The human dental apical papilla promotes spinal cord repair through a paracrine mechanism.

Traumatic spinal cord injury is an overwhelming condition that strongly and suddenly impacts the patient's life and her/his entourage. There are currently no predictable treatments to repair the spinal cord, while many strategies are proposed and evaluated by researchers throughout the world. One of the most promising avenues is the transplantation of stem cells, although its therapeutic efficiency is limited by several factors, among which cell survival at the lesion site. In our previous study, we showed that the implantation of a human dental apical papilla, residence of stem cells of the apical papilla (SCAP), supported functional recovery in a rat model of spinal cord hemisection. In this study, we employed protein multiplex, immunohistochemistry, cytokine arrays, RT- qPCR, and RNAseq technology to decipher the mechanism by which the dental papilla promotes repair of the injured spinal cord. We found that the apical papilla reduced inflammation at the lesion site, had a neuroprotective effect on motoneurons, and increased the apoptosis of activated macrophages/ microglia. This therapeutic effect is likely driven by the secretome of the implanted papilla since it is known to secrete an entourage of immunomodulatory or pro-angiogenic factors. Therefore, we hypothesize that the secreted molecules were mainly produced by SCAP, and that by anchoring and protecting them, the human papilla provides a protective niche ensuring that SCAP could exert their therapeutic actions. Therapeutic abilities of the papilla were demonstrated in the scope of spinal cord injury but could very well be beneficial to other types of tissue.

Fungal adaptation to contemporary fungicide applications: the case of Botrytis cinerea populations from Champagne vineyards (France).

In addition to being one of the most acute problems impeding chemical control of fungal diseases, the evolution of fungicide resistance is an emblematic case of local adaptation to spatially heterogeneous and temporally variable selection pressures. Here we dissected the adaptation of Botrytis cinerea (the causal agent of grey mould) populations on grapes to several fungicides. We carried out a 2-year survey (four collection dates) on three treated/untreated pairs of plots from vineyards in Champagne (France) and monitored the frequency of four resistant phenotypes that are unambiguously associated with four distinct genotypes. For two loci under selection by currently used fungicides (MDR1 and MDR2), the frequencies of resistant mutations at vintage were greater in treated plots compared to untreated plots, showing that the effect of selection is detectable even at the plot scale. This effect was not detectable for two other loci under selection by previously used fungicides (BenR1 and ImiR1). We also found that treatment with currently used fungicides reduced B. cinerea effective population size, leading to a significant decrease in genic diversity and allelic richness in treated vs. untreated plots. We further highlight that even under ample drift and migration, fungal populations can present an efficient response to selection. Finally, for the four studied loci, the costs of fungicide resistance were estimated by modelling the decrease in the frequency of resistant mutations in the absence of treatment. We discuss the importance of these estimates for defining strategies for limiting or counteracting the local adaptation of pests to fungicides.

Adaptation of genetically monomorphic bacteria: evolution of copper resistance through multiple horizontal gene transfers of complex and versatile mobile genetic elements.

Copper-based antimicrobial compounds are widely used to control plant bacterial pathogens. Pathogens have adapted in response to this selective pressure. Xanthomonas citri pv. citri, a major citrus pathogen causing Asiatic citrus canker, was first reported to carry plasmid-encoded copper resistance in Argentina. This phenotype was conferred by the copLAB gene system. The emergence of resistant strains has since been reported in Réunion and Martinique. Using microsatellite-based genotyping and copLAB PCR, we demonstrated that the genetic structure of the copper-resistant strains from these three regions was made up of two distant clusters and varied for the detection of copLAB amplicons. In order to investigate this pattern more closely, we sequenced six copper-resistant X. citri pv. citri strains from Argentina, Martinique and Réunion, together with reference copper-resistant Xanthomonas and Stenotrophomonas strains using long-read sequencing technology. Genes involved in copper resistance were found to be strain dependent with the novel identification in X. citri pv. citri of copABCD and a cus heavy metal efflux resistance-nodulation-division system. The genes providing the adaptive trait were part of a mobile genetic element similar to Tn3-like transposons and included in a conjugative plasmid. This indicates the system's great versatility. The mining of all available bacterial genomes suggested that, within the bacterial community, the spread of copper resistance associated with mobile elements and their plasmid environments was primarily restricted to the Xanthomonadaceae family.

