Harmonic Imaging of Stem Cells in Whole Blood at GHz Pixel Rate.

The pre-clinical validation of cell therapies requires monitoring the biodistribution of transplanted cells in tissues of host organisms. Real-time detection of these cells in the circulatory system and identification of their aggregation state is a crucial piece of information, but necessitates deep penetration and fast imaging with high selectivity, subcellular resolution, and high throughput. In this study, multiphoton-based in-flow detection of human stem cells in whole, unfiltered blood is demonstrated in a microfluidic channel. The approach relies on a multiphoton microscope with diffractive scanning in the direction perpendicular to the flow via a rapidly wavelength-swept laser. Stem cells are labeled with metal oxide harmonic nanoparticles. Thanks to their strong and quasi-instantaneous second harmonic generation (SHG), an imaging rate in excess of 10 000 frames per second is achieved with pixel dwell times of 1 ns, a duration shorter than typical fluorescence lifetimes yet compatible with SHG. Through automated cell identification and segmentation, morphological features of each individual detected event are extracted and cell aggregates are distinguished from isolated cells. This combination of high-speed multiphoton microscopy and high-sensitivity SHG nanoparticle labeling in turbid media promises the detection of rare cells in the bloodstream for assessing novel cell-based therapies.

Label-free 3D characterization of cardiac fibrosis in muscular dystrophy using SHG imaging of cleared tissue.

BACKGROUND INFORMATION: Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the gene encoding dystrophin. It leads to repeated cycles of muscle fiber necrosis and regeneration and progressive replacement of fibers by fibrotic and adipose tissue, with consequent muscle weakness and premature death. Fibrosis and, in particular, collagen accumulation are important pathological features of dystrophic muscle. A better understanding of the development of fibrosis is crucial to enable better management of DMD. Three-dimensional (3D) characterization of collagen organization by second harmonic generation (SHG) microscopy has already proven a highly informative means of studying the fibrotic network in tissue. RESULTS: Here, we combine for the first-time tissue clearing with SHG microscopy to characterize in depth the 3D cardiac fibrosis network from DMDmdx rat model. Heart sections (1-mm-thick) from 1-year-old wild-type (WT) and DMDmdx rats were cleared using the CUBIC protocol. SHG microscopy revealed significantly greater collagen deposition in DMDmdx versus WT sections. Analyses revealed a specific pattern of SHG+ segmented objects in DMDmdx cardiac muscle, characterized by a less elongated shape and increased density. Compared with the observed alignment of SHG+ collagen fibers in WT rats, profound fiber disorganization was observed in DMDmdx rats, in which we observed two distinct SHG+ collagen fiber profiles, which may reflect two distinct stages of the fibrotic process in DMD. CONCLUSION AND SIGNIFICANCE: The current work highlights the interest to combine multiphoton SHG microscopy and tissue clearing for 3D fibrosis network characterization in label free organ. It could be a relevant tool to characterize the fibrotic tissue remodeling in relation to the disease progression and/or to evaluate the efficacy of therapeutic strategies in preclinical studies in DMD model or others fibrosis-related cardiomyopathies diseases.

Telomere-to-telomere assembly of the genome of an individual Oikopleura dioica from Okinawa using Nanopore-based sequencing.

BACKGROUND: The larvacean Oikopleura dioica is an abundant tunicate plankton with the smallest (65-70 Mbp) non-parasitic, non-extremophile animal genome identified to date. Currently, there are two genomes available for the Bergen (OdB3) and Osaka (OSKA2016) O. dioica laboratory strains. Both assemblies have full genome coverage and high sequence accuracy. However, a chromosome-scale assembly has not yet been achieved. RESULTS: Here, we present a chromosome-scale genome assembly (OKI2018_I69) of the Okinawan O. dioica produced using long-read Nanopore and short-read Illumina sequencing data from a single male, combined with Hi-C chromosomal conformation capture data for scaffolding. The OKI2018_I69 assembly has a total length of 64.3 Mbp distributed among 19 scaffolds. 99% of the assembly is contained within five megabase-scale scaffolds. We found telomeres on both ends of the two largest scaffolds, which represent assemblies of two fully contiguous autosomal chromosomes. Each of the other three large scaffolds have telomeres at one end only and we propose that they correspond to sex chromosomes split into a pseudo-autosomal region and X-specific or Y-specific regions. Indeed, these five scaffolds mostly correspond to equivalent linkage groups in OdB3, suggesting overall agreement in chromosomal organization between the two populations. At a more detailed level, the OKI2018_I69 assembly possesses similar genomic features in gene content and repetitive elements reported for OdB3. The Hi-C map suggests few reciprocal interactions between chromosome arms. At the sequence level, multiple genomic features such as GC content and repetitive elements are distributed differently along the short and long arms of the same chromosome. CONCLUSIONS: We show that a hybrid approach of integrating multiple sequencing technologies with chromosome conformation information results in an accurate de novo chromosome-scale assembly of O. dioica's highly polymorphic genome. This genome assembly opens up the possibility of cross-genome comparison between O. dioica populations, as well as of studies of chromosomal evolution in this lineage.

