NOTCH signaling in skeletal progenitors is critical for fracture repair

Fracture nonunions develop in 10%–20% of patients with fractures, resulting in prolonged disability. Current data suggest that bone union during fracture repair is achieved via proliferation and differentiation of skeletal progenitors within periosteal and soft tissues surrounding bone, while bone marrow stromal/stem cells (BMSCs) and other skeletal progenitors may also contribute. The NOTCH signaling pathway is a critical maintenance factor for BMSCs during skeletal development, although the precise role for NOTCH and the requisite nature of BMSCs following fracture is unknown. Here, we evaluated whether NOTCH and/or BMSCs are required for fracture repair by performing nonstabilized and stabilized fractures on NOTCH-deficient mice with targeted deletion of RBPjk in skeletal progenitors, maturing osteoblasts, and committed chondrocytes. We determined that removal of NOTCH signaling in BMSCs and subsequent depletion of this population result in fracture nonunion, as the fracture repair process was normal in animals harboring either osteoblast- or chondrocyte-specific deletion of RBPjk. Together, this work provides a genetic model of a fracture nonunion and demonstrates the requirement for NOTCH and BMSCs in fracture repair, irrespective of fracture stability and vascularity

Photonic Biosensor Assays to Detect and Distinguish Subspecies of Francisella tularensis

The application of photonic biosensor assays to diagnose the category-A select agent Francisella tularensis was investigated. Both interferometric and long period fiber grating sensing structures were successfully demonstrated; both these sensors are capable of detecting the optical changes induced by either immunological binding or DNA hybridization. Detection was made possible by the attachment of DNA probes or immunoglobulins (IgG) directly to the fiber surface via layer-by-layer electrostatic self-assembly. An optical fiber biosensor was tested using a standard transmission mode long period fiber grating of length 15 mm and period 260 μm, and coated with the IgG fraction of antiserum to F. tularensis. The IgG was deposited onto the optical fiber surface in a nanostructured film, and the resulting refractive index change was measured using spectroscopic ellipsometry. The presence of F. tularensis was detected from the decrease of peak wavelength caused by binding of specific antigen. Detection and differentiation of F. tularensis subspecies tularensis (type A strain TI0902) and subspecies holarctica (type B strain LVS) was further accomplished using a single-mode multi-cavity fiber Fabry-Perot interferometric sensor. These sensors were prepared by depositing seven polymer bilayers onto the fiber tip followed by attaching one of two DNA probes: (a) a 101-bp probe from the yhhW gene unique to type-A strains, or (b) a 117-bp probe of the lpnA gene, common to both type-A and type-B strains. The yhhW probe was reactive with the type-A, but not the type-B strain. Probe lpnA was reactive with both type-A and type-B strains. Nanogram quantities of the target DNA could be detected, highlighting the sensitivity of this method for DNA detection without the use of PCR. The DNA probe reacted with 100% homologous target DNA, but did not react with sequences containing 2-bp mismatches, indicating the high specificity of the assay. These assays will fill an important void that exists for rapid, culture-free, and field-compatible diagnosis of F. tularensis

Immature mice are more susceptible to the detrimental effects of high fat diet on cancellous bone in the distal femur

AbstractWith the increasing prevalence of obesity among children and adolescents, it is imperative to understand the implications of early diet-induced obesity on bone health. We hypothesized that cancellous bone of skeletally immature mice is more susceptible to the detrimental effects of a high fat diet (HFD) than mature mice, and that removing excess dietary fat will reverse these adverse effects. Skeletally immature (5weeks old) and mature (20weeks old) male C57BL/6J mice were fed either a HFD (60% kcal fat) or low fat diet (LFD; 10% kcal fat) for 12weeks, at which point, the trabecular bone structure in the distal femoral metaphysis and third lumbar vertebrae were evaluated by micro-computed tomography. The compressive strength of the vertebrae was also measured. In general, the HFD led to deteriorations in cancellous bone structure and compressive biomechanical properties in both age groups. The HFD-fed immature mice had a greater decrease in trabecular bone volume fraction (BVF) in the femoral metaphysis, compared to mature mice (p=0.017 by 2-way ANOVA). In the vertebrae, however, the HFD led to similar reductions in BVF and compressive strength in the two age groups. When mice on the HFD were switched to a LFD (HFD:LFD) for an additional 12weeks, the femoral metaphyseal BVF in immature mice showed no improvements, whereas the mature mice recovered their femoral metaphyseal BVF to that of age-matched lean controls. The vertebral BVF and compressive strength of HFD:LFD mouse bones, following diet correction, were equivalent to those of LFD:LFD mice in both age groups. These data suggest that femoral cancellous metaphyseal bone is more susceptible to the detrimental effects of HFD before skeletal maturity and is less able to recover after correcting the diet. Negative effects of HFD on vertebrae are less severe and can renormalize with LFD:LFD mice after diet correction, in both skeletally immature and mature animals

