Proline Incorporation Research Articles

Nature-Inspired Molecular-Crowding Enabling Wide-Humidity Range Applicable, Anti-Freezing, and Robust Zwitterionic Hydrogels for On-Skin Electronics.

Hydrogels are currently in the limelight for applications in soft electronics but they suffer from the tendency to lose water or freeze when exposed to dry environments or low temperatures. Molecular crowding is a prevalent occurrence in living cells, in which molecular crowding agents modify the hydrogen bonding structure, causing a significant reduction in water activity. Here, a wide-humidity range applicable, anti-freezing, and robust hydrogel is developed through the incorporation of natural amino acid proline (Pro) and conductive MXene into polyvinyl alcohol (PVA) hydrogel networks. Theoretical calculations reveal that Pro can transform "free water" into "locked water" via the molecular-crowding effect, thereby suppressing water evaporation and ice forming. Accordingly, the prepared hydrogel exhibits high water retention capability, with 77% and 55% being preserved after exposure to 20 °C, 28% relative humidity (RH)and 35 °C, 90% RH for 12h. Meanwhile, Pro lowers the freezing temperature of the hydrogel to 34 °C and enhances its stretchability and strength. Finally, the PVA/Pro/MXene hydrogels are assembled as multifunctional on-skin strain sensors and conductive electrodes to monitor human motions and detect tiny electrophysiological signals. Collectively, this work provides a molecular crowding strategy that will motivate researchers to develop more advanced hydrogels for versatile applications.

Loss of attachment promotes proline accumulation and excretion in cancer cells.

Previous studies have revealed a role for proline metabolism in supporting cancer development and metastasis. In this study, we show that many cancer cells respond to loss of attachment by accumulating and secreting proline. Detached cells display reduced proliferation accompanied by a general decrease in overall protein production and de novo amino acid synthesis compared to attached cells. However, proline synthesis was maintained under detached conditions. Furthermore, while overall proline incorporation into proteins was lower in detached cells compared to other amino acids, there was an increased production of the proline-rich protein collagen. The increased excretion of proline from detached cells was also shown to be used by macrophages, an abundant and important component of the tumor microenvironment. Our study suggests that detachment induced accumulation and secretion of proline may contribute to tumor progression by supporting increased production of extracellular matrix and providing proline to surrounding stromal cells.

Identification of new pentapeptides as potential inhibitors of amyloid–β42 aggregation using virtual screening and molecular dynamics simulations

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease mainly characterized by extracellular accumulation of amyloid-β (Aβ) peptide. Previous studies reported pentapeptide RIIGL as an effective inhibitor of Aβ aggregation and neurotoxicity induced by Aβ aggregates. In this work, a library of 912 pentapeptides based on RIIGL has been designed and assessed for their efficacy to inhibit Aβ42 aggregation using computational techniques. The top hit pentapeptides revealed by molecular docking were further assessed for their binding affinity with Aβ42 monomer using MM-PBSA (molecular mechanics Poisson-Boltzmann surface area) method. The MM-PBSA analysis identified RLAPV, RVVPI, and RIAPA, which bind to Aβ42 monomer with a higher binding affinity −55.80, −46.32, and −44.26 kcal/mol, respectively, as compared to RIIGL (ΔGbinding = −41.29 kcal/mol). The residue-wise binding free energy predicted hydrophobic contacts between Aβ42 monomer and pentapeptides. The secondary structure analysis of the conformational ensembles generated by molecular dynamics (MD) depicted remarkably enhanced sampling of helical and no β–sheet conformations in Aβ42 monomer on the incorporation of RVVPI and RIAPA. Notably, RVVPI and RIAPA destabilized the D23–K28 salt bridge in Aβ42 monomer, which plays a crucial role in Aβ42 oligomer stability and fibril formation. The MD simulations highlighted that the incorporation of proline and arginine in pentapeptides contributed to their strong binding with Aβ42 monomer. Furthermore, RVVPI and RIAPA prevented conformational conversion of Aβ42 monomer to aggregation-prone structures, which, in turn, resulted in a lower aggregation tendency of Aβ42 monomer.

Integration of Metal Catalysis and Organocatalysis in a Metal Nanocluster with Anchored Proline.

