular gland (SMG) samples by bulk RNA sequencing (RNA-seq) and examine the molecular underpinnings of the heterogeneous cell populations by single-cell (sc) RNA-seq. Our results from scRNA-seq highlight the remarkable diversity of clusters of epithelial and nonepithelial cells that reside in the SMG that is also faithfully recapitulated by deconvolution of the bulk-RNA data sets. Our analyses reveal complex transcriptomic heterogeneity within both the ductal and acinar subpopulations and identify atypical SMG cell types, such as mucoacinar cells that are unique to humans and ionocytes that have been recently described in the mouse. We use CellChat to explore ligand-receptor interactome predictions that likely mediate crucial cell-cell communications between the various cell clusters. Finally, we apply a trajectory inference method to investigate specific cellular branching points and topology that offers insights into the dynamic and complex differentiation process of the adult SMG. The data sets and the analyses herein comprise an extensive wealth of high-resolution information and a valuable resource for a deeper mechanistic understanding of human SMG biology and pathophysiology.

Youth with disabilities' perspectives of the environment and participation: a qualitative meta-synthesis.

Meta-syntheses can enhance our knowledge regarding the impact of the environment on the participation of youth with disabilities and generate theoretical frameworks to inform policy and best practices. The purpose of this study was to describe school-aged youth with disabilities' perspectives regarding the impact of the environment and modifications on their participation. A meta-synthesis systematically integrates qualitative evidence from multiple studies. Six databases were searched and 1287 citations reviewed for inclusion by two independent raters; 15 qualitative articles were selected for inclusion. Two independent reviewers evaluated the quality of each study and coded the results section. Patterns between codes within and across articles were examined using a constant comparative approach. Environments may be more or less inclusive for youth with disabilities depending upon others' understanding of individual abilities and needs, youth involvement in decisions about accommodations, and quality of services and policies. Youth implemented strategies to negotiate environmental barriers and appraised the quality of their participation based on the extent to which they engaged alongside peers. This meta-synthesis generated a framework illustrating the relationship between the environment, modifications and participation, and provided a conceptualization of participation grounded in the lived experiences of youth with disabilities. Findings reveal gaps in current knowledge and highlight the importance of involving youth with disabilities in decision making.

Characterization of alpha1(IV) collagen mutations in Caenorhabditis elegans and the effects of alpha1 and alpha2(IV) mutations on type IV collagen distribution.

Type IV collagen is a major component of basement membranes. We have characterized 11 mutations in emb-9, the alpha1(IV) collagen gene of Caenorhabditis elegans, that result in a spectrum of phenotypes. Five are substitutions of glycines in the Gly-X-Y domain and cause semidominant, temperature-sensitive lethality at the twofold stage of embryogenesis. One is a glycine substitution that causes recessive, non-temperature-sensitive larval lethality. Three putative null alleles, two nonsense mutations and a deletion, all cause recessive, non-temperature-sensitive lethality at the threefold stage of embryogenesis. The less severe null phenotype indicates that glycine substitution containing mutant chains dominantly interfere with the function of other molecules. The emb-9 null mutants do not stain with anti-EMB-9 antisera and show intracellular accumulation of the alpha2(IV) chain, LET-2, indicating that LET-2 assembly and/or secretion requires EMB-9. Glycine substitutions in either EMB-9 or LET-2 cause intracellular accumulation of both chains. The degree of intracellular accumulation differs depending on the allele and temperature and correlates with the severity of the phenotype. Temperature sensitivity appears to result from reduced assembly/secretion of type IV collagen, not defective function in the basement membrane. Because the dominant interference of glycine substitution mutations is maximal when type IV collagen secretion is totally blocked, this interference appears to occur intracellularly, rather than in the basement membrane. We suggest that the nature of dominant interference caused by mutations in type IV collagen is different than that caused by mutations in fibrillar collagens.

Genetic identification, sequence, and alternative splicing of the Caenorhabditis elegans alpha 2(IV) collagen gene.

