Programmed genomic instability regulates neural transdifferentiation of human brain microvascular pericytes.

BACKGROUND: Transdifferentiation describes transformation in vivo of specialized cells from one lineage into another. While there is extensive literature on forced induction of lineage reprogramming in vitro, endogenous mechanisms that govern transdifferentiation remain largely unknown. The observation that human microvascular pericytes transdifferentiate into neurons provided an opportunity to explore the endogenous molecular basis for lineage reprogramming. RESULTS: We show that abrupt destabilization of the higher-order chromatin topology that chaperones lineage memory of pericytes is driven by transient global transcriptional arrest. This leads within minutes to localized decompression of the repressed competing higher-order chromatin topology and expression of pro-neural genes. Transition to neural lineage is completed by probabilistic induction of R-loops in key myogenic loci upon re-initiation of RNA polymerase activity, leading to depletion of the myogenic transcriptome and emergence of the neurogenic transcriptome. CONCLUSIONS: These findings suggest that the global transcriptional landscape not only shapes the functional cellular identity of pericytes, but also stabilizes lineage memory by silencing the competing neural program within a repressed chromatin state.

Cannibalized erythroblasts accelerate developmental neurogenesis by regulating mitochondrial dynamics.

Metabolic support was long considered to be the only developmental function of hematopoiesis, a view that is gradually changing. Here, we disclose a mechanism triggered during neurulation that programs brain development by donation of sacrificial yolk sac erythroblasts to neuroepithelial cells. At embryonic day (E) 8.5, neuroepithelial cells transiently integrate with the endothelium of yolk sac blood vessels and cannibalize intravascular erythroblasts as transient heme-rich endosymbionts. This cannibalistic behavior instructs precocious neuronal differentiation of neuroepithelial cells in the proximity of blood vessels. By experiments in vitro, we show that access to erythroblastic heme accelerates the pace of neurogenesis by induction of a truncated neurogenic differentiation program from a poised state. Mechanistically, the poised state is invoked by activation of the mitochondrial electron transport chain that leads to amplified production of reactive oxygen species in addition to omnipresent guanosine triphosphate (GTP) with consequential upregulation of pro-differentiation ß-catenin.

Bi-modal reprogramming of cell cycle by MiRNA-4673 amplifies human neurogenic capacity.

Molecular mechanisms that inform heterochronic adaptations of neurogenesis in Homo sapiens remain largely unknown. Here, we uncover a signature in the cell cycle that amplifies the proliferative capacity of human neural progenitors by input from microRNA4673 encoded in Notch-1. The miRNA instructs bimodal reprogramming of the cell cycle, leading to initial synchronization of neural precursors at the G0 phase of the cell cycle followed by accelerated progression through interphase. The key event in G0 synchronization is transient inhibition by miR4673 of cyclin-dependent kinase-18, a member of an ancient family of cyclins that license M-G1 transition. In parallel, autophagic degradation of p53/p21 and transcriptional silencing of XRCC3/BRCA2 relax G1/S cell cycle checkpoint and accelerate interphase by ≈2.8-fold. The resultant reprogrammed cell cycle amplifies the proliferative capacity and delays the differentiation of human neural progenitors.

Neural microvascular pericytes contribute to human adult neurogenesis.

Consistent adult neurogenic activity in humans is observed in specific niches within the central nervous system. However, the notion of an adult neurogenic niche is challenged by accumulating evidence for ectopic neurogenic activity in other cerebral locations. Herein we interface precision of ultrastructural resolution and anatomical simplicity of accessible human dental pulp neurogenic zone to address this conflict. We disclose a basal level of adult neurogenic activity characterized by glial invasion of terminal microvasculature followed by release of individual platelet-derived growth factor receptor-ß mural pericytes and subsequent reprogramming into NeuN+ local interneurons. Concomitant angiogenesis, a signature of adult neurogenic niches, accelerates the rate of neurogenesis by amplifying release and proliferation of the mural pericyte population by ≈10-fold. Subsequent in vitro and in vivo experiments confirmed gliogenic and neurogenic capacities of human neural pericytes. Findings foreshadow the bimodal nature of the glio-vascular assembly where pericytes, under instruction from glial cells, can stabilize the quiescent microvasculature or enrich local neuronal microcircuits upon differentiation.

miR4673 improves fitness profile of neoplastic cells by induction of autophagy.