Chiral metal nanoclusters have recently been attracting great attention. It is challenging to realize asymmetric catalysis via atomically precise metal nanoclusters. Herein, we report the synthesis and total structure determination of chiral clusters [Au7Ag8(dppf)3(l-/d-proline)6](BF4)2 (l-/d-Au7Ag8). Superatomic clusters l-/d-Au7Ag8 display intense and mirror-image Cotton effects in their CD spectra. Density functional theory (DFT) calculations were carried out to understand the correlation between electronic structures and the optical activity of the enantiomeric pair. Surprisingly, the incorporation of proline in a metal nanocluster can significantly promote the catalytic efficiency in asymmetric Aldol reactions. The increase of catalytic activity of Au7Ag8 in comparison with organocatalysis by proline is attributed to the cooperative effect of the metal core and prolines, showing the advantages of the integration of metal catalysis and organocatalysis in a metal nanocluster.

Polyethylene glycol induced somatic embryogenesis and plant regeneration from cotyledons of Vigna radiata (L.) Wilczek

• Developed a high-frequency regeneration protocol via Polyethylene glycol (PEG)–induced somatic embryogenesis. • 3-day old cotyledonary explants used for the development of early-stage- somatic embryos. • Incorporation of ABA and proline was effective in the maturation of somatic embryos. • Regenerated plants are phenotypically identical to the mother plants. • Genetic fidelity analysis of in vitro regenerated Vigna radiata plants was reported newly. The results show that the in vitro regenerated plants are true-to-type with no somaclonal variation. • This protocol could be useful for genetic transformation studies as well as large-scale production strategies of true-to-type plants without any somoclonal variation. An efficient plantlet regeneration protocol of green gram [ Vigna radiata (L.) Wilczek] via somatic embryogenesis (SE) using Polyethylene glycol (PEG) is reported in this study. Embryogenic calli were induced using 3-day-old cotyledonary explants on MS medium supplemented with picloram (5 µM). Early-stage somatic embryos were generated by treating these 3-day-old cotyledonary explants or calli derived from those explants with osmolytes such as, sorbitol, mannitol and PEG, among which PEG was found to be the ideal. Early-stage embryoids were subsequently developed into torpedo and cotyledonary stage embryoids in liquid cultures supplemented with abscisic acid (5 μM), proline (130 μM). Furthermore, plantlet conversion was achieved in MS medium with 6-Benzylaminopurine (BAP) 1 μM + gibberellic acid (GA 3 ) 12 μM with a ca . 35% regeneration frequency. Plantlets were established under field conditions after the confirmation of their survival. The survival rate of plantlets post-transplantation was ca. 99%. The field-grown plants were confirmed to be phenotypically identical to the mother plant. According to RAPD profiles, in vitro regenerated plants were monomorphic and similar to the control/mother plants. The regeneration protocol developed in this study could be efficiently utilized for genetic transformation studies and large-scale production strategies of true-to-type plants without any somaclonal variation.

BMS-202, a PD-1/PD-L1 inhibitor, decelerates the pro-fibrotic effects of fibroblasts derived from scar tissues via ERK and TGFβ1/Smad signaling pathways.

IntroductionHypertrophic scar (HS), a fibroproliferative disorder of the skin with some tumor‐like properties, is closely related to dysregulated inflammation. PD‐1/PD‐L1 inhibitor is a promising medication for cancer therapy as its potent functions on adaptive immune response; whether it could be a candidate for HS therapy has aroused our interest. This study aimed to explore the effect and the mechanism of BMS‐202, a PD‐1/PD‐L1 inhibitor, in HS.MethodsTen HS and adjacent normal skin tissues collected from HS patients were used to detect α‐SMA, collagen I, and PD‐L1 expression by Quantitative reverse transcription‐polymerase chain reaction and western blot (WB) analysis. Fibroblasts derived from HS tissues (HFBs) were exposed to diverse concentrations of BMS‐202, of which proliferation, migration, apoptosis, and collagen synthesis were evaluated by Cell Counting Kit‐8, wound healing, terminal deoxynucleotidyl transferase (TdT) dUTP Nick‐End labeling, and [3H]‑proline incorporation assays, respectively. The effect of BMS‐202 on α‐SMA and collagen I expression, and transforming growth factor beta 1 (TGFβ1)/Smad signaling in HFBs was also determined by WB and enzyme‐linked immunosorbent assay.ResultsThe expression level of PD‐L1 was significantly elevated in both HS tissues and HFBs, which was positively correlated with α‐SMA and collagen I expressions. BMS‐202 exerted a significant suppression effect on the cell proliferation, migration, collagen synthesis, and α‐SMA and collagen I expression of HFBs in a concentration‐dependent way; but did not affect apoptosis. Finally, BMS‐202 could reduce the phosphorylation of ERK1/2, Smad2, and Smad3, and the TGFβ1 expression once its concentration reached 2.5 nM.ConclusionBMS‐202 effectively suppressed proliferation, migration, and extracellular matrix deposition of HFBs, potentially through the regulation of the ERK and TGFβ1/Smad signaling pathways.

