Lsr2, a pleiotropic regulator at the core of the infectious strategy of Mycobacterium abscessus.

Mycobacterium abscessus is a non-tuberculous mycobacterium, causing lung infections in cystic fibrosis patients. During pulmonary infection, M. abscessus switches from smooth (Mabs-S) to rough (Mabs-R) morphotypes, the latter being hyper-virulent. Previously, we isolated the lsr2 gene as differentially expressed during S-to-R transition. lsr2 encodes a pleiotropic transcription factor that falls under the superfamily of nucleoid-associated proteins. Here, we used two functional genomic methods, RNA-seq and chromatin immunoprecipitation-sequencing (ChIP-seq), to elucidate the molecular role of Lsr2 in the pathobiology of M. abscessus. Transcriptomic analysis shows that Lsr2 differentially regulates gene expression across both morphotypes, most of which are involved in several key cellular processes of M. abscessus, including host adaptation and antibiotic resistance. These results were confirmed through quantitative real-time PCR, as well as by minimum inhibitory concentration tests and infection tests on macrophages in the presence of antibiotics. ChIP-seq analysis revealed that Lsr2 extensively binds the M. abscessus genome at AT-rich sequences and appears to form long domains that participate in the repression of its target genes. Unexpectedly, the genomic distribution of Lsr2 revealed no distinctions between Mabs-S and Mabs-R, implying more intricate mechanisms at play for achieving target selectivity.IMPORTANCELsr2 is a crucial transcription factor and chromosome organizer involved in intracellular growth and virulence in the smooth and rough morphotypes of Mycobacterium abscessus. Using RNA-seq and chromatin immunoprecipitation-sequencing (ChIP-seq), we investigated the molecular role of Lsr2 in gene expression regulation along with its distribution on M. abscessus genome. Our study demonstrates the pleiotropic regulatory role of Lsr2, regulating the expression of many genes coordinating essential cellular and molecular processes in both morphotypes. In addition, we have elucidated the role of Lsr2 in antibiotic resistance both in vitro and in vivo, where lsr2 mutant strains display heightened sensitivity to antibiotics. Through ChIP-seq, we reported the widespread distribution of Lsr2 on M. abscessus genome, revealing a direct repressive effect due to its extensive binding on promoters or coding sequences of its targets. This study unveils the significant regulatory role of Lsr2, intricately intertwined with its function in shaping the organization of the M. abscessus genome.

Enhanced Visible Light Absorption in Heteroleptic Cuprous Phenanthrolines.

This work presents a series of Cu(I) heteroleptic 1,10-phenanthroline chromophores featuring enhanced UVA and visible-light-harvesting properties manifested through vectorial control of the copper-to-phenanthroline charge-transfer transitions. The molecules were prepared using the HETPHEN strategy, wherein a sterically congested 2,9-dimesityl-1,10-phenanthrolne (mesPhen) ligand was paired with a second phenanthroline ligand incorporating extended π-systems in their 4,7-positions. The combination of electrochemistry, static and time-resolved electronic spectroscopy, 77 K photoluminescence spectra, and time-dependent density functional theory calculations corroborated all of the experimental findings. The model chromophore, [Cu(mesPhen)(phen)]+ (1), lacking 4,7-substitutions preferentially reduces the mesPhen ligand in the lowest energy metal-to-ligand charge-transfer (MLCT) excited state. The remaining cuprous phenanthrolines (2-4) preferentially reduce their π-conjugated ligands in the low-lying MLCT excited state. The absorption cross sections of 2-4 were enhanced (εMLCTmax = 7430-9980 M-1 cm-1) and significantly broadened across the UVA and visible regions of the spectrum compared to 1 (εMLCTmax = 6494 M-1 cm-1). The excited-state decay mechanism mirrored those of long-lived homoleptic Cu(I) phenanthrolines, yielding three distinguishable time constants in ultrafast transient absorption experiments. These represent pseudo-Jahn-Teller distortion (τ1), singlet-triplet intersystem crossing (τ2), and the relaxed MLCT excited-state lifetime (τ3). Effective light-harvesting from Cu(I)-based chromophores can now be rationalized within the HETPHEN strategy while achieving directionality in their respective MLCT transitions, valuable for integration into more complex donor-acceptor architectures and longer-lived photosensitizers.

