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Telocytes (TCs) are CD34-positive interstitial cells that have long cytoplasmic projections, called telopodes; they have been identified in several organs and in various species. These cells establish a complex communication network between different stromal and epithelial cell types, and there is growing evidence that they play a key role in physiology and pathology. In many tissues, TC network impairment has been implicated in the onset and progression of pathological conditions, which makes the study of TCs of great interest for the development of novel therapies. In this review, we summarise the main methods involved in the characterisation of these cells as well as their inherent difficulties and then discuss the functional assays that are used to uncover the role of TCs in normal and pathological conditions, from the most traditional to the most recent. Furthermore, we provide future perspectives in the study of TCs, especially regarding the establishment of more precise markers, commercial lineages and means for drug delivery and genetic editing that directly target TCs. [ABSTRACT FROM AUTHOR]

Fibrotic diseases account for 45% of all deaths in the developed world, and progressive scarring conditions, such as dystrophic epidermolysis bullosa (EB), result in a significant reduction in quality of life. There is thus a need for novel approaches in fibrosis prevention. Herein, it is shown that iota carrageenan, a low‐cost, regulatory approved polysaccharide, is effective at preventing scarring by inhibiting collagen fibrillogenesis, abrogating the transforming growth factor beta 1 (TGFβ1) pathway, and lubricating the epithelium. It is demonstrated that iota carrageenan, and other anionic polysaccharides, can prevent collagen fibril formation, a key step in scar formation. It also binds TGFβ1 to prevent myofibroblast transdifferentiation, evidenced by a significant reduction in both transcription and translation of collagen 1 and alpha smooth muscle actin. Iota carrageenan may be formulated as a microparticle suspension, to allow spraying for even coverage on hard‐to‐reach anatomical sites, like the oral mucosa. This formulation provides good lubrication, to reduce the shear forces that initiate the progressive oral scarring in EB. This study not only provides a novel therapy for fibrosis prevention, but also demonstrates the potential of self‐delivering immunomodulatory polysaccharides as a safe, cost‐effective, and stable platform, which can be more easily translated for clinical benefit. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) are a promising cell population for regenerative medicine applications, where paracrine signalling through the extracellular vesicles (EVs) regulates bone tissue homeostasis and development. MSCs are known to reside in low oxygen tension, which promotes osteogenic differentiation via hypoxia-inducible factor-1α activation. Epigenetic reprogramming has emerged as a promising bioengineering strategy to enhance MSC differentiation. Particularly, the process of hypomethylation may enhance osteogenesis through gene activation. Therefore, this study aimed to investigate the synergistic effects of inducing hypomethylation and hypoxia on improving the therapeutic efficacy of EVs derived from human bone marrow MSCs (hBMSCs). The effects of the hypoxia mimetic agent deferoxamine (DFO) and the DNA methyltransferase inhibitor 5-azacytidine (AZT) on hBMSC viability was assessed by quantifying the DNA content. The epigenetic functionality was evaluated by assessing histone acetylation and histone methylation. hBMSC mineralisation was determined by quantifying alkaline phosphate activity, collagen production and calcium deposition. EVs were procured from AZT, DFO or AZT/DFO-treated hBMSCs over a two-week period, with EV size and concentration defined using transmission electron microscopy, nanoflow cytometry and dynamic light scattering. The effects of AZT-EVs, DFO-EVs or AZT/DFO-EVs on the epigenetic functionality and mineralisation of hBMSCs were evaluated. Moreover, the effects of hBMSC-EVs on human umbilical cord vein endothelial cells (HUVECs) angiogenesis was assessed by quantifying pro-angiogenic cytokine release. DFO and AZT caused a time–dose dependent reduction in hBMSC viability. Pre-treatment with AZT, DFO or AZT/DFO augmented the epigenetic functionality of the MSCs through increases in histone acetylation and hypomethylation. AZT, DFO and AZT/DFO pre-treatment significantly enhanced extracellular matrix collagen production and mineralisation in hBMSCs. EVs derived from AZT/DFO-preconditioned hBMSCs (AZT/DFO-EVs) enhanced the hBMSC proliferation, histone acetylation and hypomethylation when compared to EVs derived from AZT-treated, DFO-treated and untreated hBMSCs. Importantly, AZT/DFO-EVs significantly increased osteogenic differentiation and mineralisation of a secondary hBMSC population. Furthermore, AZT/DFO-EVs enhanced the pro-angiogenic cytokine release of HUVECs. Taken together, our findings demonstrate the considerable utility of synergistically inducing hypomethylation and hypoxia to improve the therapeutic efficacy of the MSC-EVs as a cell-free approach for bone regeneration. [ABSTRACT FROM AUTHOR]

