A comparative analysis of the microarchitecture of cortical membranous and cortical endochondral onlay bone grafts in the craniofacial skeleton

Previous work in this laboratory established that an onlay bone graft's survival is determined primarily by its relative cortical and cancellous composition rather than its embryologic origin. A volumetric analysis of external bone graft resorption, however, does not explain the internal microarchitectural changes that may be occurring as these grafts become incorporated. To expand the knowledge of bone graft dynamics beyond volumetric parameters, a better understanding of the internal processes of bone graft remodeling is needed. In this comparative study of cortical onlay bone graft microarchitecture, the authors propose to show that cortical onlay bone grafts undergo measurable internal microarchitectural changes as they become incorporated into the surrounding craniofacial skeleton. In addition, the authors propose to further demonstrate similarities between the internal microarchitecture of cortical onlay bone grafts of different embryologic origin over time. Twenty-five adult New Zealand White rabbits were used for this study. They were divided into two groups of eight animals and one group of nine. The groups were killed at 3, 8, and 16 weeks. Cortical membranous and endochondral bone grafts were placed subperiosteally onto each rabbit's cranium. In addition, five ungrafted cortical endochondral and membranous bone specimens were used as controls. Microcomputed tomography (MCT) scanning and histomorphometric analysis were performed on all of the specimens to obtain detailed information regarding the microarchitecture of the cortical bone grafts. The parameters of bone volume fraction, bone surface area to volume, mean trabecular number, and anisotropy were used to give quantitative information about a bone's micro-organization. The results showed that there is no statistically significant difference between the cortical endochondral and the cortical membranous bone grafts for bone volume fraction, bone surface to volume, mean trabecular number, and anisotropy measurements for all time points. There were, however, statistically significant differences when comparing the control and 3-week groups to the 16-week group for all parameters. The advanced MCT technology and histomorphometric techniques proved to be effective in providing a qualitative and quantitative ultrastructural comparison of cortical endochondral and membranous onlay bone grafts over time. In this study, a statistically significant change in the internal microarchitecture of cortical onlay bone grafts of different embryologic origins was seen as they were remodeled and resorbed at all time points. Specifically, the onlay cortical bone grafts developed a less dense, more trabecular, and less organized internal ultrastructure. In addition, no difference in the three-dimensional ultrastructure of cortical endochondral and membranous bone was found. These results challenge some of the currently accepted theories of bone-graft dynamics and may eventually lead to a change in the way clinicians approach bone-graft selection for craniofacial surgery.     

Neurons expressing the highest levels of gamma-synuclein are unaffected by targeted inactivation of the gene

Homologous recombination in ES cells was employed to generate mice with targeted deletion of the first three exons of the gamma-synuclein gene. Complete inactivation of gene expression in null mutant mice was confirmed on the mRNA and protein levels. Null mutant mice are viable, are fertile, and do not display evident phenotypical abnormalities. The effects of gamma-synuclein deficiency on motor and peripheral sensory neurons were studied by various methods in vivo and in vitro. These two types of neurons were selected because they both express high levels of gamma-synuclein from the early stages of mouse embryonic development but later in the development they display different patterns of intracellular compartmentalization of the protein. We found no difference in the number of neurons between wild-type and null mutant animals in several brain stem motor nuclei, in lumbar dorsal root ganglia, and in the trigeminal ganglion. The survival of gamma-synuclein-deficient trigeminal neurons in various culture conditions was not different from that of wild-type neurons. There was no difference in the numbers of myelinated and nonmyelinated fibers in the saphenous nerves of these animals, and sensory reflex thresholds were also intact in gamma-synuclein null mutant mice. Nerve injury led to similar changes in sensory function in wild-type and mutant mice. Taken together, our data suggest that like alpha-synuclein, gamma-synuclein is dispensable for the development and function of the nervous system.     

Analysis of the overexpression of a newly found gene <Emphasis Type="Italic">toothrin</Emphasis> in <Emphasis Type="Italic">Drosophila</Emphasis>

A newly found locus of the Drosophila melanogaster genome, named toothrin (tth) has been used to study the role of the conserved the 2/3 domain of genes from the d4 family. In contrast to all vertebrates studied (including humans), in which the 2/3 domain is always accompanied by the d4 domain, the tth gene contains the sequence encoding the 2/3 domain but lacks that encoding the d4 domain. The tth gene overexpression has been studied using the two-component system UAS-GAL4. It has been demonstrated that the tth overexpression at the third-instar larval (prepupal) stage decreases survival rate, simultaneously causing a substantial deceleration of development in Drosophila. It is known that the change of developmental stages in Drosophila is controlled by the rates of the expression of ecdysteroid and juvenile hormones (JHs). It is supposed that the overexpression of the tth gene causes either a shift in the ecdysterone-to-JH ratio (through a decreased JH decay rate or a delayed initiation of ecdysone synthesis) or a deceleration of the release of ecdysterones synthesized.Translated from Genetika, Vol. 41, No. 2, 2005, pp. 196–202.Original Russian Text Copyright © 2005 by Simonova, Kulikova, Mertsalov, Umnova, Bashkirov, Buchman, Korochkin.     