Therapeutic resistance of neoplasms is mainly attributed to gradual evolution of mutational profile1. Here, we demonstrate a microRNA-mediated mechanism that effectively improves fitness of SKBR3 mammary carcinoma cells by cytoplasmic reprogramming. The reprogramming is triggered by endogenous miR4673 transcribed from notch-1 locus. The miRNA downregulates cdk-18, a cyclin-dependent kinase that regulates M-G1 transition in cycling cells2,3. Suppression of cdk-18 triggers mitophagy and autophagy. Due to high autophagic flux, oestrogen receptor-1+/progesterone receptor+/p53+ (Esr1+/Pr+/p53+) SKBR3 cells are coerced into an Esr1-/Prlow/p53-profile. Increased mitophagy in combination with proteasomal degradation of p53 transiently arrests the cycling cells at G0 and enhances radio-resistance of the SKBR3 population. These findings highlight the impact on cancer therapy of non-encoded neoplastic resistance, arising as a consequence of miRNA-mediated autophagic reprogramming that uncouples phenotype and genotype.

Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis.

Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two ß-propeller domains and a C-terminal ß-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity.

Bacterial profile of dentine caries and the impact of pH on bacterial population diversity.

Dental caries is caused by the release of organic acids from fermentative bacteria, which results in the dissolution of hydroxyapatite matrices of enamel and dentine. While low environmental pH is proposed to cause a shift in the consortium of oral bacteria, favouring the development of caries, the impact of this variable has been overlooked in microbial population studies. This study aimed to detail the zonal composition of the microbiota associated with carious dentine lesions with reference to pH. We used 454 sequencing of the 16S rRNA gene (V3-V4 region) to compare microbial communities in layers ranging in pH from 4.5-7.8 from 25 teeth with advanced dentine caries. Pyrosequencing of the amplicons yielded 449,762 sequences. Nine phyla, 97 genera and 409 species were identified from the quality-filtered, de-noised and chimera-free sequences. Among the microbiota associated with dentinal caries, the most abundant taxa included Lactobacillus sp., Prevotella sp., Atopobium sp., Olsenella sp. and Actinomyces sp. We found a disparity between microbial communities localised at acidic versus neutral pH strata. Acidic conditions were associated with low diversity microbial populations, with Lactobacillus species including L. fermentum, L. rhamnosus and L. crispatus, being prominent. In comparison, the distinctive species of a more diverse flora associated with neutral pH regions of carious lesions included Alloprevotella tanerrae, Leptothrix sp., Sphingomonas sp. and Streptococcus anginosus. While certain bacteria were affected by the pH gradient, we also found that ∼ 60% of the taxa associated with caries were present across the investigated pH range, representing a substantial core. We demonstrated that some bacterial species implicated in caries progression show selective clustering with respect to pH gradient, providing a basis for specific therapeutic strategies.

pH gradient and distribution of streptococci, lactobacilli, prevotellae, and fusobacteria in carious dentine.

