Impact of Anti-Mycotic Drugs on the Osteogenic Response of Bone Marrow Mesenchymal Stem Cells In Vitro.

The treatment of fungal bone infections and infected non-unions is a huge challenge in modern trauma and orthopedics, which normally contain the local and systemic administration of anti-fungal drugs. Although frequently used, little is known about the impact of systemic and locally administered fungicides on the osteogenic regenerative capabilities of infected bone tissue, especially upon the osteogenesis of human bone marrow mesenchymal stem cells (BM-hMSCs). This study evaluates the effects of the three most common fungicides for the systemic treatment of bone infections, Voriconazole (VOR), liposomal Amphotericin B (LAMB), and Fluconazole (FLU), as well as the effects of VOR and LAMB-loaded Polymethylmethacrylate (PMMA) cement chips in different concentrations upon the osteogenic response of BM-hMSCs in vitro. Within this study, we compared the ability of BM-hMSC to differentiate into osteoblast-like cells and synthesize hydroxyapatite as assessed by radioactive 99mTechnetium-Hydroxydiphosphonate (99mTc-HDP) labeling, cell proliferation, and analyses of supernatants upon various osteogenic parameters. Our results revealed that VOR added to the cell culture medium affects the osteogenic potential of BM-hMSC negatively, while there were no detectable effects of LAMB and FLU. Moreover, we showed dose-dependent negative effects of high- and extended-dose fungicide-loaded PMMA cement due to cytotoxicity, with a higher cytotoxic potential of VOR than LAMB, while low-dose fungicide-loaded PMMA had no significant effect on the osteogenic potential of BM-hMSC in vitro.

Impact of Antibiotic-Loaded PMMA Spacers on the Osteogenic Potential of hMSCs.

Antibiotic-loaded PMMA bone cement is frequently used in modern trauma and orthopedic surgery. Although many of the antibiotics routinely applied are described to have cytotoxic effects in the literature, clinical experience shows no adverse effects for bone healing. To determine the effects of antibiotic-loaded PMMA spacers on osteogenesis in vitro, we cultivated human bone marrow mesenchymal stem cells (BM-hMSCs) in the presence of PMMA spacers containing Gentamicin, Vancomycin, Gentamicin + Clindamycin as well as Gentamicin + Vancomycin in addition to a blank control (agarose) and PMMA containing no antibiotics. The cell number was assessed with DAPI staining, and the osteogenic potential was evaluated by directly measuring the amount of hydroxyapatite synthesized using radioactive 99mTc-HDP labelling as well as measuring the concentration of calcium and phosphate in the cell culture medium supernatant. The results showed that Gentamicin and Vancomycin as well as their combination show a certain amount of cytotoxicity but no negative effect on osteogenic potential. The combination of Gentamicin and Clindamycin, on the other hand, led to a drastic reduction in both the cell count and the osteogenic potential.

Fear circuit-based neurobehavioral signatures mirror resilience to chronic social stress in mouse.

Consistent evidence from human data points to successful threat-safety discrimination and responsiveness to extinction of fear memories as key characteristics of resilient individuals. To promote valid cross-species approaches for the identification of resilience mechanisms, we establish a translationally informed mouse model enabling the stratification of mice into three phenotypic subgroups following chronic social defeat stress, based on their individual ability for threat-safety discrimination and conditioned learning: the Discriminating-avoiders, characterized by successful social threat-safety discrimination and extinction of social aversive memories; the Indiscriminate-avoiders, showing aversive response generalization and resistance to extinction, in line with findings on susceptible individuals; and the Non-avoiders displaying impaired aversive conditioned learning. To explore the neurobiological mechanisms underlying the stratification, we perform transcriptome analysis within three key target regions of the fear circuitry. We identify subgroup-specific differentially expressed genes and gene networks underlying the behavioral phenotypes, i.e., the individual ability to show threat-safety discrimination and respond to extinction training. Our approach provides a translationally informed template with which to characterize the behavioral, molecular, and circuit bases of resilience in mice.

99mTc-HDP Labeling-A Non-Destructive Method for Real-Time Surveillance of the Osteogenic Differentiation Potential of hMSC during Ongoing Cell Cultures.

