Generation of Stable Isopentenyl Monophosphate Aryloxy Triester Phosphoramidates as Activators of Vγ9Vδ2 T Cells.

Aryloxy triester phosphoramidate prodrugs of the monophosphate derivatives of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) were synthesized as lipophilic derivatives that can improve cell uptake. Despite the structural similarity of IPP and DMAPP, it was noted that their phosphoramidate prodrugs exhibited distinct stability profiles in aqueous environments, which we show is due to the position of the allyl bond in the backbones of the IPP and DMAPP monophosphates. As the IPP monophosphate aryloxy triester phosphoramidates showed favorable stability, they were subsequently investigated for their ability to activate Vγ9/Vδ2 T cells and they showed promising activation of this subset of T cells. Together, these findings represent the first report of IPP and DMAPP monophosphate prodrugs and the ability of IPP aryloxy triester phosphoramidate prodrugs to activate Vγ9/Vδ2 T cells highlighting their potential as possible immunotherapeutics.

Isovaleric acid ameliorates ovariectomy-induced osteoporosis by inhibiting osteoclast differentiation.

Osteoclasts (OCs) play important roles in bone remodelling and contribute to bone loss by increasing bone resorption activity. Excessively activated OCs cause diverse bone disorders including osteoporosis. Isovaleric acid (IVA), also known as 3-methylbutanoic acid is a 5-carbon branched-chain fatty acid (BCFA), which can be generated by bacterial fermentation of a leucine-rich diet. Here, we find that IVA suppresses differentiation of bone marrow-derived macrophages into OCs by RANKL. IVA inhibited the expression of OC-related genes. IVA-induced inhibitory effects on OC generation were attenuated by pertussis toxin but not by H89, suggesting a Gi -coupled receptor-dependent but protein kinase A-independent response. Moreover, IVA stimulates AMPK phosphorylation, and treatment with an AMPK inhibitor blocks IVA-induced inhibition of OC generation. In an ovariectomized mouse model, addition of IVA to the drinking water resulted in significant decrease of body weight gain and inhibited the expression of not only OC-related genes but also fusogenic genes in the bone tissue. IVA exposure also blocked bone destruction and OC generation in the bone tissue of ovariectomized mice. Collectively, the results demonstrate that IVA is a novel bioactive BCFA that inhibits OC differentiation, suggesting that IVA can be considered a useful material to control osteoclast-associated bone disorders, including osteoporosis.

A new anti-austerity agent, 4'-O-methylgrynullarin from Derris scandens induces PANC-1 human pancreatic cancer cell death under nutrition starvation via inhibition of Akt/mTOR pathway.

An ethanolic extract of Derris scandens flowers showed potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrient-deprived condition, with a PC50 value of 0.7 µg/mL. Phytochemical investigation of this active extract led to the isolation of four prenylated isoflavones (1-4) including a new compound named 4'-O-methylgrynullarin (1). The structure elucidation of the new compound was achieved by HRFABMS and NMR spectroscopic analysis. The isolated compounds exhibited potent anti-austerity activity against four different human pancreatic cancer cell lines under nutrient-deprived conditions. The new compound 4'-O-methylgrynullarin (1) was also found to inhibit PANC-1 cell migration and colony formation under nutrient-rich condition. Mechanistically, compound 1 inhibited key survival proteins in the Akt/mTOR signaling pathway. Therefore, 4'-O-methylgrynullarin (1) can be considered as a potential lead compound for the anticancer drug development based on the anti-austerity strategy.

Behavioural and antennal responses of Aedes aegypti (l.) (Diptera: Culicidae) gravid females to chemical cues from conspecific larvae.