OBJECTIVES: Caries process comprises acidogenic and aciduric bacteria that are responsible for lowering the pH and subsequent destruction of hydroxyapatite matrix in enamel and dentine. The aim of this study was to identify the correlation between the pH gradient of a carious lesion and proportion and distribution of four bacterial genera; lactobacilli, streptococci, prevotellae, and fusobacteria with regard to total load of bacteria. MATERIALS AND METHODS: A total of 25 teeth with extensive dentinal caries were sampled in sequential layers. Using quantitative real-time PCR of 16S rRNA gene, we quantified the total load of bacteria as well as the proportion of the above-mentioned genera following pH measurement of each sample with a fine microelectrode. RESULTS: We demonstrated the presence of a pH gradient across the lesion with a strong association between the quantity of lactobacilli and the lowest pH range (pH 4.5-5.0; p = 0.003). Streptococci had a tendency to occupy the most superficial aspect of the carious lesion but showed no correlation to any pH value. Prevotellae showed clear preference for the pH range 5.5-6.0 (p = 0.042). The total representation of these four genera did not reach more than one quarter of the total bacterial load in most carious samples. CONCLUSION: We revealed differential colonization behavior of bacteria with respect to pH gradient and a lower than expected abundance of lactobacilli and streptococci in established carious lesions. The data indicate the numerical importance of relatively unexplored taxa within the lesion of dentinal caries. CLINICAL RELEVANCE: The gradient nature of pH in the lesion as well as colonization difference of examined bacterial taxa with reference to pH provides a new insight in regard to conservative caries management.

Structural analysis of reactionary dentin formed in response to polymicrobial invasion.

In response to microbial invasion of dentin odontoblasts secrete an altered calcified matrix termed reactionary dentin (Rd). 3D reconstruction of focused-ion-beam scanning electron microscopy (FIB-SEM) image slices revealed helical tubular structures in Rd that contrasted with regular cylindrical tubules characteristic of dentin from healthy teeth and affected so-called physiological dentin (Pd) lying exterior to Rd. This helical structure in Rd provided effective constriction of tubule lumen diameter that formed a barrier to bacterial advance towards the dental pulp. SEM of resin cast preparations revealed altered extension of odontoblast processes through Rd. The distribution of key mineral elements was studied by combination of 3D reconstruction of focused-ion-beam based X-ray microanalysis (FIB-EDS), laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). There was a marked redistribution of calcium and phosphorous in Rd together with an increase of diffusely deposited magnesium compatible with the mineral deposition phase of synthesis of this altered matrix. Changes in tubule structure and mineral content characteristic of Rd are consistent with reduced hardness and lower elastic modulus reported for this matrix. Findings provide insight into the unique structure of Rd synthesised as a primary response to infection.

Directed glia-assisted angiogenesis in a mature neurosensory structure: pericytes mediate an adaptive response in human dental pulp that maintains blood-barrier function.

The specialized tightly controlled microcirculation of craniofacial neurosensory organs is an essential evolutionary adaptation and yet a dilemma where angiogenic remodeling occurs. Despite extreme plasticity of neurosensory structures, the capacity to reconcile barrier phenotype of the microcirculation with an angiogenic cascade is not known. Here we provide primary evidence for such a response in an elemental neurosensory structure, human dental pulp, following chronic carious insult. In response to hypoxic challenge neurosensory odontoblasts express hypoxia-inducible factor-1α and notch-1. Associated radial rearrangement of astrocyte-like telacytes that communicate through a cell-poor zone with the microvasculature is observed. Activated pericytes characterized by expression of α-smooth muscle actin are located adjacent to the telacyte attachment to the vasculature. In this location, endothelial expression of sonic hedgehog parallels expression of notch-1 by pericytes. The angiogenic response is initiated by pericyte contraction and altered endothelial polarity and proliferation leading to intussusception of endothelial cells and extensive remodeling of basement membrane with upregulation of laminin-8 and laminin-5. These responses guide intravascular loop formation that maintains both intact basement membrane and tight junctions. This initial phase is followed by formation of anastomoses that enhance the hemodynamic capacity of the intravascular loops. The formation of anastomoses is mediated by extension of cytonemes from pericytes guided by MHC-II(+)/CD-163(+) microglia aligned with the telacytes. The cytonemes seek out pericytes on adjacent intravascular loops to initiate migration of endothelial cells. These findings support a new paradigm for understanding angiogenic capacity of neurosensory structures and aberrations of this response manifest as neurovasculopathies.

Streptococcus gordonii FSS2 Challisin affects fibrin clot formation by digestion of the αC region and cleavage of the N -terminal region of the Bß chains of fibrinogen.