Sepsis-induced apoptosis causes progressive profound depletion of B and CD4+ T lymphocytes in humans

Patients with sepsis have impaired host defenses that contribute to the lethality of the disorder. Recent work implicates lymphocyte apoptosis as a potential factor in the immunosuppression of sepsis. If lymphocyte apoptosis is an important mechanism, specific subsets of lymphocytes may be more vulnerable. A prospective study of lymphocyte cell typing and apoptosis was conducted in spleens from 27 patients with sepsis and 25 patients with trauma. Spleens from 16 critically ill nonseptic (3 prospective and 13 retrospective) patients were also evaluated. Immunohistochemical staining showed a caspase-9-mediated profound progressive loss of B and CD4 T helper cells in sepsis. Interestingly, sepsis did not decrease CD8 T or NK cells. Although there was no overall effect on lymphocytes from critically ill nonseptic patients (considered as a group), certain individual patients did exhibit significant loss of B and CD4 T cells. The loss of B and CD4 T cells in sepsis is especially significant because it occurs during life-threatening infection, a state in which massive lymphocyte clonal expansion should exist. Mitochondria-dependent lymphocyte apoptosis may contribute to the immunosuppression in sepsis by decreasing the number of immune effector cells. Similar loss of lymphocytes may be occurring in critically ill patients with other disorders.     

Evolutionary dynamics of methicillin-resistant Staphylococcus aureus within a healthcare system

BackgroundIn the past decade, several countries have seen gradual replacement of endemic multi-resistant healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) with clones that are more susceptible to antibiotic treatment. One example is Singapore, where MRSA ST239, the dominant clone since molecular profiling of MRSA began in the mid-1980s, has been replaced by ST22 isolates belonging to EMRSA-15, a recently emerged pandemic lineage originating from Europe.ResultsWe investigated the population structure of MRSA in Singaporean hospitals spanning three decades, using whole genome sequencing. Applying Bayesian phylogenetic methods we report that prior to the introduction of ST22, the ST239 MRSA population in Singapore originated from multiple introductions from the surrounding region; it was frequently transferred within the healthcare system resulting in a heterogeneous hospital population. Following the introduction of ST22 around the beginning of the millennium, this clone spread rapidly through Singaporean hospitals, supplanting the endemic ST239 population. Coalescent analysis revealed that although the genetic diversity of ST239 initially decreased as ST22 became more dominant, from 2007 onwards the genetic diversity of ST239 began to increase once more, which was not associated with the emergence of a sub-clone of ST239. Comparative genomic analysis of the accessory genome of the extant ST239 population identified that the Arginine Catabolic Mobile Element arose multiple times, thereby introducing genes associated with enhanced skin colonization into this population.ConclusionsOur results clearly demonstrate that, alongside clinical practice and antibiotic usage, competition between clones also has an important role in driving the evolution of nosocomial pathogen populations.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0643-z) contains supplementary material, which is available to authorized users.     

Single-molecule sequencing reveals the molecular basis of multidrug-resistance in ST772 methicillin-resistant Staphylococcus aureus

Background

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-associated infection, but there is growing awareness of the emergence of multidrug-resistant lineages in community settings around the world. One such lineage is ST772-MRSA-V, which has disseminated globally and is increasingly prevalent in India. Here, we present the complete genome sequence of DAR4145, a strain of the ST772-MRSA-V lineage from India, and investigate its genomic characteristics in regards to antibiotic resistance and virulence factors.

Results

Sequencing using single-molecule real-time technology resulted in the assembly of a single continuous chromosomal sequence, which was error-corrected, annotated and compared to nine draft genome assemblies of ST772-MRSA-V from Australia, Malaysia and India. We discovered numerous and redundant resistance genes associated with mobile genetic elements (MGEs) and known core genome mutations that explain the highly antibiotic resistant phenotype of DAR4145. Staphylococcal toxins and superantigens, including the leukotoxin Panton-Valentinin Leukocidin, were predominantly associated with genomic islands and the phage φ-IND772PVL. Some of these mobile resistance and virulence factors were variably present in other strains of the ST772-MRSA-V lineage.

Conclusions

The genomic characteristics presented here emphasize the contribution of MGEs to the emergence of multidrug-resistant and highly virulent strains of community-associated MRSA. Antibiotic resistance was further augmented by chromosomal mutations and redundancy of resistance genes. The complete genome of DAR4145 provides a valuable resource for future investigations into the global dissemination and phylogeography of ST772-MRSA-V.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1599-9) contains supplementary material, which is available to authorized users.