Comparison of genomic-enabled cross selection criteria for the improvement of inbred line breeding populations.

A crucial step in inbred plant breeding is the choice of mating design to derive high-performing inbred varieties while also maintaining a competitive breeding population to secure sufficient genetic gain in future generations. In practice, the mating design usually relies on crosses involving the best parental inbred lines to ensure high mean progeny performance. This excludes crosses involving lower performing but more complementary parents in terms of favorable alleles. We predicted the ability of crosses to produce putative outstanding progenies (high mean and high variance progeny distribution) using genomic prediction models. This study compared the benefits and drawbacks of 7 genomic cross selection criteria (CSC) in terms of genetic gain for 1 trait and genetic diversity in the next generation. Six CSC were already published, and we propose an improved CSC that can estimate the proportion of progeny above a threshold defined for the whole mating plan. We simulated mating designs optimized using different CSC. The 835 elite parents came from a real breeding program and were evaluated between 2000 and 2016. We applied constraints on parental contributions and genetic similarities between selected parents according to usual breeder practices. Our results showed that CSC based on progeny variance estimation increased the genetic value of superior progenies by up to 5% in the next generation compared to CSC based on the progeny mean estimation (i.e. parental genetic values) alone. It also increased the genetic gain (up to 4%) and/or maintained more genetic diversity at QTLs (up to 4% more genic variance when the marker effects were perfectly estimated).

Mycobacterium abscessus Opsonization Allows an Escape from the Defensin Bactericidal Action in Drosophila.

Mycobacterium abscessus, an intracellular nontuberculous mycobacterium, is considered the most pathogenic species among the group of rapidly growing mycobacteria. The resistance of M. abscessus to the host innate response contributes to its pathogenicity in addition to several virulence factors. We have recently shown in Drosophila that antimicrobial peptides (AMPs), whose production is induced by M. abscessus, are unable to control mycobacterial infection. This could be due to their inability to kill mycobacteria and/or the hidden location of the pathogen in phagocytic cells. Here, we demonstrate that the rapid internalization of M. abscessus by Drosophila macrophages allows it to escape the AMP-mediated humoral response. By depleting phagocytes in AMP-deficient flies, we found that several AMPs were required for the control of extracellular M. abscessus. This was confirmed in the Tep4 opsonin-deficient flies, which we show can better control M. abscessus growth and have increased survival through overproduction of some AMPs, including Defensin. Furthermore, Defensin alone was sufficient to kill extracellular M. abscessus both in vitro and in vivo and control its infection. Collectively, our data support that Tep4-mediated opsonization of M. abscessus allows its escape and resistance toward the Defensin bactericidal action in Drosophila. IMPORTANCE Mycobacterium abscessus, an opportunistic pathogen in cystic fibrosis patients, is the most pathogenic species among the fast-growing mycobacteria. How M. abscessus resists the host innate response before establishing an infection remains unclear. Using Drosophila, we have recently demonstrated that M. abscessus resists the host innate response by surviving the cytotoxic lysis of the infected phagocytes and the induced antimicrobial peptides (AMPs), including Defensin. In this work, we demonstrate that M. abscessus resists the latter response by being rapidly internalized by Drosophila phagocytes. Indeed, by combining in vivo and in vitro approaches, we show that Defensin is able to control extracellular M. abscessus infection through a direct bactericidal action. In conclusion, we report that M. abscessus escapes the host AMP-mediated humoral response by taking advantage of its internalization by the phagocytes.

Surface Immobilization of a Re(I) Tricarbonyl Phenanthroline Complex to Si(111) through Sonochemical Hydrosilylation.