99-Metastabil Technetium (99mTc) is a radiopharmaceutical widely used in skeletal scintigraphy. Recent publications show it can also be used to determine the osteogenic potential of human mesenchymal stem cells (hMSCs) by binding to hydroxyapatite formed during bone tissue engineering. This field lacks non-destructive methods to track live osteogenic differentiation of hMSCs. However, no data about the uptake kinetics of 99mTc and its effect on osteogenesis of hMSCs have been published yet. We therefore evaluated the saturation time of 99mTc by incubating hMSC cultures for different periods, and the saturation concentration by using different amounts of 99mTc activity for incubation. The influence of 99mTc on osteogenic potential of hMSCs was then evaluated by labeling a continuous hMSC culture three times over the course of 3 weeks, and comparing the findings to cultures labeled once. Our findings show that 99mTc saturation time is less than 0.25 h, and saturation concentration is between 750 and 1000 MBq. Repeated exposure to γ-radiation emitted by 99mTc had no negative effects on hMSC cultures. These new insights can be used to make this highly promising method broadly available to support researchers in the field of bone tissue engineering using this method to track and evaluate, in real-time, the osteogenic differentiation of hMSC, without any negative influence on the cell viability, or their osteogenic differentiation potential.

Enabling technologies towards personalization of scaffolds for large bone defect regeneration.

Additive manufacturing (AM) can deliver personalized scaffolds to support large volume defect tissue regeneration - a major clinical challenge in many medical disciplines. The freedom in scaffold design and composition (biomaterials and biologics) offered by AM yields a plethora of possibilities but is confronted with a heterogenous biological regeneration potential across individuals. A key challenge is to make the right choice for individualized scaffolds that match biology, anatomy, and mechanics of patients. This review provides an overview of state-of-the-art technologies, that is, in silico modelling for scaffold design, omics and bioinformatics to capture patient biology and information technology for data management, that, when combined in a synergistic way with AM, have great potential to make personalized tissue regeneration strategies available to all patients, empowering precision medicine.

Early life adversity targets the transcriptional signature of hippocampal NG2+ glia and affects voltage gated sodium (Nav) channels properties.

The precise mechanisms underlying the detrimental effects of early life adversity (ELA) on adult mental health remain still elusive. To date, most studies have exclusively targeted neuronal populations and not considered neuron-glia crosstalk as a crucially important element for the integrity of stress-related brain function. Here, we have investigated the impact of ELA, in the form of a limited bedding and nesting material (LBN) paradigm, on a glial subpopulation with unique properties in brain homeostasis, the NG2+ cells. First, we have established a link between maternal behavior, activation of the offspring's stress response and heterogeneity in the outcome to LBN manipulation. We further showed that LBN targets the hippocampal NG2+ transcriptome with glucocorticoids being an important mediator of the LBN-induced molecular changes. LBN altered the NG2+ transcriptome and these transcriptional effects were correlated with glucocorticoids levels. The functional relevance of one LBN-induced candidate gene, Scn7a, could be confirmed by an increase in the density of voltage-gated sodium (Nav) channel activated currents in hippocampal NG2+ cells. Scn7a remained upregulated until adulthood in LBN animals, which displayed impaired cognitive performance. Considering that Nav channels are important for NG2+ cell-to-neuron communication, our findings provide novel insights into the disruption of this process in LBN mice.

DNA Methyltransferase 1 (DNMT1) Function Is Implicated in the Age-Related Loss of Cortical Interneurons.

Increased life expectancy in modern society comes at the cost of age-associated disabilities and diseases. Aged brains not only show reduced excitability and plasticity, but also a decline in inhibition. Age-associated defects in inhibitory circuits likely contribute to cognitive decline and age-related disorders. Molecular mechanisms that exert epigenetic control of gene expression contribute to age-associated neuronal impairments. Both DNA methylation, mediated by DNA methyltransferases (DNMTs), and histone modifications maintain neuronal function throughout lifespan. Here we provide evidence that DNMT1 function is implicated in the age-related loss of cortical inhibitory interneurons. Dnmt1 deletion in parvalbumin-positive interneurons attenuates their age-related decline in the cerebral cortex. Moreover, conditional Dnmt1-deficient mice show improved somatomotor performance and reduced aging-associated transcriptional changes. A decline in the proteostasis network, responsible for the proper degradation and removal of defective proteins, is implicated in age- and disease-related neurodegeneration. Our data suggest that DNMT1 acts indirectly on interneuron survival in aged mice by modulating the proteostasis network during life-time.