Clinically Administered Doses of Pitavastatin and Rosuvastatin.

Clinical studies have indicated that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, also known as statins, can potentially inhibit chronic heart failure. In the Stat-LVDF study, a difference was noted in terms of the effect of lipophilic pitavastatin (PTV) and hydrophilic rosuvastatin (RSV) on plasma BNP, suggesting that statin lipophilicity and pharmacokinetics change the pleiotropic effect on heart failure in humans. Therefore, we assessed the beneficial effects of PTV on hypertrophy in cardiac myocytes compared with RSV at clinically used doses. Cultured cardiomyocytes were stimulated with 30 μM phenylephrine (PE) in the presence of PTV (250 nM) or RSV (50 nM). These doses were calculated based on the maximum blood concentration of statins used in clinical situations in Japan. The results showed that PTV, but not RSV, significantly inhibits the PE-induced increase in cell size and leucine incorporation without causing cell toxicity. In addition, PTV significantly suppressed PE-induced mRNA expression of hypertrophic response genes. PE-induced ERK phosphorylation was inhibited by PTV, but not by RSV. Furthermore, PTV significantly suppressed the angiotensin-II-induced proline incorporation in primary cultured cardiac fibroblasts. In conclusion, a clinical dose of PTV was noted to directly inhibit cardiomyocyte hypertrophy and cardiac fibrosis, suggesting that lipophilic PTV can be a potential drug candidate against chronic heart failure.

Zerumbone prevents pressure overload-induced left ventricular systolic dysfunction by inhibiting cardiac hypertrophy and fibrosis

BackgroundCardiac hypertrophy and fibrosis are hallmarks of cardiac remodeling and are involved functionally in the development of heart failure (HF). However, it is unknown whether Zerumbone (Zer) prevents left ventricular (LV) systolic dysfunction by inhibiting cardiac hypertrophy and fibrosis. PurposeThis study investigated the effect of Zer on cardiac hypertrophy and fibrosis in vitro and in vivo. Study Design/methodsIn primary cultured cardiac cells from neonatal rats, the effect of Zer on phenylephrine (PE)-induced hypertrophic responses and transforming growth factor beta (TGF-β)-induced fibrotic responses was observed. To determine whether Zer prevents the development of pressure overload-induced HF in vivo, a transverse aortic constriction (TAC) mouse model was utilized. Cardiac function was evaluated by echocardiography. The changes of cardiomyocyte surface area were observed using immunofluorescence staining and histological analysis (HE and WGA staining). Collagen synthesis and fibrosis formation were measured by scintillation counter and picrosirius staining, respectively. The total mRNA levels of genes associated with hypertrophy (ANF and BNP) and fibrosis (Postn and α-SMA) were measured by qRT-PCR. The protein expressions (Akt and α-SMA) were assessed by western blotting. ResultsZer significantly suppressed PE-induced increase in cell size, mRNA levels of ANF and BNP, and Akt phosphorylation in cardiomyocytes. The TGF-β-induced increase in proline incorporation, mRNA levels of Postn and α-SMA, and protein expression of α-SMA were decreased by Zer in cultured cardiac fibroblasts. In the TAC male C57BL/6 mice, echocardiography results demonstrated that Zer improved cardiac function by increasing LV fractional shortening and reducing LV wall thickness compared with the vehicle group. ZER significantly reduced the level of phosphorylated Akt both in cultured cardiomyocytes treated with PE and in the hearts of TAC. Finally, Zer inhibited the pressure overload-induced cardiac hypertrophy and cardiac fibrosis. ConclusionZer ameliorates pressure overload-induced LV dysfunction, at least in part by suppressing both cardiac hypertrophy and fibrosis.