A sonochemical-based hydrosilylation method was employed to covalently attach a rhenium tricarbonyl phenanthroline complex to silicon(111). fac-Re(5-(p-Styrene)-phen)(CO)3Cl (5-(p-styrene)-phen = 5-(4-vinylphenyl)-1,10-phenanthroline) was reacted with hydrogen-terminated silicon(111) in an ultrasonic bath to generate a hybrid photoelectrode. Subsequent reaction with 1-hexene enabled functionalization of remaining atop Si sites. Attenuated total reflectance-Fourier transform infrared spectroscopy confirms attachment of the organometallic complex to silicon without degradation of the organometallic core, supporting hydrosilylation as a strategy for installing coordination complexes that retain their molecular integrity. Detection of Re(I) and nitrogen by X-ray photoelectron spectroscopy (XPS) further support immobilization of fac-Re(5-(p-styrene)-phen)(CO)3Cl. Cyclic voltammetry and electrochemical impedance spectroscopy under white light illumination indicate that fac-Re(5-(p-styrene)-phen)(CO)3Cl undergoes two electron reductions. Mott-Schottky analysis indicates that the flat band potential is 239 mV more positive for p-Si(111) co-functionalized with both fac-Re(5-(p-styrene)-phen)(CO)3Cl and 1-hexene than when functionalized with 1-hexene alone. XPS, ultraviolet photoelectron spectroscopy, and Mott-Schottky analysis show that functionalization with fac-Re(5-(p-styrene)-phen)(CO)3Cl and 1-hexene introduces a negative interfacial dipole, facilitating reductive photoelectrochemistry.

Micropatterning of electrochemiluminescent polymers based on multipolar Ru-complex two-photon initiators.

In this work, we present an original stereolithography strategy based on multibranched Ru-complexes with a high two-photon initiating ability allowing the 'one-pot' direct laser writing of ECL-active materials deposited onto electro-active surfaces at the µm scale.

Two-Photon Absorption Cooperative Effects within Multi-Dipolar Ruthenium Complexes: The Decisive Influence of Charge Transfers.

One- and two-photon characterizations of a series of hetero- and homoleptic [RuL3-n(bpy)n]2+ (n = 0, 1, 2) complexes carrying bipyridine π-extended ligands (L), have been carried out. These π-extended D-π-A-A-π-D-type ligands (L), where the electron donor units (D) are based on diphenylamine, carbazolyl, or fluorenyl units, have been designed to modulate the conjugation extension and the donating effect. Density functional theory calculations were performed in order to rationalize the observed spectra. Calculations show that the electronic structure of the π-extended ligands has a pronounced effect on the composition of HOMO and LUMO and on the metallic contribution to frontier MOs, resulting in strikingly different nonlinear properties. This work demonstrates that ILCT transitions are the keystone of one- and two-photon absorption bands in the studied systems and reveals how much MLCT and LLCT charge transfers play a decisive role on the two-photon properties of both hetero- and homoleptic ruthenium complexes through cooperative or suppressive effects.

Neuroligin 1 expression is linked to plasticity of behavioral and neuronal responses to sex pheromone in the male moth Agrotis ipsilon.

In the moth Agrotis ipsilon, the behavioral response of males to the female-emitted sex pheromone increases throughout adult life and following a prior exposure to sex pheromone, whereas it is temporally inhibited after the onset of mating. This behavioral flexibility is paralleled with changes in neuronal sensitivity to pheromone signal within the primary olfactory centers, the antennal lobes. In the present study, we tested the hypothesis that neuroligins, post-synaptic transmembrane proteins known to act as mediators of neuronal remodeling, are involved in the olfactory modulation in A. ipsilon males. We cloned a full-length cDNA encoding neuroligin 1, which is expressed predominantly in brain and especially in antennal lobes. The level of neuroligin 1 expression in antennal lobes gradually raised from day-2 until day-4 of adult life, as well as at 24 h, 48 h and 72 h following pre-exposure to sex pheromone, and the temporal dynamic of these changes correlated with increased sex pheromone responsiveness. By contrast, there was no significant variation in antennal lobe neuroligin 1 expression during the post-mating refractory period. Taken together, these results highlight that age- and odor experience-related increase in sex pheromone responsiveness is linked to the overexpression of neuroligin 1 in antennal lobes, thus suggesting a potential role played by this post-synaptic cell-adhesion molecule in mediating the plasticity of the central olfactory system in A. ipsilon.