Mass trapping of gravid females represents one promising strategy for the development of sustainable tools against Aedes aegypti. However, this technique requires the development of effective odorant lures that can compete with natural breeding sites. The presence of conspecific larvae has been shown to stimulate oviposition. Hence, we evaluated the role of four major molecules previously identified from Ae. aegypti larvae (isovaleric, myristoleic, myristic [i.e. tetradecanoic], and pentadecanoic acids) on the oviposition of conspecific females, as well as their olfactory perception to evaluate their range of detection. Using flight cage assays, the preference of gravid females to oviposit in water that previously contained larvae (LHW) or containing the four larval compounds was evaluated. Then, compounds and doses inducing the highest stimulation were challenged for their efficacy against LHW. Only isovaleric acid elicited antennal response, suggesting that the other compounds may act as taste cues. Pentadecanoic acid induced significant oviposition stimulation, especially when dosed at 10 ppm. Myristoleic acid and isovaleric acid deterred oviposition at 10 and 100 ppm, while no effect on oviposition was observed with myristic acid irrespectively of the dose tested. When the four compounds were pooled to mimic larvae's chemical signature, they favored oviposition at 1 ppm but negatively affected egg-laying at higher concentrations. When properly dosed, pentadecanoic acid and the blend of compounds may be promising lures for ovitraps as they could compete with LHW. Due to their low volatility, their effect should be further evaluated under field conditions, in addition with long-range attractants for developing effective tools against gravid females.

Isoprene and ß-caryophyllene confer plant resistance via different plant internal signalling pathways.

Isoprene and other terpenoids are important biogenic volatile organic compounds in terms of atmospheric chemistry. Isoprene can aid plant performance under abiotic stresses, but the fundamental biological reasons for the high emissions are not completely understood. Here, we provide evidence of a previously unrecognized ecological function for isoprene and for the sesquiterpene, ß-caryophyllene. We show that isoprene and ß-caryophyllene act as core components of plant signalling networks, inducing resistance against microbial pathogens in neighbouring plants. We challenged Arabidopsis thaliana with Pseudomonas syringae, after exposure to pure volatile terpenoids or to volatile emissions of transformed poplar or Arabidopsis plants. The data suggest that isoprene induces a defence response in receiver plants that is similar to that elicited by monoterpenes and depended on salicylic acid (SA) signalling. In contrast, the sesquiterpene, ß-caryophyllene, induced resistance via jasmonic acid (JA)-signalling. The experiments in an open environment show that natural biological emissions are enough to induce resistance in neighbouring Arabidopsis. Our results show that both isoprene and ß-caryophyllene function as allelochemical components in complex plant signalling networks. Knowledge of this system may be used to boost plant immunity against microbial pathogens in various crop management schemes.

Covalent modification of Keap1 at Cys77 and Cys434 by pubescenoside a suppresses oxidative stress-induced NLRP3 inflammasome activation in myocardial ischemia-reperfusion injury.

Background and Purpose: Kelch ECH-associating protein 1 (Keap1) is a crucial chaperonin for E3 ubiquitin ligases. Modification of the key reactive cysteine residues in Keap1 affects the interaction between Keap1 and its substrate nuclear factor erythroid 2-related factor 2 (Nrf2), subsequently regulating oxidative stress and NLPR3 inflammasome activation, which are important factors for myocardial ischemia-reperfusion injury (MI/RI). Pubescenoside A (PBA), an active compound from Ilex pubescens, has antithrombotic and anti-inflammatory effects. However, the effect of PBA on MI/RI is still unknown. In the present study, we aimed to determine whether PBA can protect the heart against MI/RI and clarify the direct target and the underlying mechanism of PBA. Methods: The left anterior descending artery (LAD) ligation-induced MI/RI mice model or oxygen and glucose deprivation/reperfusion (OGD/R) were used to evaluate the cardioprotective effect of PBA. Pull-down assays, co-immunoprecipitation (Co-IP) assays, LC/MS/MS, isothermal calorimetry (ITC) experiments and covalent docking were used to identify the target of PBA. Results: PBA protected cardiomyocytes against OGD/R in vitro and LAD-induced MI/RI in vivo. PBA suppressed NLRP3 inflammation activation and induced the Nrf2 signaling pathway. Interestingly, PBA targeted Keap1 by selectively covalently binding to conserved cysteine residues, cysteine 77 (Cys77) in the BTB domain and cysteine 434 (Cys434) in the Kelch domain of Keap1, subsequently inhibiting ubiquitination of Nrf2 and activating antioxidant enzymes. Additionally, the cysteines of Keap1 has different degree of activation by PBA as follows: Cys77 > Cys434 > Cys23 > Cys38 > Cys226 > Cys273, which further elucidates the cysteine sensitivity of Keap1. Conclusions: Our results indicated that PBA might be a new Nrf2 activator that covalently binds to two critical domains of Keap1, and shows cardioprotective activities against ischemia-reperfusion injury.