The nematode Caenorhabditis elegans has two type IV collagen genes homologous to the mammalian alpha 1(IV) and alpha 2(IV) collagen genes. We demonstrate by transgenic rescue of mutant animals that the genetic locus encoding the C. elegans alpha 2(IV) collagen gene is let-2 on the X chromosome. The most severe effect of mutations in let-2 is temperature-sensitive embryonic lethality. The embryonic lethal phenotype is similar to that seen in animals with mutations in the alpha 1(IV) collagen gene, emb-9. The sequence of the entire C. elegans alpha 2(IV) collagen gene is presented. Comparisons with mammalian type IV collagen sequences show high amino acid sequence conservation in the C-terminal NCl domain and of crosslinking residues (Cys and Lys) in the N-terminal 7S domain. RT-PCR analysis shows that transcripts of the C. elegans alpha 2(IV) collagen gene are alternatively spliced. Transcripts contain one of two mutually exclusive exons, exon 9 or 10. These exons encode very similar products, differing primarily in the sequence of a 9-10 amino acid Gly-X-Y interruption. The expression of these alternatively spliced alpha 2(IV) collagen transcripts is developmentally regulated. In embryos over 90% of the alpha 2(IV) collagen mRNA contains exon 9, while larval and adult RNAs contain 80-90% exon 10. This shift in expression of alternative alpha 2(IV) collagen transcripts suggests that C. elegans embryos may require a different form of alpha 2(IV) collagen than do larvae and adults.

The NC1/endostatin domain of Caenorhabditis elegans type XVIII collagen affects cell migration and axon guidance.

Type XVIII collagen is a homotrimeric basement membrane molecule of unknown function, whose COOH-terminal NC1 domain contains endostatin (ES), a potent antiangiogenic agent. The Caenorhabditis elegans collagen XVIII homologue, cle-1, encodes three developmentally regulated protein isoforms expressed predominantly in neurons. The CLE-1 protein is found in low amounts in all basement membranes but accumulates at high levels in the nervous system. Deletion of the cle-1 NC1 domain results in viable fertile animals that display multiple cell migration and axon guidance defects. Particular defects can be rescued by ectopic expression of the NC1 domain, which is shown to be capable of forming trimers. In contrast, expression of monomeric ES does not rescue but dominantly causes cell and axon migration defects that phenocopy the NC1 deletion, suggesting that ES inhibits the promigratory activity of the NC1 domain. These results indicate that the cle-1 NC1/ES domain regulates cell and axon migrations in C. elegans.

Type IV collagen is detectable in most, but not all, basement membranes of Caenorhabditis elegans and assembles on tissues that do not express it.

Type IV collagen in Caenorhabditis elegans is produced by two essential genes, emb-9 and let-2, which encode alpha1- and alpha2-like chains, respectively. The distribution of EMB-9 and LET-2 chains has been characterized using chain-specific antisera. The chains colocalize, suggesting that they may function in a single heterotrimeric collagen molecule. Type IV collagen is detected in all basement membranes except those on the pseudocoelomic face of body wall muscle and on the regions of the hypodermis between body wall muscle quadrants, indicating that there are major structural differences between some basement membranes in C. elegans. Using lacZ/green fluorescent protein (GFP) reporter constructs, both type IV collagen genes were shown to be expressed in the same cells, primarily body wall muscles, and some somatic cells of the gonad. Although the pharynx and intestine are covered with basement membranes that contain type IV collagen, these tissues do not express either type IV collagen gene. Using an epitope-tagged emb-9 construct, we show that type IV collagen made in body wall muscle cells can assemble into the pharyngeal, intestinal, and gonadal basement membranes. Additionally, we show that expression of functional type IV collagen only in body wall muscle cells is sufficient for C. elegans to complete development and be partially fertile. Since type IV collagen secreted from muscle cells only assembles into some of the basement membranes that it has access to, there must be a mechanism regulating its assembly. We propose that interaction with a cell surface-associated molecule(s) is required to facilitate type IV collagen assembly.

Transcriptomic and Single-Cell Analysis of the Murine Parotid Gland.