The combined administration of LNC-encapsulated retinoic acid and calcitriol stimulates oligodendrocyte progenitor cell differentiation in vitro and in vivo after intranasal administration.

Intranasal administration is an efficient strategy for bypassing the BBB, favoring drug accumulation in the brain, and improving its efficiency. Lipid nanocapsules (LNC) are suitable nanocarriers for the delivery of lipophilic drugs via this route and can be used to encapsulate lipophilic molecules such as retinoic acid (RA) and calcitriol (Cal). As the hallmarks of multiple sclerosis (MS) are neuroinflammation and oligodendrocyte loss, our hypothesis was that by combining two molecules known for their pro-differentiating properties, encapsulated in LNC, and delivered by intranasal administration, we would stimulate oligodendrocyte progenitor cells (OPC) differentiation into oligodendrocytes and provide a new pro-remyelinating therapy. LNC loaded with RA (LNC-RA) and Cal (LNC-Cal) were stable for at least 8 weeks. The combination of RA and Cal was more efficient than the molecules alone, encapsulated or not, on OPC differentiation in vitro and decreased microglia cell activation in a dose-dependent manner. After the combined intranasal administration of LNC-RA and LNC-Cal in a mouse cuprizone model of demyelination, increased MBP staining was observed in the corpus callosum. In conclusion, intranasal delivery of lipophilic drugs encapsulated in LNC is a promising strategy for myelinating therapies.

Using neutral cline decay to estimate contemporary dispersal: a generic tool and its application to a major crop pathogen.

Dispersal is a key parameter of adaptation, invasion and persistence. Yet standard population genetics inference methods hardly distinguish it from drift and many species cannot be studied by direct mark-recapture methods. Here, we introduce a method using rates of change in cline shapes for neutral markers to estimate contemporary dispersal. We apply it to the devastating banana pest Mycosphaerella fijiensis, a wind-dispersed fungus for which a secondary contact zone had previously been detected using landscape genetics tools. By tracking the spatio-temporal frequency change of 15 microsatellite markers, we find that σ, the standard deviation of parent-offspring dispersal distances, is 1.2 km/generation(1/2) . The analysis is further shown robust to a large range of dispersal kernels. We conclude that combining landscape genetics approaches to detect breaks in allelic frequencies with analyses of changes in neutral genetic clines offers a powerful way to obtain ecologically relevant estimates of dispersal in many species.

Multicentric evaluation of a new real-time PCR assay for quantification of Cryptosporidium spp. and identification of Cryptosporidium parvum and Cryptosporidium hominis.

Cryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum. The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidium-positive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium-seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum, were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed.

Recent range expansion and agricultural landscape heterogeneity have only minimal effect on the spatial genetic structure of the plant pathogenic fungus Mycosphaerella fijiensis.

Understanding how geographical and environmental features affect genetic variation at both the population and individual levels is crucial in biology, especially in the case of pathogens. However, distinguishing between these factors and the effects of historical range expansion on spatial genetic structure remains challenging. In the present study, we investigated the case of Mycosphaerella fijiensis-a plant pathogenic fungus that has recently colonized an agricultural landscape characterized by the presence of potential barriers to gene flow, including several commercial plantations in which disease control practises such as the use of fungicides are applied frequently, and low host density areas. We first genotyped 300 isolates sampled at a global scale on untreated plants in two dimensions over a 50 × 80-km area. Using two different clustering algorithms, no genetic structure was detected in the studied area, suggesting expansion of large populations and/or no influence of potential barriers. Second, we investigated the potential effect of disease control practises on M. fijiensis diversity by comparing populations sampled in commercial vs food-crop plantations. At this local scale, we detected significantly higher allelic richness inside commercial plantations compared with the surrounding food-crop plantation populations. Analysis of molecular variance indicated that 99% of the total genetic variance occurred within populations. We discuss the suggestion that high population size and/or high migration rate between populations might be responsible for the absence of any effect of disease control practises on genetic diversity and differentiation.