Involvement of Methoprene-tolerant and Krüppel homolog 1 in juvenile hormone-signaling regulating the maturation of male accessory glands in the moth Agrotis ipsilon.

Male accessory glands (MAGs) produce seminal fluid proteins that are essential for the fertility and also influence the reproductive physiology and behavior of mated females. In many insect species, and especially in the moth Agrotis ipsilon, juvenile hormone (JH) promotes the maturation of the MAGs but the underlying molecular mechanisms in this hormonal regulation are not yet well identified. Here, we examined the role of the JH receptor, Methoprene-tolerant (Met) and the JH-inducible transcription factor, Krüppel homolog 1 (Kr-h1) in transmitting the JH signal that upregulates the growth and synthetic activity of the MAGs in A. ipsilon. We cloned two full length cDNAs encoding Met1 and Met2 which are co-expressed with Kr-h1 in the MAGs where their expression levels increase with age in parallel with the length and protein content of the MAGs. RNAi-mediated knockdown of either Met1, Met2, or Kr-h1 resulted in reduced MAG length and protein amount. Moreover, injection of JH-II into newly emerged adult males induced the transcription of Met1, Met2 and Kr-h1 associated to an increase in the length and protein content of the MAGs. By contrast, JH deficiency decreased Met1, Met2 and Kr-h1 mRNA levels as well as the length and protein reserves of the MAGs of allatectomized old males and these declines were partly compensated by a combined injection of JH-II in operated males. Taken together, our results highlighted an involvement of the JH-Met-Kr-h1 signaling pathway in the development and secretory activity of the MAGs in A. ipsilon.

Unexpected disruption of the dimensionality-driven two-photon absorption enhancement within a multipolar polypyridyl ruthenium complex series.

The dimensionality-driven two-photon absorption (2PA) enhancement effect is investigated in a series of functionalized bipyridyl Ru-complexes. Our design strategy leads to very high 2PA responses up to ∼1500 GM. However, we highlight that the 2PA performance vs. dimensionality correlation reaches an unexpected limit stemming from 'anti-cooperative' interchromophoric couplings.

A critical role for Dop1-mediated dopaminergic signaling in the plasticity of behavioral and neuronal responses to sex pheromone in a moth.

Most animal species, including insects, are able to modulate their responses to sexual chemosignals and this flexibility originates from the remodeling of olfactory areas under the influence of the dopaminergic system. In the moth Agrotis ipsilon, the behavioral response of males to the female-emitted sex pheromone increases throughout adult life and after a prior exposure to pheromone signal, and this change is accompanied by an increase in neuronal sensitivity within the primary olfactory centers, the antennal lobes (ALs). To identify the underlying neuromodulatory mechanisms, we examined whether this age- and experience-dependent olfactory plasticity is mediated by dopamine (DA) through the Dop1 receptor, an ortholog of the vertebrate D1-type dopamine receptors, which is positively coupled to adenylyl cyclase. We cloned A. ipsilon Dop1 (AiDop1), which is expressed predominantly in brain and especially in ALs; its knockdown induced a decrease in AL cAMP and altered sex pheromone-orientated flight. The levels of DA, AiDop1 expression and cAMP in ALs increased from the third day of adult life and at 24 and 48 h following pre-exposure to sex pheromone, and the dynamic of these changes correlated with the increased responsiveness to sex pheromone. These results demonstrate that Dop1 is required for the display of male sexual behavior and that age- and experience-related neuronal and behavioral changes are sustained by DA-Dop1 signaling that operates within ALs, probably through cAMP-dependent mechanisms in A. ipsilon Thus, this study expands our understanding of the neuromodulatory mechanisms underlying olfactory plasticity, mechanisms that appear to be highly conserved between insects and mammals.