The salivary complex of mammals consists of 3 major pairs of glands: the parotid, submandibular, and sublingual glands. While the 3 glands share similar functional properties, such as saliva secretion, their differences are largely based on the types of secretions they produce. While recent studies have begun to shed light on the underlying molecular differences among the glands, few have examined the global transcriptional repertoire over various stages of gland maturation. To better elucidate the molecular nature of the parotid gland, we have performed RNA sequencing to generate comprehensive and global gene expression profiles of this gland at different stages of maturation. Our transcriptomic characterization and hierarchical clustering analysis with adult organ RNA sequencing data sets has identified a number of molecular players and pathways that are relevant for parotid gland biology. Moreover, our detailed analysis has revealed a unique parotid gland-specific gene signature that may represent important players that could impart parotid gland-specific biological properties. To complement our transcriptomic studies, we have performed single-cell RNA sequencing to map the transcriptomes of parotid epithelial cells. Interrogation of the single-cell transcriptomes revealed the degree of molecular and cellular heterogeneity of the various epithelial cell types within the parotid gland. Moreover, we uncovered a mixed-lineage population of cells that may reflect molecular priming of differentiation potentials. Overall our comprehensive studies provide a powerful tool for the discovery of novel molecular players important in parotid gland biology.

Calyculin-A improves chromosome condensation for cytogenetic analysis of blastomeres from bovine and murine eight-cell stage embryos.

This study evaluated the serine/threonine phosphatase inhibitor calyculin-A for rapid, efficient induction of premature chromosome condensation (PCC) in blastomeres obtained from Day 3 bovine and Day 2 murine eight-cell stage embryos, and its potential for use in cytogenetic analysis. Experiment 1 tested calyculin-A duration (0, 60, 120, and 180min) to induce PCC in bovine blastomeres. More blastomeres that underwent PCC had chromosomes suitable for cytogenetic analysis if treated for 120 or 180min (P<0.005). Experiment 2 compared doses of calyculin-A (0, 10, 50, and 100nM) on bovine blastomeres; calyculin-A (50nM, 120min) induced PCC suitable for cytogenetic analysis in the greatest number of blastomeres when compared to other doses (52.5%; P<0.005). Effects of calyculin-A (50nM) on murine blastomeres at durations of 0, 60, 90, and 120min to induce PCC were tested in Experiment 3, with 90min inducing the highest frequency of condensed chromosomes suitable for cytogenetic analysis (34%; P<0.05). Finally, Experiment 4 evaluated calyculin-A treated bovine embryos under optimal conditions (50nM, 120min) for use in gender and cytogenetic analysis. Whole chromosome paint probes were successfully hybridized to chromosomes along with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI) counterstaining, allowing detection of embryo gender (54% F:46% M) and ploidy of individual blastomeres within embryos (64% diploid:36% mixoploid embryos). In conclusion, we inferred that calyculin-A was useful for rapid induction of PCC, producing chromosome spreads suitable for cytogenetic analysis of blastomeres in G1 or G2/M phase of the cell cycle.

Recent Advances in the Etiology and Treatment of Burning Mouth Syndrome.

Burning mouth syndrome (BMS) is a debilitating condition that has a striking female predilection. Although the oral mucosa is normal in appearance, patients with BMS experience oral burning that most commonly localizes to the lips and tongue. BMS is a diagnosis of exclusion, and all underlying pathoses associated with allodynia must be ruled out prior to rendering the diagnosis. The etiopathogenesis of BMS remains poorly understood, and thus patient management is challenging. Data indicate that oral and systemic factors both contribute to the development and persistence of the condition. Of particular interest, emerging work identifies structural and functional deficits within the nervous system that may lead to a more mechanistic understanding of BMS pathology. In addition, several novel findings suggest that circadian rhythm dysfunction may be a previously unappreciated yet clinically significant driver of disease. Circadian rhythm controls pain perception, mood, and sleep and plays a key role in the regulation of the hypothalamic-pituitary-adrenal axis. Since these are altered in patients with BMS, this may be reflective of underlying circadian dysfunction. While evidence-based treatment strategies for BMS are lacking, current treatment approaches consist of local and systemic medications, such as clonazepam, alpha lipoic acid, capsaicin, low-level laser therapy, gabapentin, and amitriptylin. In addition, the use of cognitive behavioral therapy is reported. This review provides an overview of the recent literature related to the etiology and treatment of BMS and identifies current challenges facing researchers and clinicians alike.

Making spectral shape measurements in inverse Compton scattering a tool for advanced diagnostic applications.