Motor neuron translatome reveals deregulation of SYNGR4 and PLEKHB1 in mutant TDP-43 amyotrophic lateral sclerosis models.

Amyotrophic lateral sclerosis (ALS) is an incurable neurological disease with progressive loss of motor neuron (MN) function in the brain and spinal cord. Mutations in TARDBP, encoding the RNA-binding protein TDP-43, are one cause of ALS, and TDP-43 mislocalization in MNs is a key pathological feature of >95% of ALS cases. While numerous studies support altered RNA regulation by TDP-43 as a major cause of disease, specific changes within MNs that trigger disease onset remain unclear. Here, we combined translating ribosome affinity purification (TRAP) with RNA sequencing to identify molecular changes in spinal MNs of TDP-43-driven ALS at motor symptom onset. By comparing the MN translatome of hTDP-43A315T mice to littermate controls and to mice expressing wild type hTDP-43, we identified hundreds of mRNAs that were selectively up- or downregulated in MNs. We validated the deregulated candidates Tex26, Syngr4, and Plekhb1 mRNAs in an independent TRAP experiment. Moreover, by quantitative immunostaining of spinal cord MNs, we found corresponding protein level changes for SYNGR4 and PLEKHB1. We also observed these changes in spinal MNs of an independent ALS mouse model caused by a different patient mutant allele of TDP-43, suggesting that they are general features of TDP-43-driven ALS. Thus, we identified SYNGR4 and PLEKHB1 to be deregulated in MNs at motor symptom onset in TDP-43-driven ALS models. This spatial and temporal pattern suggests that these proteins could be functionally important for driving the transition to the symptomatic phase of the disease.

DNA Methylation-Mediated Modulation of Endocytosis as Potential Mechanism for Synaptic Function Regulation in Murine Inhibitory Cortical Interneurons.

The balance of excitation and inhibition is essential for cortical information processing, relying on the tight orchestration of the underlying subcellular processes. Dynamic transcriptional control by DNA methylation, catalyzed by DNA methyltransferases (DNMTs), and DNA demethylation, achieved by ten-eleven translocation (TET)-dependent mechanisms, is proposed to regulate synaptic function in the adult brain with implications for learning and memory. However, focus so far is laid on excitatory neurons. Given the crucial role of inhibitory cortical interneurons in cortical information processing and in disease, deciphering the cellular and molecular mechanisms of GABAergic transmission is fundamental. The emerging relevance of DNMT and TET-mediated functions for synaptic regulation irrevocably raises the question for the targeted subcellular processes and mechanisms. In this study, we analyzed the role dynamic DNA methylation has in regulating cortical interneuron function. We found that DNMT1 and TET1/TET3 contrarily modulate clathrin-mediated endocytosis. Moreover, we provide evidence that DNMT1 influences synaptic vesicle replenishment and GABAergic transmission, presumably through the DNA methylation-dependent transcriptional control over endocytosis-related genes. The relevance of our findings is supported by human brain sample analysis, pointing to a potential implication of DNA methylation-dependent endocytosis regulation in the pathophysiology of temporal lobe epilepsy, a disease characterized by disturbed synaptic transmission.

The secreted BMP antagonist ERFE is required for the development of a functional circulatory system in Xenopus.

The hormone Erythroferrone (ERFE) is a member of the C1q/TNF-related protein family that regulates iron homeostasis through the suppression of hamp. In a gain of function screen in Xenopus embryos, we identified ERFE as a potent secondary axis-inducing agent. Experiments in Xenopus embryos and ectodermal explants revealed that ERFE functions as a selective inhibitor of the BMP pathway and the conserved C1q domain is not required for this activity. Inhibition occurs at the extracelluar level, through the interaction of ERFE with the BMP ligand. During early Xenopus embryogenesis, erfe is first expressed in the ventral blood islands where initial erythropoiesis occurs and later in circulating blood cells. ERFE knockdown does not alter the expression of etv.2, aplnr and flt1 in tailbud stage embryos indicating endothelial cell specification is independent of ERFE. However, in tadpole embryos, defects of the vascular network and primitive blood circulation are observed as well as edema formation. RNAseq analysis of ERFE morphant embryos also revealed the inhibition of gja4 indicating disruption of dorsal aorta formation.