Bacteria within endocarditis vegetations are encased in fibrin matrix that is resistant to resolution. We have previously shown that FSS2 Challisin, a serine protease from Streptococcus gordonii, is able to hydrolyse the Aα and Bß chains of fibrinogen and has potent angiotensin converting enzyme (ACE) activity. The alteration in the structure of fibrin formed from FSS2 Challisin-degraded fibrinogen may therefore contribute to the resistant fibrin matrix. To this end, we have investigated the specific interactions of FSS2 Challisin with fibrinogen. FSS2 Challisin extensively degrades the αC region of fibrinogen Aα chains, hydrolysing both the αC-domain and αC-connnector. Additionally, the N-terminal region of the Bß chains is cleaved twice, at Leu19 and Ser28, removing the B fibrinopeptides and 'B' knobs. Substrate analysis indicates FSS2 Challisin has specific requirement for proline two residues before the cleavage point and a neutral or basic un-branched amino acid preceding the cleavage point. Fibrin formation by thrombin was modified and the initiation of fibrinolysis extended, in FSS2 Challisin-treated plasma clots. Digestion of fibrinogen by FSS2 Challisin prior to thrombin action increased fiber density and fiber branch point density. The velocity of fibrinolysis was significantly slower for fibrin formed from FSS2 Challisin-treated fibrinogen but was faster when data was normalised for the increased fibrin density. Thromboelastography of whole blood treated with FSS2 Challisin indicated reduced clot coagulation time and increased shear resistance. Combined ACE and fibrinogenase activities of FSS2 Challisin suggest a pro-coagulant effect of this virulence factor which is conserved in the viridans streptococci.

Ligation of CD24 expressed by oral epithelial cells induces kinase dependent decrease in paracellular permeability mediated by tight junction proteins.

In previous studies we demonstrated uniform strong expression of CD24 in the epithelial attachment to the tooth and in the migrating epithelium of the periodontitis lesion. Titers of serum antibodies auto-reactive with CD24 peptide correlated with reduced severity of periodontal disease. In the present study an epithelial culture model with close correspondence for expression patterns for tight junction components in periodontal epithelia was used. Ligation of CD24 expressed by oral epithelial cells with an anti-CD24 antibody induced formation of tight junctions and live-cell imaging confirmed that paracellular diffusion of fluorochrome-labeled dextran was reduced. Expression of mRNA and protein for zona occludens-1, -2, junction adhesion molecule-A (JAM-A), occludin and claudins-1, -4, -8, -15, -18 was significantly increased following ligation of CD24 but only claudins-4 and -15, JAM-A, occludin and zona occludens-1 and -2 were increased at cell contacts. This change in expression patterns reflected that observed between the epithelium of the periodontal lesion and that of the healthy gingival attachment. In the model system, response profiles to kinase inhibitors indicated a key role of c-Src kinase activation in the development of diffusion-limiting tight junction complexes. Activation was confirmed by demonstrating concomitant phosphorylation of the kinase. Pre-incubation with antibodies against JAM-A and claudin-15 prevented barrier-enhancing effects of anti-CD24 antibodies while pre-incubation with antibody to claudin-4 was partially effective. It is concluded that antibodies to CD24 facilitate expression and location of JAM-A, claudins-4 and -15 that mediate enhanced epithelial barrier function in a protective response against bacterial products.

Blueprint of an ancestral neurosensory organ revealed in glial networks in human dental pulp.