Epigenetics plays a critical role in regulating mesenchymal stem cells' (MSCs) fate for tissue repair and regeneration. There is increasing evidence that the inhibition of histone deacetylase (HDAC) isoform 3 can enhance MSC osteogenesis. This study investigated the potential of using a selective HDAC2 and 3 inhibitor, MI192, to promote human dental pulp stromal cells (hDPSCs) bone-like tissue formation in vitro and in vivo within porous Bombyx Mori silk scaffolds. Both 2 and 5 wt% silk scaffolds were fabricated and characterised. The 5 wt% scaffolds possess thicker internal lamellae, reduced scaffold swelling and degradation rates, whilst increased compressive modulus in comparison to the 2 wt% silk scaffold. MI192 pre-treatment of hDPSCs on 5 wt% silk scaffold significantly enhanced hDPSCs alkaline phosphatase activity (ALP). The expression of osteoblast-related genes (RUNX2, ALP, Col1a, OCN) was significantly upregulated in the MI192 pre-treated cells. Histological analysis confirmed that the MI192 pre-treated hDPSCs-silk scaffold constructs promoted bone extracellular matrix (ALP, Col1a, OCN) deposition and mineralisation compared to the untreated group. Following 6 weeks of subcutaneous implantation in nude mice, the MI192 pre-treated hDPSCs-silk scaffold constructs enhanced the vascularisation and extracellular matrix mineralisation compared to untreated control. In conclusion, these findings demonstrate the potential of using epigenetic reprogramming and silk scaffolds to promote hDPSCs bone formation efficacy, which provides evidence for clinical translation of this technology for bone augmentation. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are emerging in tissue engineering as promising acellular tools, circumventing many of the limitations associated with cell‐based therapies. Epigenetic regulation through histone deacetylase (HDAC) inhibition has been shown to increase differentiation capacity. Therefore, this study aimed to investigate the potential of augmenting osteoblast epigenetic functionality using the HDAC inhibitor Trichostatin A (TSA) to enhance the therapeutic efficacy of osteoblast‐derived EVs for bone regeneration. TSA was found to substantially alter osteoblast epigenetic function through reduced HDAC activity and increased histone acetylation. Treatment with TSA also significantly enhanced osteoblast alkaline phosphatase activity (1.35‐fold), collagen production (2.8‐fold) and calcium deposition (1.55‐fold) during osteogenic culture (P ≤ 0.001). EVs derived from TSA‐treated osteoblasts (TSA‐EVs) exhibited reduced particle size (1‐05‐fold) (P > 0.05), concentration (1.4‐fold) (P > 0.05) and protein content (1.16‐fold) (P ≤ 0.001) when compared to untreated EVs. TSA‐EVs significantly enhanced the proliferation (1.13‐fold) and migration (1.3‐fold) of human bone marrow stem cells (hBMSCs) when compared to untreated EVs (P ≤ 0.05). Moreover, TSA‐EVs upregulated hBMSCs osteoblast‐related gene and protein expression (ALP, Col1a, BSP1 and OCN) when compared to cells cultured with untreated EVs. Importantly, TSA‐EVs elicited a time‐dose dependent increase in hBMSCs extracellular matrix mineralisation. MicroRNA profiling revealed a set of differentially expressed microRNAs from TSA‐EVs, which were osteogenic‐related. Target prediction demonstrated these microRNAs were involved in regulating pathways such as 'endocytosis' and 'Wnt signalling pathway'. Moreover, proteomics analysis identified the enrichment of proteins involved in transcriptional regulation within TSA‐EVs. Taken together, our findings suggest that altering osteoblasts' epigenome accelerates their mineralisation and promotes the osteoinductive potency of secreted EVs partly due to the delivery of pro‐osteogenic microRNAs and transcriptional regulating proteins. As such, for the first time we demonstrate the potential to harness epigenetic regulation as a novel engineering approach to enhance EVs therapeutic efficacy for bone repair. [ABSTRACT FROM AUTHOR]