Human muscle stem cell responses to mechanical stress into tunable 3D alginate matrices.

Preclinical data acquired for human muscle stem (hMuStem) cells indicate their great repair capacity in the context of muscle injury. However, their clinical potential is limited by their moderate ability to survive after transplantation. To overcome these limitations, their encapsulation within protective environment would be beneficial. In this study, tunable calcium-alginate hydrogels obtained through molding method using external or internal gelation were investigated as a new strategy for hMuStem cell encapsulation. The mechanical properties of these hydrogels were characterized in their fully hydrated state by compression experiments using Atomic Force Microscopy. Measured elastic moduli strongly depended on the gelation mode and calcium/alginate concentrations. Values ranged from 1 to 12.5 kPa and 3.9 to 25 kPa were obtained for hydrogels prepared following internal and external gelation, respectively. Also, differences in mechanical properties of hydrogels resulted from their internal organization, with an isotropic structure for internal gelation, while external mode led to anisotropic one. It was further shown that viability, morphological and myogenic differentiation characteristics of hMuStem cells incorporated within alginate hydrogels were preserved after their release. These results highlight that hMuStem cells encapsulated in calcium-alginate hydrogels maintain their functionality, thus allowing to develop muscle regeneration protocols to improve their therapeutic efficacy.

Human MuStem cells are competent to fuse with nonhuman primate myofibers in a clinically relevant transplantation context: A proof-of-concept study.

We previously reported that human muscle-derived stem cells (hMuStem cells) contribute to tissue repair after local administration into injured skeletal muscle or infarcted heart in immunodeficient rodent models. However, extrapolation of these findings to a clinical context is problematic owing to the considerable differences often seen between in vivo findings in humans versus rodents. Therefore, we investigated whether the muscle regenerative behavior of hMuStem cells is maintained in a clinically relevant transplantation context. Human MuStem cells were intramuscularly administered by high-density microinjection matrices into nonhuman primates receiving tacrolimus-based immunosuppression thereby reproducing the protocol that has so far produced the best results in clinical trials of cell therapy in myopathies. Four and 9 weeks after administration, histological analysis of cell injection sites revealed large numbers of hMuStem cell-derived nuclei in all cases. Most graft-derived nuclei were distributed in small myofiber groups in which no signs of a specific immune response were observed. Importantly, hMuStem cells contributed to simian tissue repair by fusing mainly with host myofibers, demonstrating their capacity for myofiber regeneration in this model. Together, these findings obtained in a valid preclinical model provide new insights supporting the potential of hMuStem cells in future cell therapies for muscle diseases.

The Salmonella virulence protein PagN contributes to the advent of a hyper-replicating cytosolic bacterial population.

Salmonella enterica subspecies enterica serovar Typhimurium is an intracellular pathogen that invades and colonizes the intestinal epithelium. Following bacterial invasion, Salmonella is enclosed within a membrane-bound vacuole known as a Salmonella-containing vacuole (SCV). However, a subset of Salmonella has the capability to prematurely rupture the SCV and escape, resulting in Salmonella hyper-replication within the cytosol of epithelial cells. A recently published RNA-seq study provides an overview of cytosolic and vacuolar upregulated genes and highlights pagN vacuolar upregulation. Here, using transcription kinetics, protein production profile, and immunofluorescence microscopy, we showed that PagN is exclusively produced by Salmonella in SCV. Gentamicin protection and chloroquine resistance assays were performed to demonstrate that deletion of pagN affects Salmonella replication by affecting the cytosolic bacterial population. This study presents the first example of a Salmonella virulence factor expressed within the endocytic compartment, which has a significant impact on the dynamics of Salmonella cytosolic hyper-replication.

Dual neutrophil subsets exacerbate or suppress inflammation in tuberculosis via IL-1ß or PD-L1.

Neutrophils can be beneficial or deleterious during tuberculosis (TB). Based on the expression of MHC-II and programmed death ligand 1 (PD-L1), we distinguished two functionally and transcriptionally distinct neutrophil subsets in the lungs of mice infected with mycobacteria. Inflammatory [MHC-II-, PD-L1lo] neutrophils produced inflammasome-dependent IL-1ß in the lungs in response to virulent mycobacteria and "accelerated" deleterious inflammation, which was highly exacerbated in IFN-γR-/- mice. Regulatory [MHC-II+, PD-L1hi] neutrophils "brake" inflammation by suppressing T-cell proliferation and IFN-γ production. Such beneficial regulation, which depends on PD-L1, is controlled by IFN-γR signaling in neutrophils. The hypervirulent HN878 strain from the Beijing genotype curbed PD-L1 expression by regulatory neutrophils, abolishing the braking function and driving deleterious hyperinflammation in the lungs. These findings add a layer of complexity to the roles played by neutrophils in TB and may explain the reactivation of this disease observed in cancer patients treated with anti-PD-L1.

Specific and label-free endogenous signature of dystrophic muscle by Synchrotron deep ultraviolet radiation.