The inhibitory effect of volatile organic compounds produced by Bacillus subtilis CL2 on pathogenic fungi of wolfberry.

Bacillus subtilis strain CL2 is antagonistic to wolfberry postharvest pathogenic fungi. In this study, we isolated and screened this strain for in vitro experiments. The result of the two-sealed-base-plates method revealed that volatile organic compounds (VOCs) emitted from the strain CL2 inhibited the hyphal growth of four pathogenic fungi Mucor circinelloides LB1, Fusarium arcuatisporum LB5, Alternaria iridiaustralis LB7, and Colletotrichum fioriniae LB8. After exposure to VOCs for 5 days, the hyphal growth of the pathogen C. fioriniae LB8 was inhibited by 73%. Scanning electron microscopy revealed that the VOCs produced by B. subtilis CL2 caused the mycelium morphology of the pathogenic fungi to deform, twist, fold, and shrink. In the in vivo experiments, we noticed that VOCs could significantly reduce the weight loss rate of wolfberry fruits caused by the pathogenic fungus M. circinelloides LB1 and that the decay incidence rate were caused by the pathogenic fungi F. arcuatisporum LB5, A. iridiaustralis LB7, and C. fioriniae LB8. On the basis of the headspace-gas chromatography-ion mobility spectrometry analysis, seven VOCs produced by strain CL2 were identified. Among them, 2,3-butanedione and 3-methylbutyric acid are the main antifungal active substances. This study investigated the antifungal properties of VOCs produced by the strain CL2 on postharvest pathogenic fungi isolated from wolfberry fruits both in vivo and in vitro, thereby providing the theoretical basis for its future applications.

Nine prenylated acylphloroglucinols with potential anti-depressive and hepatoprotective activities from Hypericum scabrum.

In our screening program for new biologically active secondary metabolites, nine new polycyclic polyprenyled acylphloroglucinols, hyperscabins D-L, together with three known compounds, were obtained from the aerial parts of Hypericum scabrum. The chemical structures of 1-9 were characterized by extensive spectroscopic analyses, nuclear magnetic resonance calculation with DP4+ probability analysis, and the electronic circular dichroism spectra were calculated. Compound 1 was an unusual prenylated acylphloroglucinol decorated with a 5-oxaspiro [4,5] deca-1,9-dione skeleton. Compound 2 was a newly identified spirocyclic polyprenylated acylphloroglucinol possessing a rare 5,5-spiroketal segment. Compounds 3, 8, and 10 (10 µM) exhibited pronounced hepatoprotective activity against d-galactosamine-induced WB-F344 cell damage in vitro assays. All test compounds (1, 3, and 7-12) demonstrated potential inhibitory effects at 10 µM against noradrenalinet ([3H]-NE) reuptake in rat brain synaptosome.

Short chain fatty acids produced by Cutibacterium acnes inhibit biofilm formation by Staphylococcus epidermidis.

Biofilm formation by bacterial pathogens is associated with numerous human diseases and can confer resistance to both antibiotics and host defenses. Many strains of Staphylococcus epidermidis are capable of forming biofilms and are important human pathogens. Since S. epidermidis coexists with abundant Cutibacteria acnes on healthy human skin and does not typically form a biofilm in this environment, we hypothesized that C. acnes may influence biofilm formation of S. epidermidis. Culture supernatants from C. acnes and other species of Cutibacteria inhibited S. epidermidis but did not inhibit biofilms by Pseudomonas aeruginosa or Bacillus subtilis, and inhibited biofilms by S. aureus to a lesser extent. Biofilm inhibitory activity exhibited chemical properties of short chain fatty acids known to be produced from C. acnes. The addition of the pure short chain fatty acids propionic, isobutyric or isovaleric acid to S. epidermidis inhibited biofilm formation and, similarly to C. acnes supernatant, reduced polysaccharide synthesis by S. epidermidis. Both short chain fatty acids and C. acnes culture supernatant also increased sensitivity of S. epidermidis to antibiotic killing under biofilm-forming conditions. These observations suggest the presence of C. acnes in a diverse microbial community with S. epidermidis can be beneficial to the host and demonstrates that short chain fatty acids may be useful to limit formation of a biofilm by S. epidermidis.