Risk factors associated with exposure to bovine respiratory disease pathogens during the peri-weaning period in dairy bull calves

peer-reviewedBackground Bovine respiratory disease (BRD) remains among the leading causes of death of cattle internationally. The objective of this study was to identify risk factors associated with exposure to BRD pathogens during the peri-weaning period (day (d)-14 to d 14 relative to weaning at 0) in dairy bull calves using serological responses to these pathogens as surrogate markers of exposure. Clinically normal Holstein-Friesian and Jersey breed bull calves (n = 72) were group housed in 4 pens using a factorial design with calves of different breeds and planes of nutrition in each pen. Intrinsic, management and clinical data were collected during the pre-weaning (d − 56 to d − 14) period. Calves were gradually weaned over 14 days (d − 14 to d 0). Serological analysis for antibodies against key BRD pathogens (BRSV, BPI3V, BHV-1, BHV-4, BCoV, BVDV and H. somni) was undertaken at d − 14 and d 14. Linear regression models (for BVDV, BPI3V, BHV-1, BHV-4, BCoV and H. somni) and a single mixed effect random variable model (for BRSV) were used to identify risk factors for changes in antibody levels to these pathogens. Results BRSV was the only pathogen which demonstrated clustering by pen. Jersey calves experienced significantly lower changes in BVDV S/P than Holstein-Friesian calves. Animals with a high maximum respiratory score (≥8) recorded significant increases in H. somni S/P during the peri-weaning period when compared to those with respiratory scores of ≤3. Haptoglobin levels of between 1.32 and 1.60 mg/ml at d − 14 were significantly associated with decreases in BHV-1 S/N during the peri-weaning period. Higher BVDV S/P ratios at d − 14 were significantly correlated with increased changes in serological responses to BHV-4 over the peri-weaning period. Conclusions Haptoglobin may have potential as a predictor of exposure to BHV-1. BRSV would appear to play a more significant role at the ‘group’ rather than ‘individual animal’ level. The significant associations between the pre-weaning levels of antibodies to certain BRD pathogens and changes in the levels of antibodies to the various pathogens during the peri-weaning period may reflect a cohort of possibly genetically linked ‘better responders’ among the study population

The Generation of Successive Unmarked Mutations and Chromosomal Insertion of Heterologous Genes in Actinobacillus pleuropneumoniae Using Natural Transformation

We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol) and sacB (which allows counter-selection using sucrose) flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331), which is the most prevalent in the UK, and 15 (HS143). By screening clinical isolates of other serovars, it should be possible to identify other amenable strains

Horizontal gene transfer in Histophilus somni and its role in the evolution of pathogenic strain 2336, as determined by comparative genomic analyses

<p>Abstract</p> <p>Background</p> <p>Pneumonia and myocarditis are the most commonly reported diseases due to <it>Histophilus somni</it>, an opportunistic pathogen of the reproductive and respiratory tracts of cattle. Thus far only a few genes involved in metabolic and virulence functions have been identified and characterized in <it>H. somni </it>using traditional methods. Analyses of the genome sequences of several <it>Pasteurellaceae </it>species have provided insights into their biology and evolution. In view of the economic and ecological importance of <it>H. somni</it>, the genome sequence of pneumonia strain 2336 has been determined and compared to that of commensal strain 129Pt and other members of the <it>Pasteurellaceae</it>.</p> <p>Results</p> <p>The chromosome of strain 2336 (2,263,857 bp) contained 1,980 protein coding genes, whereas the chromosome of strain 129Pt (2,007,700 bp) contained only 1,792 protein coding genes. Although the chromosomes of the two strains differ in size, their average GC content, gene density (total number of genes predicted on the chromosome), and percentage of sequence (number of genes) that encodes proteins were similar. The chromosomes of these strains also contained a number of discrete prophage regions and genomic islands. One of the genomic islands in strain 2336 contained genes putatively involved in copper, zinc, and tetracycline resistance. Using the genome sequence data and comparative analyses with other members of the <it>Pasteurellaceae</it>, several <it>H. somni </it>genes that may encode proteins involved in virulence (<it>e.g</it>., filamentous haemaggutinins, adhesins, and polysaccharide biosynthesis/modification enzymes) were identified. The two strains contained a total of 17 ORFs that encode putative glycosyltransferases and some of these ORFs had characteristic simple sequence repeats within them. Most of the genes/loci common to both the strains were located in different regions of the two chromosomes and occurred in opposite orientations, indicating genome rearrangement since their divergence from a common ancestor.</p> <p>Conclusions</p> <p>Since the genome of strain 129Pt was ~256,000 bp smaller than that of strain 2336, these genomes provide yet another paradigm for studying evolutionary gene loss and/or gain in regard to virulence repertoire and pathogenic ability. Analyses of the complete genome sequences revealed that bacteriophage- and transposon-mediated horizontal gene transfer had occurred at several loci in the chromosomes of strains 2336 and 129Pt. It appears that these mobile genetic elements have played a major role in creating genomic diversity and phenotypic variability among the two <it>H. somni </it>strains.</p