Interaction of relativistic electron beams with high power lasers can both serve as a secondary light source and as a novel diagnostic tool for various beam parameters. For both applications, it is important to understand the dynamics of the inverse Compton scattering mechanism and the dependence of the scattered light's spectral properties on the interacting laser and electron beam parameters. Measurements are easily misinterpreted due to the complex interplay of the interaction parameters. Here we report the potential of inverse Compton scattering as an advanced diagnostic tool by investigating two of the most influential interaction parameters, namely the laser intensity and the electron beam emittance. Established scaling laws for the spectral bandwidth and redshift of the mean scattered photon energy are refined. This allows for a quantitatively well matching prediction of the spectral shape. Driving the interaction to a nonlinear regime, we spectrally resolve the rise of higher harmonic radiation with increasing laser intensity. Unprecedented agreement with 3D radiation simulations is found, showing the good control and characterization of the interaction. The findings advance the interpretation of inverse Compton scattering measurements into a diagnostic tool for electron beams from laser plasma acceleration.

Recombinant ob protein reduces feeding and body weight in the ob/ob mouse.

To determine whether the product of the recently cloned ob gene functions as an adipose-related satiety factor, recombinant murine ob protein was administered intraperitoneally to ob/ob mice. Monomeric ob protein given as single morning injections to groups of three animals at seven doses ranging from 5 to 100 micrograms reduced 24-h chow consumption in a dose-dependent manner from values of 81 +/- 6.8% of control (10-micrograms dose, P = 0.04) to 29 +/- 7.7% of control (100-micrograms dose, P < 0.0001). Daily injections of 80 micrograms of ob protein into six ob/ob mice for 2 wk led to an 11 +/- 1.6% decrease in body weight (P = 0.0009) and suppressed feeding to 26 +/- 4.9% of baseline (P < 0.0001), with significant reduction of serum insulin and glucose levels. The effect of recombinant ob protein on feeding was not augmented by cofactors secreted by adipose tissue, nor did exposure of adipose tissue to ob protein affect intracellular ob mRNA levels. Posttranslational modification of ob protein was not required for activity; however, addition of a hexahistidine tag to the amino terminus of the mature ob protein resulted in prolonged suppression of feeding after injection into ob/ob mice. These results demonstrate a direct effect of the ob protein to suppress feeding in the ob/ob mouse and suggest that this molecule plays a critical role in regulating total body fat content.

In vitro mutagenesis of Caenorhabditis elegans cuticle collagens identifies a potential subtilisin-like protease cleavage site and demonstrates that carboxyl domain disulfide bonding is required for normal function but not assembly.

The importance of conserved amino acids in the amino and carboxyl non-Gly-X-Y domains of Caenorhabditis elegans cuticle collagens was examined by analyzing site-directed mutations of the sqt-1 and rol-6 collagen genes in transgenic animals. Altered collagen genes on transgenic arrays were shown to produce appropriate phenotypes by injecting in vivo cloned mutant alleles. Equivalent alterations in sqt-1 and rol-6 generally produced the same phenotypes, indicating that conserved amino acids in these two collagens have similar functions. Serine substitutions for either of two conserved carboxyl domain cysteines produced LRol phenotypes. Substitution for both cysteines in sqt-1 also resulted in an LRol phenotype, demonstrating that disulfide bonding is important for normal function but not required for assembly. Arg-1 or Arg-4 to Cys mutations in homology block A (HBA; consensus, 1-RXRRQ-5; in the amino non-Gly-X-Y domain) caused RRol phenotypes, while the same alteration at Arg-3 had no effect, indicating that Arg-3 is functionally different from Arg-1 and Arg-4. Substitutions of Arg-4 with Ser, Leu, or Glu also produced the RRol phenotype, while Lys substitutions for Arg-1 or Arg-4 did not generate any abnormal phenotypes. His substitutions for Arg-1 or Arg-4 caused somewhat less severe RRol phenotypes. Therefore, strong positively charged residues, Arg or Lys, are required at positions 1 and 4 for normal function. The conserved pattern of arginines in HBA matches the cleavage sites of the subtilisin-like endoproteinases. HBA may be a cleavage site for a subtilisin-like protease, and cleavage may be important for cuticle collagen processing.