Sensory function of human dental pulp has long been known. A composite role has been suggested for odontoblasts as sensory cells in addition to the synthesis of dentinal matrix. However, the neural basis for such a composite sensory activity remains enigmatic. Here, we aimed to probe the question by pursuing an evolutionary logic; if dental pulp is a vestigial sensory organ co-opted to a function of synthesis of mineralized matrix, essential elements of neurosensory organs may persist in dental pulp. Through structural analysis by confocal laser scanning microscopy, three distinct cell populations adjacent to odontoblasts, glial fibrillary acidic protein (GFAP)(+) seracytes, S100(+) telacytes, and HLA-II(+) alacytes were identified in peripheral human dental pulp. Subsequent molecular fingerprinting by quantitative reverse transcriptase-polymerase chain reaction established these cells as analogous to radial glia (GFAP(+) cells), astrocytes (S100(+) cells), and microglia (HLA-II(+) cells) of central nervous system organs. In the cell-rich zone of the pulp, S100(+) cells formed a network, ensheathed unmyelinated axons, and extended end-feet around the capillaries. The microcirculation adjacent to the glial cells in the cell-rich zone possessed ultrastructural features and a gene expression profile typical of the blood-brain barrier system. These novel findings support a new paradigm for understanding sensory functionality of dental pulp by the demonstration of a sophisticated neural structure in the human dental pulp that is analogous to other central sensory organs. Further, the structure that is revealed informs the concept of the evolutionary origin of the dental pulp, suggesting that a neurosensory organ was the precursor structure of teeth.

Adaptive calcified matrix response of dental pulp to bacterial invasion is associated with establishment of a network of glial fibrillary acidic protein+/glutamine synthetase+ cells.

We report evidence for anatomical and functional changes of dental pulp in response to bacterial invasion through dentin that parallel responses to noxious stimuli reported in neural crest-derived sensory tissues. Sections of resin-embedded carious adult molar teeth were prepared for immunohistochemistry, in situ hybridization, ultrastructural analysis, and microdissection to extract mRNA for quantitative analyses. In odontoblasts adjacent to the leading edge of bacterial invasion in carious teeth, expression levels of the gene encoding dentin sialo-protein were 16-fold greater than in odontoblasts of healthy teeth, reducing progressively with distance from this site of the carious lesion. In contrast, gene expression for dentin matrix protein-1 by odontoblasts was completely suppressed in carious teeth relative to healthy teeth. These changes in gene expression were related to a gradient of deposited reactionary dentin that displayed a highly modified structure. In carious teeth, interodontoblastic dentin sialo-protein(-) cells expressing glutamine synthetase (GS) showed up-regulation of glial fibrillary acidic protein (GFAP). These cells extended processes that associated with odontoblasts. Furthermore, connexin 43 established a linkage between adjacent GFAP(+)/GS(+) cells in carious teeth only. These findings indicate an adaptive pulpal response to encroaching caries that includes the deposition of modified, calcified, dentin matrix associated with networks of GFAP(+)/GS(+) interodontoblastic cells. A regulatory role for the networks of GFAP(+)/GS(+) cells is proposed, mediated by the secretion of glutamate to modulate odontoblastic response.

Lactobacilli are prominent in the initial stages of polymicrobial infection of dental pulp.

In earlier studies we used molecular methods to identify the major bacterial consortia associated with advanced dentin caries. These consortia are dominated by bacteria from the families Lactobacillaceae, Streptococcaceae, Veillonellaceae (formerly Acidaminococcaceae), Eubacteriaceae, and Lachnospiraceae from the phylum Firmicutes; Coriobacteriaceae, Bifidobacteriaceae, and Propionibacteriaceae from the phylum Actinobacteria; and Prevotellaceae from the phylum Bacteroidetes, as well as fusobacteria. The phases of infection of vital pulp tissue by dentin microorganisms remain obscure. In the present study, fluorescence in situ hybridization was performed on sections of tissue embedded in resin. Probes for 16S rRNA corresponding to the major taxa of bacteria in carious dentin were used to provide information on the characteristics of pulp infection. Lactobacilli were prominent in 7 of 8 pulps determined to be at a limited stage of infection. Established infection (6 pulps) showed a more complex profile, with lactobacilli persisting in all of the lesions and with invasion of the necrotic regions of tissue by Bacteroidetes, fusobacteria, Lachnospiraceae, and Coriobacteriaceae in particular. Advanced infections (7 pulps) were characterized by mixed anaerobic species, with a strong representation by Coriobacteriaceae and Lachnospiraceae. Lactobacilli were not represented at this stage. Typically, groups of organisms were spatially isolated within the pulp tissue. Analysis indicated that lactobacilli could invade vital pulp tissue to achieve a very high biomass that was not associated with a detectable local inflammatory infiltrate. The findings establish that invasion of the dental pulp can be associated with a pronounced selection from the complex microbial populations within carious dentin, suggesting specific pathogenicity.