Hydroxylated Ethacrylic and Tiglic Acid Derivatives from the Stems and Branches of Enkianthus chinensis and Their Potential Anti-inflammatory Activities.

Two new hydroxylated ethacrylic acid derivatives (compounds 1 and 2) and 11 new hydroxylated tiglic acid derivatives (compounds 3-13), together with one known compound (compound 14), were isolated from the stems and branches of Enkianthus chinensis. Their structures were established by extensive spectroscopic analyses, while their absolute configurations were determined by X-ray crystallographic methods (compounds 1 and 2), Mo2(OAc)4-induced electronic circular dichroism experiments (compounds 3 and 4), and chemical methods (compounds 5-11). This study is the first investigation on the secondary metabolites of this species. The anti-inflammatory activities of all isolated compounds were evaluated in an LPS-induced mouse peritoneal macrophage model. Notably, compounds 3 and 12 both exerted potent inhibitory effects on NO production with IC50 values of 2.9 and 1.2 µM, respectively.

Overexpression of Isoprene Synthase Affects ABA- and Drought-Related Gene Expression and Enhances Tolerance to Abiotic Stress.

Isoprene is the most abundant single biogenic volatile compound emitted by plants. Despite the relevance of this molecule to plant abiotic resistance and its impact on global atmospheric chemistry, little is known about the details of its mechanism of action. Here, we characterized through both physiological and molecular methods the mechanisms of action of isoprene using model transgenic arabidopsis lines overexpressing a monocot isoprene synthase gene. Our results demonstrated the effect that isoprene had on ABA signaling at different tissue-specific, spatial, and temporal scales. In particular, we found that isoprene enhanced stomatal sensitivity to ABA through upregulation of RD29B signaling gene. By contrast, isoprene decreased sensitivity to ABA in germinating seeds and roots, suggesting tissue-specific mechanisms of action. In leaves, isoprene caused the downregulation of COR15A and P5CS genes, suggesting that the enhanced tolerance to water-deprivation stress observed in isoprene-emitting plants may be mediated chiefly by an enhanced membrane integrity and tolerance to osmotic stress.

Ammonia uptake by transmembrane pH gradient poly(isoprene)-block-poly(ethylene glycol) polymersomes.

Transmembrane pH gradient poly(isoprene)-block-poly(ethylene glycol) (PI-b-PEG) polymersomes were investigated for their potential use in the detoxification of ammonia, a metabolite that is excessively present in patients suffering from urea cycle disorders and advanced liver diseases, and which causes neurotoxic effects (e.g., hepatic encephalopathy). Polymers varying in PI and PEG block length were synthesized via nitroxide-mediated polymerization and screened for their ability to self-assemble into polymersomes in aqueous media. Ammonia sequestration by the polymersomes was investigated in vitro. While most vesicular systems were able to capture ammonia in simulated intestinal fluids, uptake was lost in partially dehydrated medium mimicking conditions in the colon. Polymeric crosslinking of residual olefinic bonds in the PI block increased polymersome stability, partially preserving the ammonia capture capacity in the simulated colon environment. These more stable vesicular systems hold promise for the chronic oral treatment of hyperammonemia.

Alpaca (Vicugna pacos), the first nonprimate species with a phosphoantigen-reactive Vγ9Vδ2 T cell subset.