Glomerular Mesangial Cell pH Homeostasis Mediates Mineralocorticoid Receptor-Induced Cell Proliferation

Mineralocorticoids (e.g., aldosterone) support chronic inflammatory tissue damage, including glomerular mesangial injury leading to glomerulosclerosis. Furthermore, aldosterone leads to activation of the extracellular signal-regulated kinases (ERK1/2) in rat glomerular mesangial cells (GMC). Because ERK1/2 can affect cellular pH homeostasis via activation of Na+/H+-exchange (NHE) and the resulting cellular alkalinization may support proliferation, we tested the hypothesis that aldosterone affects pH homeostasis and thereby cell proliferation as well as collagen secretion also in primary rat GMC. Cytoplasmic pH and calcium were assessed by single-cell fluorescence ratio imaging, using the dyes BCECF or FURA2, respectively. Proliferation was determined by cell counting, thymidine incorporation and collagen secretion by collagenase-sensitive proline incorporation and ERK1/2-phosphorylation by Western blot. Nanomolar aldosterone induces a rapid cytosolic alkalinization which is prevented by NHE inhibition (10 µmol/L EIPA) and by blockade of the mineralocorticoid receptor (100 nmol/L spironolactone). pH changes were not affected by inhibition of HCO3− transporters and were not dependent on HCO3−. Aldosterone enhanced ERK1/2 phosphorylation and inhibition of ERK1/2-phosphorylation (10 µmol/L U0126) prevented aldosterone-induced alkalinization. Furthermore, aldosterone induced proliferation of GMC and collagen secretion, both of which were prevented by U0126 and EIPA. Cytosolic calcium was not involved in this aldosterone action. In conclusion, our data show that aldosterone can induce GMC proliferation via a MR and ERK1/2-mediated activation of NHE with subsequent cytosolic alkalinization. GMC proliferation leads to glomerular hypercellularity and dysfunction. This effect presents a possible mechanism contributing to mineralocorticoid receptor-induced pathogenesis of glomerular mesangial injury during chronic kidney disease.

Antifibrotic Effects of (-)-Epicatechin on High Glucose Stimulated Cardiac Fibroblasts.

Cardiac fibrosis is one of the hallmarks of a diabetic cardiomyopathy. When activated, cardiac fibroblasts (CFs) increase the production of extracellular matrix proteins. Transforming growth factor (TGF)-β1 is known to mediate cardiac fibrosis through the SMAD pathway. High glucose (HG = 25 mM) cell culture media can activate CFs using TGF-β1. There is a need to identify effective antifibrotic agents. Studies in animals indicate that treatment with (-)-epicatechin (Epi) appears capable of reducing myocardial fibrosis. Epi binds to G-protein coupled estrogen receptor (GPER) and activates downstream pathways. We evaluated the potential of Epi to mitigate the development of a profibrotic phenotype in HG stimulated CFs. CF primary cultures were isolated from young male rats and were exposed for up to 48 h HG media and treated with vehicle or 1 μM Epi. Relevant profibrotic end points were measured by the use of various biochemical assays. HG exposure of CFs increased TGF-β1 protein levels by ∼15%, fibronectin ∼25%, urea levels ∼60%, proline incorporation ∼70%, and total collagen ∼15%. Epi treatment was able to significantly block HG induced increases in TGF-β1, fibronectin, urea, proline, and total collagen protein levels. GPER levels were reduced by HG and restored in CFs treated with Epi an effect associated with the activation (i.e., phosphorylation) of c-Src. Epi treatment also reverted SMAD levels. Altogether, results demonstrate that CFs cultured in HG acquire a profibrotic phenotype, which is blocked by Epi an effect, likely mediated at least, in part, by GPER effects on the SMAD/TGF-β1 pathway.

Connective tissue growth factor promotes retinal pigment epithelium mesenchymal transition via the PI3K/AKT signaling pathway.