Nuclear Pre-snRNA Export Is an Essential Quality Assurance Mechanism for Functional Spliceosomes.

Removal of introns from pre-mRNAs is an essential step in eukaryotic gene expression, mediated by spliceosomes that contain snRNAs as key components. Although snRNAs are transcribed in the nucleus and function in the same compartment, all except U6 shuttle to the cytoplasm. Surprisingly, the physiological relevance for shuttling is unclear, in particular because the snRNAs in Saccharomyces cerevisiae were reported to remain nuclear. Here, we show that all yeast pre-snRNAs including U6 undergo a stepwise maturation process after nuclear export by Mex67 and Xpo1. Sm- and Lsm-ring attachment occurs in the cytoplasm and is important for the snRNA re-import, mediated by Cse1 and Mtr10. Finally, nuclear pre-snRNA cleavage and trimethylation of the 5'-cap finalizes shuttling. Importantly, preventing pre-snRNAs from being exported or processed results in faulty spliceosome assembly and subsequent genome-wide splicing defects. Thus, pre-snRNA export is obligatory for functional splicing and resembles an essential evolutionarily conserved quality assurance step.

The IL-1 Pathway Is Hyperactive in Hidradenitis Suppurativa and Contributes to Skin Infiltration and Destruction.

Hidradenitis suppurativa (HS) (also designated acne inversa) is a chronic inflammatory disease characterized by painful purulent skin lesions and progressive destruction of skin architecture. Despite the high burden for the patients, pathogenetic pathways underlying HS alterations remain obscure. When we examined the HS cytokine pattern, IL-1ß turned out to be a highly prominent cytokine, overexpressed even compared with psoriatic lesions. Analyses of IL-1ß-induced transcriptome in various cell types showed overlapping profiles, with upregulations of molecules causing immune cell infiltration and extracellular matrix degradation, and of specific cytokines including IL-6, IL-32, and IL-36. Matching cellular IL-1 receptor levels, dermal fibroblasts showed both the strongest and broadest IL-1ß response, which was not clearly shared or strengthened by other cytokines. The IL-1ß signature was specifically present in HS lesions and could be reversed by application of IL-1 receptor antagonist. Search for blood parameters associated with IL-1ß pathway activity in HS identified serum amyloid A, which was synergistically induced by IL-1ß and IL-6 in hepatocytes. Consequently, strongly elevated blood serum amyloid A levels in HS correlated positively with the extent of inflammatory skin alterations. In summary, the IL-1ß pathway represents a pathogenetic cascade, whose activity may be therapeutically targeted and monitored by blood SAA levels.

Functional equivalence of germ plasm organizers.

The proteins Oskar (Osk) in Drosophila and Bucky ball (Buc) in zebrafish act as germ plasm organizers. Both proteins recapitulate germ plasm activities but seem to be unique to their animal groups. Here, we discover that Osk and Buc show similar activities during germ cell specification. Drosophila Osk induces additional PGCs in zebrafish. Surprisingly, Osk and Buc do not show homologous protein motifs that would explain their related function. Nonetheless, we detect that both proteins contain stretches of intrinsically disordered regions (IDRs), which seem to be involved in protein aggregation. IDRs are known to rapidly change their sequence during evolution, which might obscure biochemical interaction motifs. Indeed, we show that Buc binds to the known Oskar interactors Vasa protein and nanos mRNA indicating conserved biochemical activities. These data provide a molecular framework for two proteins with unrelated sequence but with equivalent function to assemble a conserved core-complex nucleating germ plasm.

Immortalization of common marmoset monkey fibroblasts by piggyBac transposition of hTERT.