Effects of DEHP on post-embryonic development, nuclear receptor expression, metabolite and ecdysteroid concentrations of the moth Spodoptera littoralis.

Di (2-ethylhexyl) phthalate (DEHP) is recognized in vertebrates as an Endocrine Disrupting Chemical (EDC). DEHP can alter steroid hormones production, development, reproduction and behavior in vertebrates. Only few studies investigated DEHP effects on insects. However, some recent studies on aquatic insects showed that DEHP could also act as an EDC by interfering with the signaling pathways of ecdysteroids, the main hormones involved in the control of insect post-embryonic development and physiology. The aim of the study was to investigate (1) the fate of DEHP within a terrestrial insect species by exposing larvae to food containing a wide range of DEHP concentrations and (2) the effects of this chemical on their post-embryonic development and metamorphosis, by using a multi-level approach. DEHP was shown to be present both in larvae and resulting stages, with higher concentrations in chrysalises and adults than in larvae. DEHP concentrations also decreased at the end of the last larval instar, suggesting the metabolic transformation or excretion of this chemical during this time. Only the two highest DEHP doses induced higher insect mortality, whereas low and intermediate concentrations increased larval food consumption without affecting body weight. Metabolic profiles showed that in control insects, the last three days before metamorphosis correspond to a metabolic transition, but with time-dependent changes in treated insects. Interestingly, DEHP treatments also alter both hemolymphatic ecdysteroid titers and expression levels of ecdysteroid response genes. These results confirm that DEHP can alter insect post-embryonic development and metamorphosis, by interfering with ecdysteroid pathways.

Glutathione-S-Transferases in the Olfactory Organ of the Noctuid Moth Spodoptera littoralis, Diversity and Conservation of Chemosensory Clades.

Glutathione-S-transferases (GSTs) are conjugating enzymes involved in the detoxification of a wide range of xenobiotic compounds. The expression of GSTs as well as their activities have been also highlighted in the olfactory organs of several species, including insects, where they could play a role in the signal termination and in odorant clearance. Using a transcriptomic approach, we identified 33 putative GSTs expressed in the antennae of the cotton leafworm Spodoptera littoralis. We established their expression patterns and revealed four olfactory-enriched genes in adults. In order to investigate the evolution of antennal GST repertoires in moths, we re-annotated antennal transcripts corresponding to GSTs in two moth and one coleopteran species. We performed a large phylogenetic analysis that revealed an unsuspected structural-and potentially functional-diversity of GSTs within the olfactory organ of insects. This led us to identify a conserved clade containing most of the already identified antennal-specific and antennal-enriched GSTs from moths. In addition, for all the sequences from this clade, we were able to identify a signal peptide, which is an unusual structural feature for GSTs. Taken together, these data highlight the diversity and evolution of GSTs in the olfactory organ of a pest species and more generally in the olfactory system of moths, and also the conservation of putative extracellular members across multiple insect orders.

The insect HR38 nuclear receptor, a member of the NR4A subfamily, is a synchronizer of reproductive activity in a moth.