Molecular characterization of Cryptosporidium isolates from high-excreting young dairy calves in dairy cattle herds in Western France.

Ninety-two Cryptosporidium sp.-positive fecal samples of dairy diarrheic or non-diarrheic calves from 30 cattle herds in Normandy (France) were selected. Here, the aim was to investigate the species of Cryptosporidium excreted as well as the subtypes of Cryptosporidium parvum found in 7-17-day-old dairy calves. Excretion levels were comprised between 2 × 10(4) and 4 × 10(7) oocysts per gram of feces. Here, a nested 18S SSU rRNA PCR associated with sequencing was performed for identification of Cryptosporidium species and revealed the presence of C. parvum in most cases (80/82), except for two animals which were infected with Cryptosporidium bovis. Then, C. parvum samples were submitted to gp60 PCR. For 39 samples from 24 different herds, a multilocus analysis based on four mini-microsatellites loci (MM19, MM5, MSF, and MS9-Mallon) were conducted. These results were combined with sequence analysis of the gp60 to obtain multilocus types (MLTs). Here, C. parvum gp60 genotyping identified three subtypes in the IIa zoonotic allele family: IIaA15G2R1 (88%), IIaA16G3R1 (10%), and IIaA19G2R1 (2%), and we identified 12 MLTs. The MS9-Mallon locus was reported as the most polymorphic (five alleles). The most common MLT was MLT 1 with 15 samples in 10 farms: (MS9-M: 298, MSF: 165, MM5: 264, MM19: 462, and gp60 subtype: IIaA15G2R1). When comparing diarrheic and non-diarrheic fecal samples, no difference was seen for distribution of Cryptosporidium species, C. parvum gp60 subtypes, and MLTs. Here, in a range of oocyst excretion of 10(4)-10(7) opg, both in diarrheic and non-diarrheic calves, infection was mainly due to C. parvum and to the zoonotic subtype: IIaA15G2R1.

Revisiting the historical scenario of a disease dissemination using genetic data and Approximate Bayesian Computation methodology: The case of Pseudocercospora fijiensis invasion in Africa.

The reconstruction of geographic and demographic scenarios of dissemination for invasive pathogens of crops is a key step toward improving the management of emerging infectious diseases. Nowadays, the reconstruction of biological invasions typically uses the information of both genetic and historical information to test for different hypotheses of colonization. The Approximate Bayesian Computation framework and its recent Random Forest development (ABC-RF) have been successfully used in evolutionary biology to decipher multiple histories of biological invasions. Yet, for some organisms, typically plant pathogens, historical data may not be reliable notably because of the difficulty to identify the organism and the delay between the introduction and the first mention. We investigated the history of the invasion of Africa by the fungal pathogen of banana Pseudocercospora fijiensis, by testing the historical hypothesis against other plausible hypotheses. We analyzed the genetic structure of eight populations from six eastern and western African countries, using 20 microsatellite markers and tested competing scenarios of population foundation using the ABC-RF methodology. We do find evidence for an invasion front consistent with the historical hypothesis, but also for the existence of another front never mentioned in historical records. We question the historical introduction point of the disease on the continent. Crucially, our results illustrate that even if ABC-RF inferences may sometimes fail to infer a single, well-supported scenario of invasion, they can be helpful in rejecting unlikely scenarios, which can prove much useful to shed light on disease dissemination routes.

Quality of Cure in Depth of Commercially Available Bulk-fill Composites: A Layer-by-layer Mechanical and Biological Evaluation.