Number and organization of collagen genes in Caenorhabditis elegans.

We analyzed the number and organization of collagen genes in the nematode Caenorhabditis elegans. Genomic Southern blot hybridization experiments and recombinant phage library screenings indicated that C. elegans has between 40 and 150 distinct collagen genes. A large number of recombinant phages containing collagen genes were isolated from C. elegans DNA libraries. Physical mapping studies indicated that most phage contained a single small collagen gene less than 3 kilobases in size. A few phage contained multiple collagen hybridizing regions and may contain a larger collagen gene or several tightly linked small collagen genes. No overlaps were observed between phages containing different collagen genes, implying that the genes are dispersed in the C. elegans genome. Consistent with the small size of most collagen genes, we found that the predominant class of collagen mRNA in C. elegans is 1.2 to 1.4 kilobases in length. Genomic Southern blot experiments under stringent hybridization conditions revealed considerable sequence diversity among collagen genes. Our data suggest that most collagen genes are unique or are present in only a few copies.

The Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collagen gene to determine organismal morphology, encodes a collagen.

The rol-6 gene is one of the more than 40 loci in Caenorhabditis elegans that primarily affect organismal morphology. Certain mutations in the rol-6 gene produce animals that have the right roller phenotype, i.e., they are twisted into a right-handed helix. The rol-6 gene interacts with another gene that affects morphology, sqt-1; a left roller allele of sqt-1 acts as a dominant suppressor of a right roller allele of rol-6. The sqt-1 gene has previously been shown to encode a collagen. We isolated and sequenced the rol-6 gene and found that it also encodes a collagen. The rol-6 gene was identified by physical mapping of overlapping chromosomal deficiencies that cover the gene and by identification of an allele-specific restriction site alteration. The amino acid sequence of the collagen encoded by rol-6 is more similar to that of the sqt-1 collagen than to any of the other ten C. elegans cuticle collagen sequences compared. The locations of cysteine residues flanking the Gly-X-Y repeat regions of rol-6 and sqt-1 are identical, but differ from those in the other collagens. The sequence similarities between rol-6 and sqt-1 indicate that they represent a new collagen subfamily in C. elegans. These findings suggest that these two collagens physically interact, possibly explaining the genetic interaction seen between the rol-6 and sqt-1 genes.

Splicing in Caenorhabditis elegans does not require an AG at the 3' splice acceptor site.

The dinucleotide AG, found at the 3' end of virtually all eukaryotic pre-mRNA introns, is thought to be essential for splicing. Reduction-of-function mutations in two Caenorhabditis elegans genes, the receptor tyrosine kinase gene let-23 and the collagen gene dpy-10, both alter the AG at the end of a short (ca. 50-nucleotide) intron to AA. The in vivo effects of these mutations were studied by sequencing polymerase chain reaction-amplified reverse-transcribed RNA isolated from the two mutants. As expected, we find transcripts that splice to a cryptic AG, skip an exon, and retain an unspliced intron. However, we also find significant levels of splicing at the mutated 3' splice site (AA) and at nearby non-AG dinucleotides. Our results indicate that for short C. elegans introns an AG is not required for splicing at either the correct 3' splice site or incorrect sites. Analysis of a splice site mutant involving a longer, 316-nucleotide C. elegans intron indicates that an AG is also not required there for splicing. We hypothesize that elements besides the invariant AG, e.g., an A-U-rich region, a UUUC motif, and/or a potential branch point sequence, are directing the selection of the 3' splice site and that in wild-type genes these elements cooperate so that proper splicing occurs.

Nidogen is nonessential and not required for normal type IV collagen localization in Caenorhabditis elegans.