Isolation and Mutagenesis of a Capsule-Like Complex (CLC) from Francisella tularensis, and Contribution of the CLC to F. tularensis Virulence in Mice

BACKGROUND: Francisella tularensis is a category-A select agent and is responsible for tularemia in humans and animals. The surface components of F. tularensis that contribute to virulence are not well characterized. An electron-dense capsule has been postulated to be present around F. tularensis based primarily on electron microscopy, but this specific antigen has not been isolated or characterized. METHODS AND FINDINGS: A capsule-like complex (CLC) was effectively extracted from the cell surface of an F. tularensis live vaccine strain (LVS) lacking O-antigen with 0.5% phenol after 10 passages in defined medium broth and growth on defined medium agar for 5 days at 32°C in 7% CO₂. The large molecular size CLC was extracted by enzyme digestion, ethanol precipitation, and ultracentrifugation, and consisted of glucose, galactose, mannose, and Proteinase K-resistant protein. Quantitative reverse transcriptase PCR showed that expression of genes in a putative polysaccharide locus in the LVS genome (FTL_1432 through FTL_1421) was upregulated when CLC expression was enhanced. Open reading frames FTL_1423 and FLT_1422, which have homology to genes encoding for glycosyl transferases, were deleted by allelic exchange, and the resulting mutant after passage in broth (LVSΔ1423/1422_P10) lacked most or all of the CLC, as determined by electron microscopy, and CLC isolation and analysis. Complementation of LVSΔ1423/1422 and subsequent passage in broth restored CLC expression. LVSΔ1423/1422_P10 was attenuated in BALB/c mice inoculated intranasally (IN) and intraperitoneally with greater than 80 times and 270 times the LVS LD₅₀, respectively. Following immunization, mice challenged IN with over 700 times the LD₅₀ of LVS remained healthy and asymptomatic. CONCLUSIONS: Our results indicated that the CLC may be a glycoprotein, FTL_1422 and -FTL_1423 were involved in CLC biosynthesis, the CLC contributed to the virulence of F. tularensis LVS, and a CLC-deficient mutant of LVS can protect mice against challenge with the parent strain

Escherichia coli O157 Exposure in Wyoming and Seattle: Serologic Evidence of Rural Risk

We tested the hypothesis that rural populations have increased exposure to Escherichia coli O157:H7. We measured circulating antibodies against the O157 lipopolysaccharide in rural Wyoming residents and in blood donors from Casper, Wyoming, and Seattle, Washington, by enzyme immunoassay (EIA). EIA readings were compared by analysis of variance and the least squares difference multiple comparison procedure. Rural Wyoming residents had higher antibody levels to O157 LPS than did Casper donors, who, in turn, had higher levels than did Seattle donors (respective least squares means: 0.356, 0.328, and 0.310; p<0.05, Seattle vs. Casper, p<0.001, rural Wyoming vs. either city). Lower age was significantly correlated with EIA scores; gender; and, in rural Wyoming, history of bloody diarrhea, town, duration of residence, and use of nontreated water at home were not significantly correlated. These data suggest that rural populations are more exposed to E. coli O157:H7 than urban populations

Three-dimensional bio-printing and bone tissue engineering: technical innovations and potential applications in maxillofacial reconstructive surgery

Background Bone grafting has been considered the gold standard for hard tissue reconstructive surgery and is widely used for large mandibular defect reconstruction. However, the midface encompasses delicate structures that are surrounded by a complex bone architecture, which makes bone grafting using traditional methods very challenging. Three-dimensional (3D) bioprinting is a developing technology that is derived from the evolution of additive manufacturing. It enables precise development of a scaffold from different available biomaterials that mimic the shape, size, and dimension of a defect without relying only on the surgeon’s skills and capabilities, and subsequently, may enhance surgical outcomes and, in turn, patient satisfaction and quality of life. Review This review summarizes different biomaterial classes that can be used in 3D bioprinters as bioinks to fabricate bone scaffolds, including polymers, bioceramics, and composites. It also describes the advantages and limitations of the three currently used 3D bioprinting technologies: inkjet bioprinting, micro-extrusion, and laser-assisted bioprinting. Conclusions Although 3D bioprinting technology is still in its infancy and requires further development and optimization both in biomaterials and techniques, it offers great promise and potential for facial reconstruction with improved outcome