Vγ9Vδ2 T cells are a major γδ T cell population in the human blood expressing a characteristic Vγ9JP rearrangement paired with Vδ2. This cell subset is activated in a TCR-dependent and MHC-unrestricted fashion by so-called phosphoantigens (PAgs). PAgs can be microbial [(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, HMBPP] or endogenous (isopentenyl pyrophosphate, IPP) and PAg sensing depends on the expression of B7-like butyrophilin (BTN3A, CD277) molecules. IPP increases in some transformed or aminobisphosphonate-treated cells, rendering those cells a target for Vγ9Vδ2 T cells in immunotherapy. Yet, functional Vγ9Vδ2 T cells have only been described in humans and higher primates. Using a genome-based study, we showed in silico translatable genes encoding Vγ9, Vδ2, and BTN3 in a few nonprimate mammalian species. Here, with the help of new monoclonal antibodies, we directly identified a T cell population in the alpaca (Vicugna pacos), which responds to PAgs in a BTN3-dependent fashion and shows typical TRGV9- and TRDV2-like rearrangements. T cell receptor (TCR) transductants and BTN3-deficient human 293T cells reconstituted with alpaca or human BTN3 or alpaca/human BTN3 chimeras showed that alpaca Vγ9Vδ2 TCRs recognize PAg in the context of human and alpaca BTN3. Furthermore, alpaca BTN3 mediates PAg recognition much better than human BTN3A1 alone and this improved functionality mapped to the transmembrane/cytoplasmic part of alpaca BTN3. In summary, we found remarkable similarities but also instructive differences of PAg-recognition by human and alpaca, which help in better understanding the molecular mechanisms controlling the activation of this prominent population of γδ T cells.

Suppressive activity of Vδ2+ γδ T cells on αß T cells is licensed by TCR signaling and correlates with signal strength.

Despite recent progress in the understanding of γδ T cells' roles and functions, their interaction with αß T cells still remains to be elucidated. In this study, we sought to clarify what precisely endows peripheral Vδ2+ T cells with immunosuppressive function on autologous αß T cells. We found that negatively freshly isolated Vδ2+ T cells do not exhibit suppressive behavior, even after stimulation with IL-12/IL-18/IL-15 or the sheer contact with butyrophilin-3A1-expressing tumor cell lines (U251 or SK-Mel-28). On the other hand, Vδ2+ T cells positively isolated through TCR crosslinking or after prolonged stimulation with isopentenyl pyrophosphate (IPP) mediate strong inhibitory effects on αß T cell proliferation. Stimulation with IPP in the presence of IL-15 induces the most robust suppressive phenotype of Vδ2+ T cells. This indicates that Vδ2+ T cells' suppressive activity is dependent on a TCR signal and that the degree of suppression correlates with its strength. Vδ2+ T cell immunosuppression does not correlate with their Foxp3 expression but rather with their PD-L1 protein expression, evidenced by the massive reduction of suppressive activity when using a blocking antibody. In conclusion, pharmacologic stimulation of Vδ2+ T cells via the Vδ2 TCR for activation and expansion induces Vδ2+ T cells' potent killer activity while simultaneously licensing them to suppress αß T cell responses. Taken together, the study is a further step to understand-in more detail-the suppressive activity of Vδ2+ γδ T cells.

Isoprene-Derived Secondary Organic Aerosol Induces the Expression of MicroRNAs Associated with Inflammatory/Oxidative Stress Response in Lung Cells.

Exposure to fine particulate matter (PM2.5), of which secondary organic aerosol (SOA) is a major constituent, is linked to adverse health outcomes, including cardiovascular disease, lung cancer, and preterm birth. Atmospheric oxidation of isoprene, the most abundant nonmethane hydrocarbon emitted into Earth's atmosphere primarily from vegetation, contributes to SOA formation. Isoprene-derived SOA has previously been found to alter inflammatory/oxidative stress genes. MicroRNAs (miRNAs) are epigenetic regulators that serve as post-transcriptional modifiers and key mediators of gene expression. To assess whether isoprene-derived SOA alters miRNA expression, BEAS-2B lung cells were exposed to laboratory-generated isoprene-derived SOA constituents derived from the acid-driven multiphase chemistry of authentic methacrylic acid epoxide (MAE) or isomeric isoprene epoxydiols (IEPOX) with acidic sulfate aerosol particles. These IEPOX- and MAE-derived SOA constituents have been shown to be measured in large quantities within PM2.5 collected from isoprene-rich areas affected by acidic sulfate aerosol particles derived from human activities. A total of 29 miRNAs were identified as differentially expressed when exposed to IEPOX-derived SOA and 2 when exposed to MAE-derived SOA, a number of which are inflammatory/oxidative stress associated. These results suggest that miRNAs may modulate the inflammatory/oxidative stress response to SOA exposure, thereby advancing the understanding of airway cell epigenetic response to SOA.