Highly diastereo- and enantioselective 1,3-dipolar cycloadditions between functional ketonitrones and β-substituted enals are promoted by MacMillan imidazolidinium organocatalysts. A study of the reaction scope shows that high selectivities are conserved if the N-protecting group or the ester function is varied. However, the reaction is sensitive to steric interactions with the C substituent of the nitrone. In all cases, the reaction proceeds with high exo selectivity. In most cases, a third diastereomer, incompatible with a concerted mechanism, was also observed, albeit in minute amounts. DFT calculations suggest that the cycloaddition proceeds in a nonconcerted fashion by an initial oxa-Michael-type addition of the nitrone to the double bond followed by a cyclization. This mechanism explains the formation of the observed minor diastereomers. In addition, the diastereo- and enantioselectivities of the reaction were shown to be intermediately thermodynamically controlled, and the diastereomeric ratio is modulated by the kinetics of iminium hydrolysis. [ABSTRACT FROM AUTHOR]

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An unconventional crude from biomass (biocrude) has been processed to yield a hydrocarbon stream that is not only fully processable in conventional refineries but is already close to the specification of commercial fuels such as transportation diesel. The upgrading of biocrude was carried out with a combination of hydrotreatment and catalytic cracking, yielding middle distillate as the main product. [ABSTRACT FROM AUTHOR]

Bicyclic isoxazolidines displaying one or two quaternary stereocenter(s) were formed starting from functional cyclic ketonitrones equipped with a phenyl glycinol chiral auxiliary. The products were engaged in stereocontrolled 1,3-dipolar cycloaddition reactions with a range of electron-rich and electron-poor dipolarophiles. A new reductive removal of the phenyl glycinol chiral auxiliary was introduced and was shown to afford chemoselectively a quaternary isoxazolidine derivative (of oxaprolinol-type) without cleaving the N-O isoxazolidine bond. Keeping the aldehyde function masked as a cyclic pseudo-acetal, the liberated oxy-amine function was shown to be available for a pseudo-peptide coupling with various N-protected amino acids. The isoxazolidine ring was opened by a reductive N-O bond cleavage, giving a pseudo-dipeptide that was C-terminated with an aldehyde function. [ABSTRACT FROM AUTHOR]