Despite their popularity, the use of bulk-fill composites remains controversial, both in terms of their properties and their in-depth development. The objectives of the present work were (1) to provide a more comprehensive evaluation of the quality of cure in depth of commercially available bulk-fill composites by combining various key mechanical and biological characterization methods, (2) to evaluate the inter-material differences when optimally cured, and (3) to evaluate the efficiency of an antioxidant-N-acetyl-cysteine (NAC)-to restrain the adverse effects of the leached components on cell viability. Nine bulk-fill composites (including flowable and high-viscosity materials) were investigated and compared to two conventional resin-based composites, one flowable and one high-viscosity restorative material. The materials were injected or packed into Teflon molds of various configurations, up to 6 mm material thickness. They were then light-cured from the top for 20 seconds with Bluephase G2 (Ivoclar Vivadent, irradiance = 1050 mW/cm2). The following physico-mechanical properties were measured for the upper (0-2 mm), intermediate (2-4 mm), and lower (4-6 mm) layers: degree of conversion using Raman Spectrometry (DC, in %), microhardness using a Vickers micro-indenter before (VHN dry) and after 24 hours of storage in ethanol (VHN EtOH), and flexural strength (in MPa) and flexural modulus (in GPa) using a three-point bend test. Each composite layer and an uncured layer were also stored for one week in a standard cell growth medium to generate conditioned media. Human dental pulp cells were then cultured for 24 hours with the latter and cell viability was measured using an MTS assay. A similar experiment was repeated with conditioned media produced in contact with uncured composites, with and without the addition of 4 mM NAC. The data were subjected to a Shapiro-Wilk test, then one-way ANOVA or Kruskal-Wallis test, followed either by Tukey's test (inter-material comparison) or by Dunnett's or Dunn's test (comparison between layers relative to the upper one). The level of statistical significance was set at 0.05. Some materials (EverX, X-traF, VenusBF, X-traB) did not show any significant differences (p>0.05) for any of the properties considered between the intermediate layers compared to the upper one (considered as reference). Others displayed significant differences, at least for some properties, highlighting the value of combining various key mechanical and biological characterization methods when investigating the quality of cure in depth. Significant inter-material differences (p<0.05) were observed when comparing the properties of their upper layer, considered as "optimally" polymerized. Hence, one needs to consider the absolute property values, not only their relative evolution concerning layer thickness. Finally, the use of NAC appeared as beneficial to reduce the risk of harmful effects to dental pulp cells, especially in case of excessive thickness use, and may therefore be of potential interest as an additive to composites in the future.

Inferences on pathogenic fungus population structures from microsatellite data: new insights from spatial genetics approaches.

Landscape genetics, which combines population genetics, landscape ecology and spatial statistics, has emerged recently as a new discipline that can be used to assess how landscape features or environmental variables can influence gene flow and spatial genetic variation. We applied this approach to the invasive plant pathogenic fungus Mycosphaerella fijiensis, which causes black leaf streak disease of banana. Around 880 isolates were sampled within a 50 × 50 km area located in a fragmented banana production zone in Cameroon that includes several potential physical barriers to gene flow. Two clustering algorithms and a new F(ST) -based procedure were applied to define the number of genetic entities and their spatial domain without a priori assumptions. Two populations were clearly delineated, and the genetic discontinuity appeared sharp but asymmetric. Interestingly, no landscape features matched this genetic discontinuity, and no isolation by distance (IBD) was found within populations. Our results suggest that the genetic structure observed in this production area reflects the recent history of M. fijiensis expansion in Cameroon rather than resulting from contemporary gene flow. Finally, we discuss the influence of the suspected high effective population size for such an organism on (i) the absence of an IBD signal, (ii) the characterization of contemporary gene-flow events through assignation methods of analysis and (iii) the evolution of the genetic discontinuity detected in this study.

Dental Apical Papilla as Therapy for Spinal Cord Injury.