Isovaleric acid and avicequinone-C are Chikungunya virus resistance principles in Glycosmis pentaphylla (Retz.) Correa.

BACKGROUND & OBJECTIVES: Oral administration of tender leaf extract of Glycosmis pentaphylla is traditionally known to prevent the chikungunya virus infection. Even with wide usage, the antiviral components in this plant are neither identified nor characterized. This study was carried out with the objectives of profiling the phytocompounds in this plant through LC-MS/MS and to identify the active antiviral constituents and their drug-likeliness through molecular docking. METHODS: Phytocompounds were extracted hydro-alcoholically from powdered plant parts and analyzed using LC-MS/MS. Based on mass-to-charge ratio from LC-MS/MS, compounds were identified and used as ligands for molecular docking against chikungunya target proteins. The active principles were subjected to ADME/T analysis to verify their drug-likeliness. RESULTS: The docking results and ADME/T evaluation showed that the compounds, isovaleric acid and avicequinone- C have good interaction with the protein targets and hence could be the antiviral principles of the selected plant. These compounds presented acceptable drug properties and hence could be carried forward to in vivo studies for drug development. INTERPRETATION & CONCLUSION: The antiviral properties of G. pentaphylla are known since time-immemorial. This study revealed the probable interactions after the oral administration of tender leaves of Glycosmis in preventing the chikungunya virus infection and paves the path for designing future plant-based drugs.

Delayed death in the malaria parasite Plasmodium falciparum is caused by disruption of prenylation-dependent intracellular trafficking.

Apicomplexan parasites possess a plastid organelle called the apicoplast. Inhibitors that selectively target apicoplast housekeeping functions, including DNA replication and protein translation, are lethal for the parasite, and several (doxycycline, clindamycin, and azithromycin) are in clinical use as antimalarials. A major limitation of such drugs is that treated parasites only arrest one intraerythrocytic development cycle (approximately 48 hours) after treatment commences, a phenotype known as the 'delayed death' effect. The molecular basis of delayed death is a long-standing mystery in parasitology, and establishing the mechanism would aid rational clinical implementation of apicoplast-targeted drugs. Parasites undergoing delayed death transmit defective apicoplasts to their daughter cells and cannot produce the sole, blood-stage essential metabolic product of the apicoplast: the isoprenoid precursor isopentenyl-pyrophosphate. How the isoprenoid precursor depletion kills the parasite remains unknown. We investigated the requirements for the range of isoprenoids in the human malaria parasite Plasmodium falciparum and characterised the molecular and morphological phenotype of parasites experiencing delayed death. Metabolomic profiling reveals disruption of digestive vacuole function in the absence of apicoplast derived isoprenoids. Three-dimensional electron microscopy reveals digestive vacuole fragmentation and the accumulation of cytostomal invaginations, characteristics common in digestive vacuole disruption. We show that digestive vacuole disruption results from a defect in the trafficking of vesicles to the digestive vacuole. The loss of prenylation of vesicular trafficking proteins abrogates their membrane attachment and function and prevents the parasite from feeding. Our data show that the proximate cause of delayed death is an interruption of protein prenylation and consequent cellular trafficking defects.

Isoprene: New insights into the control of emission and mediation of stress tolerance by gene expression.