Enantiopure vinyl ethers of Stericol® underwent diastereoselective thermal 1,3 dipolar cycloadditions with N-benzyl, N-benzhydryl, and N-PMB aspartate ester nitrones. Chemoselective N-debenzylation of the resulting cycloadducts afforded diastereomerically and enantiomerically pure crystalline NH-isoxazolidine, the absolute configuration of which was established by X-ray crystallography. N-Protection and N-O cleavage of this isoxazolidine gave enantioenriched quaternary aspartate derivatives bearing functionalized side chains. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) have garnered growing attention as promising acellular tools for bone repair. Although EVs' potential for bone regeneration has been shown, issues associated with their therapeutic potency and short half-life in vivo hinders their clinical utility. Epigenetic reprogramming with the histone deacetylase inhibitor Trichostatin A (TSA) has been reported to promote the osteoinductive potency of osteoblast-derived EVs. Gelatin methacryloyl (GelMA) hydrogels functionalised with the synthetic nanoclay laponite (LAP) have been shown to effectively bind, stabilise, and improve the retention of bioactive factors. This study investigated the potential of utilising a GelMA-LAP hydrogel to improve local retention and control delivery of epigenetically enhanced osteoblast-derived EVs as a novel bone repair strategy. LAP was found to elicit a dose-dependent increase in GelMA compressive modulus and shear-thinning properties. Incorporation of the nanoclay was also found to enhance shape fidelity when 3D printed compared to LAP-free gels. Interestingly, GelMA hydrogels containing LAP displayed increased mineralisation capacity (1.41-fold) (p ≤ 0.01) over 14 days. EV release kinetics from these nanocomposite systems were also strongly influenced by LAP concentration with significantly more vesicles being released from GelMA constructs as detected by a CD63 ELISA (p ≤ 0.001). EVs derived from TSA-treated osteoblasts (TSA-EVs) enhanced proliferation (1.09-fold), migration (1.83-fold), histone acetylation (1.32-fold) and mineralisation (1.87-fold) of human bone marrow stromal cells (hBMSCs) when released from the GelMA-LAP hydrogel compared to the untreated EV gels (p ≤ 0.01). Importantly, the TSA-EV functionalised GelMA-LAP hydrogel significantly promoted encapsulated hBMSCs extracellular matrix collagen production (≥1.3-fold) and mineralisation (≥1.78-fold) in a dose-dependent manner compared to untreated EV constructs (p ≤ 0.001). Taken together, these findings demonstrate the potential of combining epigenetically enhanced osteoblast-derived EVs with a nanocomposite photocurable hydrogel to promote the therapeutic efficacy of acellular vesicle approaches for bone regeneration. [ABSTRACT FROM AUTHOR]

The oxidative response induced by hypo-osmolarity is characterized in tobacco and Arabidopsis cells in order to identify the corresponding active oxygen-producing proteins. The pharmacological profiles of the oxidative responses were clearly different in the two plant materials, leading to the identification of distinct active oxygen producers in tobacco and Arabidopsis cells. In tobacco cells, a 100 kDa protein, localized in the plasma membrane, was demonstrated to produce active oxygen in the presence of NADPH. This production can be activated by fatty acids and is strongly depressed by diphenylene iodonium, as measured by an in vivo response. In Arabidopsis, 30 kDa and 34 kDa proteins localized in the cell wall were shown to be able to produce active oxygen in the presence of cofactors and the production is prevented by peroxidase inhibitors, as is the in vivo response. The two purified proteins were identified by mass spectrometry and both correspond to the peroxidase gene At5g64120. [ABSTRACT FROM PUBLISHER]

The present study examines the suggestion that in the absence of adequate bile and pancreatic juice, which support the absorption from the gut of long-chain fatty acidsinto lymph, the fatty acids are absorbed directly into the portal blood. Oleic acid (18:l) partitioning between lymph and portal blood was investigated in intact and bile- and pancreatic juice-diverted rats. In a first set of experiments, 18: 1 absorption from the gut into lymph and blood was studied by continuous recovery of the mesenteric lymph for 6 h and mesenteric portal venous blood for 1 h. In a second set of experiments, esterification processes were investigated by study of the mucosal distribution of labelled lipids and by mono- and diacylglycerol acyltransferase (EC 2.3.1.22 and EC 2.3.1.20 respectively) specific activities. In the bile- and pancreatic juice-diverted rats the absorption of labelled 18:l into lymph was significantly reduced during the first 3 h of intraluminal infusion of this substrate. In such rats a compensatory absorption of labelled 18: 1 into mesenteric portal blood was not observed. At 6 h after micellar lipid- mixture infusion, the overload of lipids both in free form and as triacylglycerols persisting in the mucosa paralleled the lower acyltransferase specific activities observed in bile- and pancreatic juice-diverted rats. These studies demonstrate the absence of a previously proposed compensatory absorption of 18: 1 into portal blood when absorption into lymph is impaired by an inadequate supply of bile and pancreatic juice. [ABSTRACT FROM PUBLISHER]