Stem cells of the apical papilla (SCAP) represent great promise regarding treatment of neural tissue damage, such as spinal cord injury (SCI). They derive from the neural crest, express numerous neurogenic markers, and mediate neurite outgrowth and axonal targeting. The goal of the present work was to investigate for the first time their potential to promote motor recovery after SCI in a rat hemisection model when delivered in their original stem cell niche-that is, by transplantation of the human apical papilla tissue itself into the lesion. Control groups consisted of animals subjected to laminectomy only (shams) and to lesion either untreated or injected with a fibrin hydrogel with or without human SCAP. Basso-Beattie-Bresnahan locomotor scores at 1 and 3 d postsurgery confirmed early functional decline in all SCI groups. This significant impairment was reversed, as seen in CatWalk analyses, after transplantation of apical papilla into the injured spinal cord wound, whereas the other groups demonstrated persistent functional impairment. Moreover, tactile allodynia did not develop as an unwanted side effect in any of the groups, even though the SCAP hydrogel group showed higher expression of the microglial marker Iba-1, which has been frequently associated with allodynia. Notably, the apical papilla transplant group presented with reduced Iba-1 expression level. Masson trichrome and human mitochondria staining showed the preservation of the apical papilla integrity and the presence of numerous human cells, while human cells could no longer be detected in the SCAP hydrogel group at the 6-wk postsurgery time point. Altogether, our data suggest that the transplantation of a human apical papilla at the lesion site improves gait in spinally injured rats and reduces glial reactivity. It also underlines the potential interest for the application of delivering SCAP in their original niche, as compared with use of a fibrin hydrogel.

High excretion of Cryptosporidium ubiquitum by peri-parturient goats in one flock in western France.

Cryptosporidium spp. is an important agent of neonatal diarrhoea in goat kids. Little is known about its molecular characterization in adult goats. A longitudinal study was set up to identify the species excreted by adult goats around parturition. Individual faecal samples were collected from 20 pregnant adult goats between 1 and 5 years old in one flock. Samplings began 3 weeks before the estimated kidding date and were done weekly until kidding and for 2 weeks after kidding. Cryptosporidium oocysts were concentrated from 15 g of faeces using a caesium chloride (CsCl) method. Oocyst output was determined using a direct immunofluorescent antibody test (IFAT). Genomic DNA was extracted from each CsCl-concentrated faecal sample positive by IFAT and submitted to a nested PCR-RFLP on the SSU rDNA gene followed by sequencing to identify the isolates at species level. According to their kidding date, goats were sampled between 4 and 8 times. Sixteen goats, out of the eighteen which kidded, were found positive at least at one sampling date. Infection was asymptomatic. Prevalence of excretion was maximal 14 days before kidding with half of the goats excreting oocysts at this date. Excretion was higher before kidding than after kidding. Unexpected levels of excretion were observed with individual oocyst excretion ranging from 6 to 2.5 × 10(5) oocysts per gram of faeces. All isolates were identified as Cryptosporidium ubiquitum.

Dynamics of excretion and molecular characterization of Cryptosporidium isolates in pre-weaned French beef calves.

Studies on excretion and molecular characterization of Cryptosporidium have been mostly conducted in dairy calves, both diarrhoeic and non-diarrhoeic. Little is known about Cryptosporidium in beef calves, especially in non-diarrhoeic ones. This study was conducted in a herd of Parthenais beef cattle (France) with no history of clinical cryptosporidiosis. Twenty-five calves were sampled once a week from birth to one month of age (age range: 5-34 days). At each sampling date, presence of clinical signs of cryptosporidiosis (diarrhoea) was recorded. Oocyst excretion was assessed using the Heine staining method and a direct immunofluorescence method (Merifluor(®) C/G) which allowed quantification (oocysts per gram of faeces, opg). All samples were subjected to a two-step nested PCR protocol to amplify the 18S rRNA gene and amplification products were sequenced. None of the calves presented diarrhoea. Twenty-three of them excreted oocysts at least one sampling date. Prevalence of excretion was maximal when calves were 27-34 days old, with a percentage of excretion of 85% in this age category [95% CI: 70; 100]. Mean excretion was maximal when calves were 20 to 26 days old, with a mean excretion of 7.6×10(5) opg (range: 0-8×10(6) opg). 32 isolates were successfully identified: 27 as Cryptosporidium bovis, 4 as Cryptosporidium ryanae and 1 as Cryptosporidium parvum. C. bovis was isolated from samples of calves between 11 and 33 days old. C. ryanae was isolated from samples of calves between 17 and 34 days old. C. parvum was isolated from one calf aged 13 days. This survey demonstrated the high infection rate of non-diarrhoeic beef calves by Cryptosporidium species other than C. parvum.