Isoprene is a volatile compound produced in large amounts by some, but not all, plants by the enzyme isoprene synthase. Plants emit vast quantities of isoprene, with a net global output of 600 Tg per year, and typical emission rates from individual plants around 2% of net carbon assimilation. There is significant debate about whether global climate change resulting from increasing CO2 in the atmosphere will increase or decrease global isoprene emission in the future. We show evidence supporting predictions of increased isoprene emission in the future, but the effects could vary depending on the environment under consideration. For many years, isoprene was believed to have immediate, physical effects on plants such as changing membrane properties or quenching reactive oxygen species. Although observations sometimes supported these hypotheses, the effects were not always observed, and the reasons for the variability were not apparent. Although there may be some physical effects, recent studies show that isoprene has significant effects on gene expression, the proteome, and the metabolome of both emitting and nonemitting species. Consistent results are seen across species and specific treatment protocols. This review summarizes recent findings on the role and control of isoprene emission from plants.

Regulating substrate mechanics to achieve odontogenic differentiation for dental pulp stem cells on TiO2 filled and unfilled polyisoprene.

We have shown that materials other than hydrogels commonly used in tissue engineering can be effective in enabling differentiation of dental pulp stem cells (DPSC). Here we demonstrate that a hydrophobic elastomer, polyisoprene (PI), a component of Gutta-percha, normally used to obturate the tooth canal, can also be used to initiate differentiation of the pulp. We showed that PI substrates without additional coating promote cell adhesion and differentiation, while their moduli can be easily adjusted either by varying the coating thickness or incorporation of inorganic particles. DPSC plated on those PI substrates were shown, using SPM and hysitron indentation, to adjust their moduli to conform to differentially small changes in the substrate modulus. In addition, optical tweezers were used to separately measure the membrane and cytoplasm moduli of DPSC, with and without Rho kinase inhibitor. The results indicated that the changes in modulus were attributed predominantly to changes within the cytoplasm, rather than the cell membrane. CLSM was used to identify cell morphology. Differentiation, as determined by qRT-PCR, of the upregulation of OCN, and COL1α1 as well as biomineralization, characterized by SEM/EDAX, was observed on hard PI substrates in the absence of induction factors, i.e. dexamethasone, with moduli 3-4 MPa, regardless of preparation. SEM showed that even though biomineralization was deposited on both spun cast thin PI and filled thick PI substrates, the minerals were aggregated into large clusters on thin PI, and uniformly distributed on filled thick PI, where it was templated within banded collagen fibers. STATEMENT OF SIGNIFICANCE: This manuscript demonstrates the potential of polyisoprene (PI), an elastomeric polymer, for use in tissue engineering. We show how dental pulp stem cells adjust their moduli continuously to match infinitesimally small changes in substrate mechanics, till a critical threshold is reached when they will differentiate. The lineage of differentiation then becomes a sensitive function of both mechanics and morphology for a given chemical composition. Since PI is a major component of Gutta-percha, the FDA approved material commonly used for obturating the root canal, this work suggests that it can easily be adapted for in vivo use in dental regeneration.

Isoprene Acts as a Signaling Molecule in Gene Networks Important for Stress Responses and Plant Growth.

Isoprene synthase converts dimethylallyl diphosphate to isoprene and appears to be necessary and sufficient to allow plants to emit isoprene at significant rates. Isoprene can protect plants from abiotic stress but is not produced naturally by all plants; for example, Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) do not produce isoprene. It is typically present at very low concentrations, suggesting a role as a signaling molecule; however, its exact physiological role and mechanism of action are not fully understood. We transformed Arabidopsis with a Eucalyptus globulus isoprene synthase The regulatory mechanisms of photosynthesis and isoprene emission were similar to those of native emitters, indicating that regulation of isoprene emission is not specific to isoprene-emitting species. Leaf chlorophyll and carotenoid contents were enhanced by isoprene, which also had a marked positive effect on hypocotyl, cotyledon, leaf, and inflorescence growth in Arabidopsis. By contrast, leaf and stem growth was reduced in tobacco engineered to emit isoprene. Expression of genes belonging to signaling networks or associated with specific growth regulators (e.g. gibberellic acid that promotes growth and jasmonic acid that promotes defense) and genes that lead to stress tolerance was altered by isoprene emission. Isoprene likely executes its effects on growth and stress tolerance through direct regulation of gene expression. Enhancement of jasmonic acid-mediated defense signaling by isoprene may trigger a growth-defense tradeoff leading to variations in the growth response. Our data support a role for isoprene as a signaling molecule.