Nicotiana tabacum L. (cv. Xanthi) suspension cultures initially grown in the dark and then exposed to continuous light followed a developmental pattern divided into a lag phase (T0–T2), a growth phase (T3–T9) and a stationary phase (T10–T21). The onset of chloroplast differentiation during the active cell division phase was associated with a rapid chlorophyll synthesis, a high rate of respiratory activity (400 μmol h−1 g−1 dry weight) and a four‐fold increase in soluble protein content compared to that of the initial inoculum. Oxygen evolution was nil until T3. It increased regularly up to a value of 30 μmol h−1 g−1 dry weight at the beginning of the stationary phase, higher values (80 μmol) being reached later in this phase. An inverse relationship between phosphoenolpyruvate carboxylase (PEPCase) and ribulose 1,5‐biphosphate carboxylase (RUBPCase) was shown during cell growth. The PEP‐Case capacity expressed on a soluble protein basis rose from an initial day 0 value of 3.5 μmol to a maximum of 7.0 μmol at T5 and then returned to the initial value during the post‐exponential and stationary phases. In contrast, RUBPCase activity was minimal (0.1 μmol) from T0 to T3 of greening, increasing to a maximum (1.7 to 2.0 μmol) at T8. The 20 fold stimulation of this enzyme capacity was related to a de novo protein synthesis. Immunodiffusion tests revealed the presence of RUBPCase molecules in soluble crude extract of dark-grown cells. The SDS-gel densitometric profile of soluble protein extract from dark-grown cells was qualitatively similar to that obtained with crude extract of green cells. The 56,000 Da polypeptide co-migrating with the large subunit (LSU) of RUBPCase appeared reduced in dark-grown cells compared to the peak height of the same polypeptide present in green cells. In both preparations, the 13,000 Da polypeptide co-migrating with the small subunit (SSU) was equally present. The current hypothesis is that the synthesis of RUBPCase at the time of greening would concern the preferential synthesis of the LSU of the enzyme.

Photoheterotrophic growth of cell suspensions of Nicotiana tabacum L. (cv. Xanthi) in organic culture medium enriched in sucrose (30 g per liter) showed a classical sigmoid growth curve. The cells developed functional chloroplast structures during the exponential growth phase, when their chlorophyll content increased steadily. A limited drop (30%) in the chlorophyll amount and structural changes of the plastids (starch accumulation) were observed during the lag phase. The measurements of photosynthetic capacities (O2 evolution and CO2 fixation) during the growth cycle revealed changes in the photosynthetic ratio (O2/CO2), which was near 1 during the lag and stationary phases and near 2 during exponential growth. During exponential growth there was also a rapid NO3- uptake. Analysis of label distribution among the products of 14CO2 fixation showed that both CO2 assimilation pathways, linked to the ribulose-biphosphate carboxylase (the autotrophic pathway) and to phosphoenolpyruvate carboxylase (the non-autotrophic pathway) were operative with an important increase of the capacity of the tatter during the exponential growth phase. Maximum rate of oxygen evolution, either endogenous or with p-benzoquinone as Hill reagent, as well as the increased CO2 fixation capacity via the non-autotrophic pathway during the exponential phase were concomitant with a high cyanide inhibited O2 uptake. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are emerging as promising nanoscale therapeutics due to their intrinsic role as mediators of intercellular communication, regulating tissue development and homeostasis. The low immunogenicity and natural cell-targeting capabilities of EVs has led to extensive research investigating their potential as novel acellular tools for tissue regeneration or for the diagnosis of pathological conditions. However, the clinical use of EVs has been hindered by issues with yield and heterogeneity. From the modification of parental cells and naturally-derived vesicles to the development of artificial biomimetic nanoparticles or the functionalisation of biomaterials, a multitude of techniques have been employed to augment EVs therapeutic efficacy. This review will explore various engineering strategies that could promote EVs scalability and therapeutic effectiveness beyond their native utility. Herein, we highlight the current state-of-the-art EV-engineering techniques with discussion of opportunities and obstacles for each. This is synthesised into a guide for selecting a suitable strategy to maximise the potential efficacy of EVs as nanoscale therapeutics. [ABSTRACT FROM AUTHOR]