CD24 regulated gene expression and distribution of tight junction proteins is associated with altered barrier function in oral epithelial monolayers.

BACKGROUND: Control of intercellular penetration of microbial products is critical for the barrier function of oral epithelia. We demonstrated that CD24 is selectively and strongly expressed in the cells of the epithelial attachment to the tooth and the epithelial lining of the diseased periodontal pocket and studies in vitro showed that CD24 regulated expression of the epithelial intercellular adhesion protein E-cadherin. RESULTS: In the present study, the barrier function of oral epithelial cell monolayers to low molecular weight dextran was assayed as a model for the normal physiological function of the epithelial attachment to limit ingress of microbial products from oral microbial biofilms. Paracellular transfer of low molecular weight dextran across monolayers of oral epithelial cells was specifically decreased following incubation with anti-CD24 peptide antibody whereas passage of dextran across the monolayer was increased following silencing of mRNA for CD24. Changes in barrier function were related to the selective regulation of the genes encoding zonula occludens-1, zonula occludens-2 and occludin, proteins implicated in tight junctions. More particularly, enhanced barrier function was related to relocation of these proteins to the cell periphery, compatible with tight junctions. CONCLUSION: CD24 has the constitutive function of maintaining expression of selected genes encoding tight junction components associated with a marginal barrier function of epithelial monolayers. Activation by binding of an external ligand to CD24 enhances this expression but is also effective in re-deployment of tight junction proteins that is aligned with enhanced intercellular barrier function. These results establish the potential of CD24 to act as a potent regulator of the intercellular barrier function of epithelia in response to local microbial ecology.

Immunohistochemistry using antibodies to the cathelicidin LL37/hCAP18 in the tammar wallaby, Macropus eugenii.

Antibodies to the human cathelicidin, hCAP18 have been used to examine epithelial tissues of adult and pouch young marsupials. Immunoreactivity was observed in skin, gastrointestinal tract, lung and mammary node of adults as well as skin, gastrointestinal tract, lung and bone marrow of pouch young. The locations of expression were similar to that reported in human tissues. Although the antibody to hCAP18 is primarily directed at the C-terminal antimicrobial peptide LL37, our observations suggest recognition of a common conserved element of this cathelicidin and lead us to conclude that the epithelial tissues of marsupials are sites of production of cathelicidin. This is consistent with observations in other mammals but is the first report of expression of these compounds in marsupials.

Differential expression of transforming growth factors-beta 1, -beta 2, -beta 3 and the type I, II, III receptors in the lining epithelia of inflamed gingiva.

AIMS: To investigate the distribution of transforming growth factor-beta isoforms in chronically inflamed periodontal tissues. METHODS: The present study determined, by immunohistochemistry, the expression patterns of TGF-betas and their receptors in the lining epithelia of inflamed gingiva. Frozen sections were obtained from 22 human gingival biopsies. RESULTS: TGF-beta 1 was not detected in gingival epithelial cells in examined sections. Detection of TGF-beta 2 indicated a progressive reduction of staining from the external oral epithelium through to gingival sulcus and the gingival attachment or pocket epithelium. TGF-beta 3 showed intense staining in all domains of both minimally inflamed gingiva and advanced periodontitis tissues. TGF-beta RI was visualised as focal staining of the spinous layer in the external oral epithelium of both periodontitis lesions and minimally inflamed tissues. TGF-beta RII was present throughout the strata, but with progressive reduction in intensity from the oral epithelium to gingival attachment or pocket epithelium respectively while, conversely, TGF-beta RIII showed an increase in diffuse staining intensity from external oral epithelium to pocket epithelium. CONCLUSIONS: A distinct expression profile was observed within different individuals for TGF-betas and the corresponding receptors. These findings provide a basis for evaluation of the role of these growth factors in the pathogenesis of periodontitis.