Following a certain type-specific number of mitotic divisions, terminally differentiated cells undergo proliferative senescence, thwarting efforts to expand different cell populations in vitro for the needs of scientific research or medical therapies. The primary cause of this phenomenon is the progressive shortening of the telomeres and the subsequent activation of cell cycle control pathways leading to a block of cell proliferation. Restoration of telomere length by transgenic expression of telomerase reverse transcriptase (TERT) usually results in bypassing of the replicative senescence and ultimately in cell immortalization. To date, there have not been any reports regarding immortalization of cells from common marmoset (Callithrix jacchus), an important non-human primate model for various human diseases, with the use of exogenous human TERT (hTERT). In this study, marmoset fibroblasts were successfully immortalized with transposon-integrated transgenic hTERT and expanded in vitro for over 500 population doublings. Calculation of population doubling levels (PDL) showed that the derived hTERT-transgenic lines had significantly higher proliferation potential than the wild-type fibroblasts, which reached only a maximum of 46 doublings. However, the immortalized cells exhibited differences in the morphology compared with the control fibroblasts and transcriptome analysis also revealed changes in the gene expression patterns. Finally, the karyotypes of all hTERT-transgenic cell lines showed various aberrations such as presence of extra Chromosome 17, isochromosome 21q, or tetraploidy. By single-cell expansion of the least affected monoclonal immortalized line, one sub-clonal line with normal karyotype was established, suggesting the possibility to derive immortal marmoset cells with normal karyotypes. The results of this study are an important step towards the development and optimization of methods for the production of immortalized cells from common marmoset monkeys.

Neurons under T Cell Attack Coordinate Phagocyte-Mediated Synaptic Stripping.

Inflammatory disorders of the CNS are frequently accompanied by synaptic loss, which is thought to involve phagocytic microglia and complement components. However, the mechanisms accounting for aberrant synaptic connectivity in the context of CD8+ T cell-driven neuronal damage are poorly understood. Here, we profiled the neuronal translatome in a murine model of encephalitis caused by CD8+ T cells targeting antigenic neurons. Neuronal STAT1 signaling and downstream CCL2 expression were essential for apposition of phagocytes, ensuing synaptic loss and neurological disease. Analogous observations were made in the brains of Rasmussen's encephalitis patients. In this devastating CD8+ T cell-driven autoimmune disease, neuronal STAT1 phosphorylation and CCL2 expression co-clustered with infiltrating CD8+ T cells as well as phagocytes. Taken together, our findings uncover an active role of neurons in coordinating phagocyte-mediated synaptic loss and highlight neuronal STAT1 and CCL2 as critical steps in this process that are amenable to pharmacological interventions.

DMRT5, DMRT3, and EMX2 Cooperatively Repress Gsx2 at the Pallium-Subpallium Boundary to Maintain Cortical Identity in Dorsal Telencephalic Progenitors.

Specification of dorsoventral regional identity in progenitors of the developing telencephalon is a first pivotal step in the development of the cerebral cortex and basal ganglia. Previously, we demonstrated that the two zinc finger doublesex and mab-3 related (Dmrt) genes, Dmrt5 (Dmrta2) and Dmrt3, which are coexpressed in high caudomedial to low rostrolateral gradients in the cerebral cortical primordium, are separately needed for normal formation of the cortical hem, hippocampus, and caudomedial neocortex. We have now addressed the role of Dmrt3 and Dmrt5 in controlling dorsoventral division of the telencephalon in mice of either sex by comparing the phenotypes of single knock-out (KO) with double KO embryos and by misexpressing Dmrt5 in the ventral telencephalon. We find that DMRT3 and DMRT5 act as critical regulators of progenitor cell dorsoventral identity by repressing ventralizing regulators. Early ventral fate transcriptional regulators expressed in the dorsal lateral ganglionic eminence, such as Gsx2, are upregulated in the dorsal telencephalon of Dmrt3;Dmrt5 double KO embryos and downregulated when ventral telencephalic progenitors express ectopic Dmrt5 Conditional overexpression of Dmrt5 throughout the telencephalon produces gene expression and structural defects that are highly consistent with reduced GSX2 activity. Further, Emx2;Dmrt5 double KO embryos show a phenotype similar to Dmrt3;Dmrt5 double KO embryos, and both DMRT3, DMRT5 and the homeobox transcription factor EMX2 bind to a ventral telencephalon-specific enhancer in the Gsx2 locus. Together, our findings uncover cooperative functions of DMRT3, DMRT5, and EMX2 in dividing dorsal from ventral in the telencephalon.SIGNIFICANCE STATEMENT We identified the DMRT3 and DMRT5 zinc finger transcription factors as novel regulators of dorsoventral patterning in the telencephalon. Our data indicate that they have overlapping functions and compensate for one another. The double, but not the single, knock-out produces a dorsal telencephalon that is ventralized, and olfactory bulb tissue takes over most remaining cortex. Conversely, overexpressing Dmrt5 throughout the telencephalon causes expanded expression of dorsal gene determinants and smaller olfactory bulbs. Furthermore, we show that the homeobox transcription factor EMX2 that is coexpressed with DMRT3 and DMRT5 in cortical progenitors cooperates with them to maintain dorsoventral patterning in the telencephalon. Our study suggests that DMRT3/5 function with EMX2 in positioning the pallial-subpallial boundary by antagonizing the ventral homeobox transcription factor GSX2.