Dystrophic muscle is characterized by necrosis/regeneration cycles, inflammation, and fibro-adipogenic development. Conventional histological stainings provide essential topographical data of this remodeling but may be limited to discriminate closely related pathophysiological contexts. They fail to mention microarchitecture changes linked to the nature and spatial distribution of tissue compartment components. We investigated whether label-free tissue autofluorescence revealed by Synchrotron deep ultraviolet (DUV) radiation could serve as an additional tool for monitoring dystrophic muscle remodeling. Using widefield microscopy with specific emission fluorescence filters and microspectroscopy defined by high spectral resolution, we analyzed samples from healthy dogs and two groups of dystrophic dogs: naïve (severely affected) and MuStem cell-transplanted (clinically stabilized) animals. Multivariate statistical analysis and machine learning approaches demonstrated that autofluorescence emitted at 420-480 nm by the Biceps femoris muscle effectively discriminates between healthy, dystrophic, and transplanted dog samples. Microspectroscopy showed that dystrophic dog muscle displays higher and lower autofluorescence due to collagen cross-linking and NADH respectively than that of healthy and transplanted dogs, defining biomarkers to evaluate the impact of cell transplantation. Our findings demonstrate that DUV radiation is a sensitive, label-free method to assess the histopathological status of dystrophic muscle using small amounts of tissue, with potential applications in regenerative medicine.

Confocal spectral microscopy, a non-destructive approach to follow contamination and biofilm formation of mCherry Staphylococcus aureus on solid surfaces.

Methods to test the safety of wood material for hygienically sensitive places are indirect, destructive and limited to incomplete microbial recovery via swabbing, brushing and elution-based techniques. Therefore, we chose mCherry Staphylococcus aureus as a model bacterium for solid and porous surface contamination. Confocal spectral laser microscope (CSLM) was employed to characterize and use the autofluorescence of Sessile oak (Quercus petraea), Douglas fir (Pseudotsuga menziesii) and poplar (Populus euramericana alba L.) wood discs cut into transversal (RT) and tangential (LT) planes. The red fluorescent area occupied by bacteria was differentiated from that of wood, which represented the bacterial quantification, survival and bio-distribution on surfaces from one hour to one week after inoculation. More bacteria were present near the surface on LT face wood as compared to RT and they persisted throughout the study period. Furthermore, this innovative methodology identified that S. aureus formed a dense biofilm on melamine but not on oak wood in similar inoculation and growth conditions. Conclusively, the endogenous fluorescence of materials and the model bacterium permitted direct quantification of surface contamination by using CSLM and it is a promising tool for hygienic safety evaluation.

Widespread use of the "ascidian" mitochondrial genetic code in tunicates.

Background: Ascidians, a tunicate class, use a mitochondrial genetic code that is distinct from vertebrates and other invertebrates. Though it has been used to translate the coding sequences from other tunicate species on a case-by-case basis, it is has not been investigated whether this can be done systematically. This is an important because a) some tunicate mitochondrial sequences are currently translated with the invertebrate code by repositories such as NCBI GenBank, and b) uncertainties about the genetic code to use can complicate or introduce errors in phylogenetic studies based on translated mitochondrial protein sequences. Methods: We collected publicly available nucleotide sequences for non-ascidian tunicates including appendicularians such as Oikopleura dioica, translated them using the ascidian mitochondrial code, and built multiple sequence alignments covering all tunicate classes. Results: All tunicates studied here appear to translate AGR codons to glycine instead of serine (invertebrates) or as a stop codon (vertebrates), as initially described in ascidians. Among Oikopleuridae, we suggest further possible changes in the use of the ATA (Ile → Met) and TGA (Trp → Arg) codons. Conclusions: We recommend using the ascidian mitochondrial code in automatic translation pipelines of mitochondrial sequences for all tunicates. Further investigation is required for additional species-specific differences.

Satellite cells fail to contribute to muscle repair but are functional in Pompe disease (glycogenosis type II).

Pompe disease, which is due to acid alpha-glucosidase deficiency, is characterized by skeletal muscle dysfunction attributed to the accumulation of glycogen-filled lysosomes and autophagic buildup. Despite the extensive tissue damages, a failure of satellite cell (SC) activation and lack of muscle regeneration have been reported in patients. However, the origin of this defective program is unknown. Additionally, whether these deficits occur gradually over the disease course is unclear. Using a longitudinal pathophysiological study of two muscles in a Pompe mouse model, here, we report that the enzymatic defect results in a premature saturating glycogen overload and a high number of enlarged lysosomes. The muscles gradually display profound remodeling as the number of autophagic vesicles, centronucleated fibers, and split fibers increases and larger fibers are lost. Only a few regenerated fibers were observed regardless of age, although the SC pool was preserved. Except for the early age, during which higher numbers of activated SCs and myoblasts were observed, no myogenic commitment was observed in response to the damage. Following in vivo injury, we established that muscle retains regenerative potential, demonstrating that the failure of SC participation in repair is related to an activation signal defect. Altogether, our findings provide new insight into the pathophysiology of Pompe disease and highlight that the activation signal defect of SCs compromises muscle repair, which could be related to the abnormal energetic supply following autophagic flux impairment.