Proliferative vitreoretinopathy (PVR) is a disease leading to the formation of contractile preretinal membranes (PRMs) and is one of the leading causes of blindness. Connective tissue growth factor (CTGF) has been identified as a possible key determinant of progressive tissue fibrosis and excessive scarring. Therefore, the present study investigated the role and mechanism of action of CTGF in PVR. Immunohistochemical staining was performed to detect the expression of CTGF, fibronectin and collagen type III in PRMs from patients with PVR. The effects and mechanisms of recombinant human CTGF and its upstream regulator, TGF-β1, on epithelial-mesenchymal transition (EMT) and the synthesis of extracellular matrix (ECM) by retinal pigment epithelium (RPE) cells were investigated using reverse transcription-quantitative PCR, western blotting and a [3H]proline incorporation assay. The data indicated that CTGF, fibronectin and collagen type III were highly expressed in PRMs. In vitro, CTGF significantly decreased the expression of the epithelial markers ZO-1 and E-cadherin and increased that of the mesenchymal markers fibronectin, N-cadherin and α-smooth muscle actin in a concentration-dependent manner. Furthermore, the expression of the ECM protein collagen type III was upregulated by CTGF. However, the trends in expression for the above-mentioned markers were reversed after knocking down CTGF. The incorporation of [3H]proline into RPE cells was also increased by CTGF. In addition, 8-Bromoadenosine cAMP inhibited CTGF-stimulated collagen synthesis and transient transfection of RPE cells with a CTGF antisense oligonucleotide inhibited TGF-β1-induced collagen synthesis. The phosphorylation of PI3K and AKT in RPE cells was promoted by CTGF and TGF-β1 and the latter promoted the expression of CTGF. The results of the present study indicated that CTGF may promote EMT and ECM synthesis in PVR via the PI3K/AKT signaling pathway and suggested that targeting CTGF signaling may have a therapeutic or preventative effect on PVR.

Old Drug, New Trick: Tilorone, a Broad-Spectrum Antiviral Drug as a Potential Anti-Fibrotic Therapeutic for the Diseased Heart.

Cardiac fibrosis is associated with most forms of cardiovascular disease. No reliable therapies targeting cardiac fibrosis are available, thus identifying novel drugs that can resolve or prevent fibrosis is needed. Tilorone, an antiviral agent, can prevent fibrosis in a mouse model of lung disease. We investigated the anti-fibrotic effects of tilorone in human cardiac fibroblasts in vitro by performing a radioisotopic assay for [3H]-proline incorporation as a proxy for collagen synthesis. Exploratory studies in human cardiac fibroblasts treated with tilorone (10 µM) showed a significant reduction in transforming growth factor-β induced collagen synthesis compared to untreated fibroblasts. To determine if this finding could be recapitulated in vivo, mice with established pathological remodelling due to four weeks of transverse aortic constriction (TAC) were administered tilorone (50 mg/kg, i.p) or saline every third day for eight weeks. Treatment with tilorone was associated with attenuation of fibrosis (assessed by Masson’s trichrome stain), a favourable cardiac gene expression profile and no further deterioration of cardiac systolic function determined by echocardiography compared to saline treated TAC mice. These data demonstrate that tilorone has anti-fibrotic actions in human cardiac fibroblasts and the adult mouse heart, and represents a potential novel therapy to treat fibrosis associated with heart failure.

Proline Homeostasis in Saccharomyces cerevisiae: How Does the Stress-Responsive Transcription Factor Msn2 Play a Role?

Overexpression of MSN2, which is the transcription factor gene in response to stress, is well-known to increase the tolerance of the yeast Saccharomyces cerevisiae cells to a wide variety of environmental stresses. Recent studies have found that the Msn2 is a feasible potential mediator of proline homeostasis in yeast. This result is based on the finding that overexpression of the MSN2 gene exacerbates the cytotoxicity of yeast to various amino acid analogs whose uptake is increased by the active amino acid permeases localized on the plasma membrane as a result of a dysfunctional deubiquitination process. Increased understanding of the cellular responses induced by the Msn2-mediated proline incorporation will provide better comprehension of how cells respond to and counteract to different kinds of stimuli and will also contribute to the breeding of industrial yeast strains with increased productivity.

DPP4 Inhibition, NPY1-36, PYY1-36, SDF-1α, and a Hypertensive Genetic Background Conspire to Augment Cell Proliferation and Collagen Production: Effects That Are Abolished by Low Concentrations of 2-Methoxyestradiol.