In the male moth, Agrotis ipsilon, the behavioural response and neuron sensitivity within the olfactory centres, the antennal lobes (ALs), to female sex pheromone increase with age, in correlation with the maturation of sex accessory glands (SAGs). By contrast, newly mated males cease to be attracted to sex pheromone and remate when their SAGs are refilled during the next night. The insect hormone receptor 38 (HR38), an ortholog of the vertebrate NR4A receptors, is a component of ecdysteroid signalling pathway which controls adult male physiology and behaviour. Here, we cloned the A. ipsilon HR38 (AiHR38) and explored its function in the coordination of reproductive events in the male. AiHR38 was detected in SAGs and ALs, and where its amount raised with age, in parallel with SAG protein content and sex pheromone responsiveness. By contrast, the AL and SAG AiHR38 expressions declined at 0-2 h after mating, in linking with depletion of SAG protein reserves and loss of sensitivity to sex pheromone. The increased AL and SAG AiHR38 expressions at 20-24 h postmating coincided with replenishing of SAGs and recovery of sensitivity to sex pheromone for a new mating. Moreover, AiHR38 knockdown resulted in reduction in SAG protein amount and disruption of sex pheromone-orientated flight. These results show that the insect HR38 is essential both for SAG activity, probably by controlling the protein synthesis, and display of male sexual behaviour, and that the concomitant regulation of its expression within SAGs and olfactory centres contributes to synchronisation between fertility and sexual activity. DATABASE: The nucleotide sequence of Agrotis ipsilon HR38 is available in the DDBJ/EMBL/GenBank databases under the accession number MF402845.

Two genomes of highly polyphagous lepidopteran pests (Spodoptera frugiperda, Noctuidae) with different host-plant ranges.

Emergence of polyphagous herbivorous insects entails significant adaptation to recognize, detoxify and digest a variety of host-plants. Despite of its biological and practical importance - since insects eat 20% of crops - no exhaustive analysis of gene repertoires required for adaptations in generalist insect herbivores has previously been performed. The noctuid moth Spodoptera frugiperda ranks as one of the world's worst agricultural pests. This insect is polyphagous while the majority of other lepidopteran herbivores are specialist. It consists of two morphologically indistinguishable strains ("C" and "R") that have different host plant ranges. To describe the evolutionary mechanisms that both enable the emergence of polyphagous herbivory and lead to the shift in the host preference, we analyzed whole genome sequences from laboratory and natural populations of both strains. We observed huge expansions of genes associated with chemosensation and detoxification compared with specialist Lepidoptera. These expansions are largely due to tandem duplication, a possible adaptation mechanism enabling polyphagy. Individuals from natural C and R populations show significant genomic differentiation. We found signatures of positive selection in genes involved in chemoreception, detoxification and digestion, and copy number variation in the two latter gene families, suggesting an adaptive role for structural variation.

Expression and modulation of neuroligin and neurexin in the olfactory organ of the cotton leaf worm Spodoptera littoralis.

Carboxylesterases are enzymes widely distributed within living organisms. In insects, they have been mainly involved in dietary metabolism and detoxification function. Interestingly, several members of this family called carboxylesterase-like adhesion molecules (CLAMs) have lost their catalytic properties and are mainly involved in neuro/developmental functions. CLAMs include gliotactins, neurotactins, glutactins, and neuroligins. The latter have for binding partner the neurexin. In insects, the function of these proteins has been mainly studied in Drosophila central nervous system or neuromuscular junction. Some studies suggested a role of neuroligins and neurexin in sensory processing but CLAM expression within sensory systems has not been investigated. Here, we reported the identification of 5 putative CLAMs expressed in the olfactory system of the model pest insect Spodoptera littoralis. One neuroligin, Slnlg4-yll and its putative binding partner neurexin SlnrxI were the most expressed in the antennae and were surprisingly associated with olfactory sensilla. In addition, both transcripts were upregulated in male antennae after mating, known to modulate the sensitivity of the peripheral olfactory system in S. littoralis, suggesting that these molecules could be involved in sensory plasticity.

An antennal carboxylesterase from Drosophila melanogaster, esterase 6, is a candidate odorant-degrading enzyme toward food odorants.