Nidogen (entactin) can form a ternary complex with type IV collagen and laminin and is thought to play a critical role in basement membrane assembly. We show that the Caenorhabditis elegans nidogen homologue nid-1 generates three isoforms that differ in numbers of rod domain endothelial growth factor repeats and are differentially expressed during development. NID-1 appears at the start of embryonic morphogenesis associated with muscle cells and subsequently accumulates on pharyngeal, intestinal, and gonad primordia. In larvae and adults NID-1 is detected in most basement membranes but accumulates most strongly around the nerve ring and developing gonad. NID-1 is concentrated under dense bodies, at the edges of muscle quadrants, and on the sublateral nerves that run under muscles. Two deletions in nid-1 were isolated: cg119 is a molecular null, whereas cg118 produces truncated NID-1 missing the G2 collagen IV binding domain. Neither deletion causes overt abnormal phenotypes, except for mildly reduced fecundity. Truncated cg118 NID-1 shows wild-type localization, demonstrating that the G2 domain is not necessary for nidogen assembly. Both nid-1 mutants assemble type IV collagen in a completely wild-type pattern, demonstrating that nidogen is not essential for type IV collagen assembly into basement membranes.

Molecular and genetic analyses of the Caenorhabditis elegans dpy-2 and dpy-10 collagen genes: a variety of molecular alterations affect organismal morphology.

We have identified and cloned the Caenorhabditis elegans dpy-2 and dpy-10 genes and determined that they encode collagens. Genetic data suggested that these genes are important in morphogenesis and possibly other developmental events. These data include the morphologic phenotypes exhibited by mutants, unusual genetic interactions with the sqt-1 collagen gene, and suppression of mutations in the glp-1 and mup-1 genes. The proximity of the dpy-2 and dpy-10 genes (3.5 kilobase) and the structural similarity of their encoded proteins (41% amino acid identity) indicate that dpy-2 and dpy-10 are the result of a gene duplication event. The genes do not, however, appear to be functionally redundant, because a dpy-10 null mutant is not rescued by the dpy-2 gene. In addition, full complementation between dpy-2 and dpy-10 can be demonstrated with all recessive alleles tested in trans. Sequence analysis of several mutant alleles of each gene was performed to determine the nature of the molecular defects that can cause the morphologic phenotypes. Glycine substitutions within the Gly-X-Y portion of the collagens can result in dumpy (Dpy), dumpy, left roller (DLRol), or temperature-sensitive DLRol phenotypes. dpy-10(cn64), a dominant temperature-sensitive DLRol allele, creates an Arg-to-Cys substitution in the amino non-Gly-X-Y portion of the protein. Three dpy-10 alleles contain Tc1 insertions in the coding region of the gene. dpy-10(cg36) (DRLol) creates a nonsense codon near the end of the Gly-X-Y region. The nature of this mutation, combined with genetic data, indicates that DLRol is the null phenotype of dpy-10. The Dpy phenotype results from reduced function of the dpy-10 collagen gene. Our results indicate that a variety of molecular defects in these collagens can result in severe morphologic changes in C. elegans.

Insulin-like growth factor (IGF) family genes are aberrantly expressed in bovine conceptuses produced in vitro or by nuclear transfer.

Embryos produced through somatic cell nuclear transfer (NT) or in vitro production (IVP) are often associated with increased abortion and abnormalities thought to arise from disruptions in normal gene expression. The insulin-like growth factor (IGF) family has a major influence on embryonic, fetal and placental development; differences in IGF expression in NT- and IVP-derived embryos may account for embryonic losses during placental attachment. In the present study, expression of IGF-I, IGF-II, IGF-I receptor (IGF-IR), and IGF-IIR mRNAs was quantitated in Day 7 and 25 bovine embryos produced in vivo, by NT, IVP, or parthenogenesis, to further understand divergent changes occurring during development. Expression of the IGF-I gene was not detected in Day 7 blastocysts for any treatment. However, there were no differences (P>0.10) among Day 7 treatments in the amounts of IGF-IR, IGF-II, and IGF-IIR mRNA. For Day 25 conceptuses, there was higher expression of IGF-I mRNA for NT and IVP embryonic tissues than for in vivo embryonic tissues (P<0.05). Furthermore, embryonic tissues from NT-derived embryos had higher expression of IGF-II mRNA than IVP embryonic tissues (P<0.05). Placental expression of IGF-IIR mRNA was greater for NT-derived than in vivo-derived embryos (P<0.05). There were no differences in IGF-IR mRNA across all treatments and tissues (P>0.10). In conclusion, these differences in growth factor gene expression during early placental attachment and rapid embryonic growth may directly or indirectly contribute to increased losses and abnormalities in IVP- and NT-derived embryos.