Detection of Cryptosporidium oocysts in fresh calf faeces: characteristics of two simple tests and evaluation of a semi-quantitative approach.

The objective of the present study was to evaluate the characteristics of two rapid tests, namely, a faecal smear staining method (Heine staining) and a commercially available immunochromatographic (IC) assay, against a direct immunofluorescent antibody test (IFAT) for the diagnosis of Cryptosporidium infections in 917 faecal samples from calves aged <3 weeks. These rapid tests were performed on non-concentrated faeces using a semi-quantitative approach using a 6-point scale (0-5) for Heine staining according to the number of oocysts per microscopic field, and a 4-point scale (0-3) for the IC assay reflecting the intensity of the positive line compared to the control line. Direct IFAT was performed following a diethyl ether concentration and results were expressed as oocysts per g of faeces (opg). Heine staining showed a sensitivity of 76.7% and a specificity of 90.7%. For faecal samples with ≥ 10,000opg, sensitivity increased to 90.0%. The sensitivity of the IC assay was lower (61.8%) but the specificity was 100%. For faeces with ≥ 100,000opg, the sensitivity of the IC assay reached 81%, indicating some limitation for clinical cryptosporidiosis diagnosis. Additional scoring (1-5) of the Heine staining correlated with the corresponding direct IFAT results, particularly for ranges of 1000 to >1,000,000opg. Additional scoring from 1 to 3 according to the thickness of the sample line for the IC test correlated with increasing levels of Cryptosporidium opg measured by IFAT in the range of 10,000 to >1,000,000opg. In conclusion, both Heine staining and the IC test can be reliably used through a simple semi-quantitative scale for grossly quantifying oocyst output in calf faecal samples.

In vitro identification of targeting ligands of human M cells by phage display.

To improve transport of vaccine-loaded nanoparticles, the phage display technology was used to identify novel lead peptides targeting human M cells. Using an in vitro model of the human follicle-associated epithelium (FAE) which contains both Caco-2 and M cells, a T7 phage display library was screened for its ability either to bind the apical cell surface of or to undergo transcytosis across Caco-2 cells or FAE. The selection for transcytosis across both enterocytes and FAE identified three different peptide sequences (CTGKSC, PAVLG and LRVG) with high frequency. CTGKSC and LRVG sequences enhanced phage transport across M-like cells. When polymeric nanoparticles were grafted with the sequences CTGKSC and LRVG, their transport by FAE was significantly enhanced. These peptides could therefore be used to enhance the transport of vaccine-loaded nanoparticles across the intestinal mucosal barrier.

Transport mechanisms of mmePEG750P(CL-co-TMC) polymeric micelles across the intestinal barrier.

Monomethylether poly(ethyleneglycol)(750)-poly(caprolactone-co-trimethylene carbonate) (mmePEG750)P(CL-co-TMC)) which spontaneously form micelles, can cross lipid bilayers via passive diffusion and demonstrate an oral bioavailability of 40% in rats. The aim of the current work was to study the transport mechanism(s) of drug-loaded mmePEG750P(CL-co-TMC) micelles across the intestinal barrier. The transport of radiolabelled polymer across Caco-2 cell monolayer was investigated by disrupting tight junctions and by inhibiting endocytosis. The polymer and drugs loaded in micelles independently crossed Caco-2 cell monolayers and did not use either the paracellular route or M-cells. The polymer did not affect P-gp pumps. This mechanistic study suggests that whereas drug-loaded micelles were absorbed by fluid-phase endocytosis, polymeric unimers diffused passively across the membrane concomitantly with micellar endocytosis.