Reception of odorant molecules within insect olfactory organs involves several sequential steps, including their transport through the sensillar lymph, interaction with the respective sensory receptors, and subsequent inactivation. Odorant-degrading enzymes (ODEs) putatively play a role in signal dynamics by rapid degradation of odorants in the vicinity of the receptors, but this hypothesis is mainly supported by in vitro results. We have recently shown that an extracellular carboxylesterase, esterase-6 (EST-6), is involved in the physiological and behavioral dynamics of the response of Drosophila melanogaster to its volatile pheromone ester, cis-vaccenyl acetate. However, as the expression pattern of the Est-6 gene in the antennae is not restricted to the pheromone responding sensilla, we tested here if EST-6 could play a broader function in the antennae. We found that recombinant EST-6 is able to efficiently hydrolyse several volatile esters that would be emitted by its natural food in vitro. Electrophysiological comparisons of mutant Est-6 null flies and a control strain (on the same genetic background) showed that the dynamics of the antennal response to these compounds is influenced by EST-6, with the antennae of the null mutants showing prolonged activity in response to them. Antennal responses to the strongest odorant, pentyl acetate, were then studied in more detail, showing that the repolarization dynamics were modified even at low doses but without modification of the detection threshold. Behavioral choice experiments with pentyl acetate also showed differences between genotypes; attraction to this compound was observed at a lower dose among the null than control flies. As EST-6 is able to degrade various bioactive odorants emitted by food and plays a role in the response to these compounds, we hypothesize a role as an ODE for this enzyme toward food volatiles.

Development of a cyclosporin-A-induced immune tolerant rat model to test marrow allograft cell type effects on bone repair.

Bone repair is an important concept in tissue engineering, and the ability to repair bone in hypotrophic conditions such as that of irradiated bone, represents a challenge for this field. Previous studies have shown that a combination of bone marrow and (BCP) was effective to repair irradiated bone. However, the origin and role played by each cell type in bone healing still remains unclear. In order to track the grafted cells, the development of an animal model that is immunotolerant to an allograft of bone marrow would be useful. Furthermore, because the immune system interacts with bone turnover, it is of critical importance to demonstrate that immunosuppressive drugs do not interfere with bone repair. After a preliminary study of immunotolerance, cyclosporin-A was chosen to be used in immunosuppressive therapy. Ten rats were included to observe qualitative and quantitative bone repair 8 days and 6 weeks after the creation of bone defects. The defects were filled with an allograft of bone marrow alone or in association with BCP under immunosuppressive treatment (cyclosporin-A). The results showed that there was no significant interaction of cyclosporin-A with osseous regeneration. The use of this new immunotolerant rat model of bone marrow allograft in future studies will provide insight on how the cells within the bone marrow graft contribute to bone healing, especially in irradiated conditions.

Determining a clinically relevant strategy for bone tissue engineering: an "all-in-one" study in nude mice.

PURPOSE: Autologous bone grafting (BG) remains the standard reconstruction strategy for large craniofacial defects. Calcium phosphate (CaP) biomaterials, such as biphasic calcium phosphate (BCP), do not yield consistent results when used alone and must then be combined with cells through bone tissue engineering (BTE). In this context, total bone marrow (TBM) and bone marrow-derived mesenchymal stem cells (MSC) are the primary sources of cellular material used with biomaterials. However, several other BTE strategies exist, including the use of growth factors, various scaffolds, and MSC isolated from different tissues. Thus, clinicians might be unsure as to which method offers patients the most benefit. For this reason, the aim of this study was to compare eight clinically relevant BTE methods in an "all-in-one" study. METHODS: We used a transgenic rat strain expressing green fluorescent protein (GFP), from which BG, TBM, and MSC were harvested. Progenitor cells were then mixed with CaP materials and implanted subcutaneously into nude mice. After eight weeks, bone formation was evaluated by histology and scanning electron microscopy, and GFP-expressing cells were tracked with photon fluorescence microscopy. RESULTS/CONCLUSIONS: Bone formation was observed in only four groups. These included CaP materials mixed with BG or TBM, in which abundant de novo bone was formed, and BCP mixed with committed cells grown in two- and three-dimensions, which yielded limited bone formation. Fluorescence microscopy revealed that only the TBM and BG groups were positive for GFP expressing-cells, suggesting that these donor cells were still present in the host and contributed to the formation of bone. Since the TBM-based procedure does not require bone harvest or cell culture techniques, but provides abundant de novo bone formation, we recommend consideration of this strategy for clinical applications.