Retinoic acid-induced expression of Hnf1b and Fzd4 is required for pancreas development in Xenopus laevis.

Retinoic acid (RA) is required for pancreas specification in Xenopus and other vertebrates. However, the gene network that is directly induced by RA signalling in this context remains to be defined. By RNA sequencing of in vitro-generated pancreatic explants, we identified the genes encoding the transcription factor Hnf1ß and the Wnt-receptor Fzd4/Fzd4s as direct RA target genes. Functional analyses of Hnf1b and Fzd4/Fzd4s in programmed pancreatic explants and whole embryos revealed their requirement for pancreatic progenitor formation and differentiation. Thus, Hnf1ß and Fzd4/Fzd4s appear to be involved in pre-patterning events of the embryonic endoderm that allow pancreas formation in Xenopus.

Expression of the DNA-Binding Factor TOX Promotes the Encephalitogenic Potential of Microbe-Induced Autoreactive CD8+ T Cells.

Infections are thought to trigger CD8+ cytotoxic T lymphocyte (CTL) responses during autoimmunity. However, the transcriptional programs governing the tissue-destructive potential of CTLs remain poorly defined. In a model of central nervous system (CNS) inflammation, we found that infection with lymphocytic choriomeningitis virus (LCMV), but not Listeria monocytogenes (Lm), drove autoimmunity. The DNA-binding factor TOX was induced in CTLs during LCMV infection and was essential for their encephalitogenic properties, and its expression was inhibited by interleukin-12 during Lm infection. TOX repressed the activity of several transcription factors (including Id2, TCF-1, and Notch) that are known to drive CTL differentiation. TOX also reduced immune checkpoint sensitivity by restraining the expression of the inhibitory checkpoint receptor CD244 on the surface of CTLs, leading to increased CTL-mediated damage in the CNS. Our results identify TOX as a transcriptional regulator of tissue-destructive CTLs in autoimmunity, offering a potential mechanistic link to microbial triggers.

The DNA Methyltransferase 1 (DNMT1) Controls the Shape and Dynamics of Migrating POA-Derived Interneurons Fated for the Murine Cerebral Cortex.

The proliferative niches in the subpallium generate a rich cellular variety fated for diverse telencephalic regions. The embryonic preoptic area (POA) represents one of these domains giving rise to the pool of cortical GABAergic interneurons and glial cells, in addition to striatal and residual POA cells. The migration from sites of origin within the subpallium to the distant targets like the cerebral cortex, accomplished by the adoption and maintenance of a particular migratory morphology, is a critical step during interneuron development. To identify factors orchestrating this process, we performed single-cell transcriptome analysis and detected Dnmt1 expression in murine migratory GABAergic POA-derived cells. Deletion of Dnmt1 in postmitotic immature cells of the POA caused defective migration and severely diminished adult cortical interneuron numbers. We found that DNA methyltransferase 1 (DNMT1) preserves the migratory shape in part through negative regulation of Pak6, which stimulates neuritogenesis at postmigratory stages. Our data underline the importance of DNMT1 for the migration of POA-derived cells including cortical interneurons.