By reducing their metabolism, dipeptidyl peptidase 4 inhibition (DPP4I) enhances the effects of numerous peptides including neuropeptide Y1–36 (NPY1–36), peptide YY1–36 (PYY1–36), and SDF-1α. Studies show that separately NPY1–36, PYY1–36 and SDF-1α stimulate proliferation of, and collagen production by, cardiac fibroblasts (CFs), preglomerular vascular smooth muscle cells (PGVSMCs), and glomerular mesangial cells (GMCs), particularly in cells isolated from genetically hypertensive rats. Whether certain combinations of these factors, in the absence or presence of DPP4I, are more profibrotic than others is unknown. Here we contrasted 24 different combinations of conditions (DPP4I, hypertensive genotype and physiologic levels [3 nM] of NPY1–36, PYY1–36, or SDF-1α) on proliferation of, and [3H]-proline incorporation by, CFs, PGVSMCs, and GMCs. In all three cell types, the various treatment conditions differentially increased proliferation and [3H]-proline incorporation, with a hypertensive genotype + DPP4I + NPY1–36 + SDF-1α being the most efficacious combination. Although the effects of this four-way combination were similar in male versus female CFs, physiologic (1 nM) concentrations of 2-methoxyestradiol (2ME; nonestrogenic metabolite of 17β-estradiol), abolished the effects of this combination in both male and female CFs. In conclusion, this study demonstrates that CFs, PGVSMCs, and GMCs are differentially activated by various combinations of NPY1–36, PYY1–36, SDF-1α, a hypertensive genetic background and DPP4I. We hypothesize that as these progrowth conditions accumulate, a tipping point would be reached that manifests in the long term as organ fibrosis and that 2ME would obviate any profibrotic effects of DPP4I, even under the most profibrotic conditions (i.e., hypertensive genotype with high NPY1–36 + SDF-1α levels and low 2ME levels).SIGNIFICANCE STATEMENTThis work elucidates combinations of factors that could contribute to long-term profibrotic effects of dipeptidyl peptidase 4 inhibitors and suggests a novel drug combination that could prevent any potential profibrotic effects of dipeptidyl peptidase 4 inhibitors while augmenting the protective effects of this class of antidiabetic agents.

Molecular Switch between Structural Compaction and Thermodynamic Stability by the Xxx-Pro Interface in Transmembrane β-Barrels.

Transmembrane β-barrel scaffolds found in outer membrane proteins are formed and stabilized by a defined pattern of interstrand intraprotein H-bonds, in hydrophobic lipid bilayers. Introducing the conformationally constrained proline in β-barrels can cause significant destabilization of these structural regions that require H-bonding, with proline additionally acting as a secondary structure breaker. Membrane protein β-barrels are therefore expected to show poor tolerance to the presence of a transmembrane proline. Here, we assign a thermodynamic measure for the extent to which a single proline can be tolerated at the C-terminal interface of the model transmembrane β-barrel PagP. We find that proline drastically destabilizes PagP by 7.0 kcal mol-1 with respect to wild-type PagP (F161 → P161). Interestingly, strategic modulation of the preceding residue can modify the measured energetics. Placing a hydrophobic or bulky side chain as the preceding residue increases the thermodynamic stability by ≤8.0 kcal mol-1. While polar substituents at the preceding residue decrease the PagP stability, these residues demonstrate stronger tertiary packing interactions in the barrel and retain the catalytic activity of native PagP. This biophysical interplay between enhanced thermodynamic stability and attaining a structurally compact functional β-barrel scaffold highlights the detrimental effect caused by proline incorporation. Our findings also reveal alternative mechanisms that protein sequences can employ to salvage the structural integrity of transmembrane protein structures.

Effect of 2nd and 3rd generation PAMAM dendrimers on proliferation, differentiation, and pro-inflammatory cytokines in human keratinocytes and fibroblasts.

BackgroundPoly(amidoamine) (PAMAM) dendrimers are of considerable interest when used as a carrier for topical drugs for the skin, although little is known about their possible side effects. Therefore, our study was about the impact of 2nd and 3rd generation PAMAM dendrimers on human keratinocytes and fibroblasts cells.MethodsThe effect of the tested compounds on collagen biosynthesis was determined using 5[3H]-proline incorporation bioassay. Morphological changes accompanying cell growth inhibition were observed using a confocal microscope. To evaluate the percentage of apoptotic/necrotic cells and the cell growth dynamic of apoptotic features, we performed Annexin V/PI double staining assay, assessed caspase activity, and performed cell cycle analysis by flow cytometry. The flow cytometry method was also used to determine the effect of dendrimers on pro-inflammatory cytokines (IL-6, IL-8 IL-1β).ResultsThe obtained results showed that as the concentration and the generation of dendrimers increased, collagen biosynthesis decreased. We also observed abnormalities in cell differentiation, which may have caused disturbed secretion of pro-inflammatory cytokines. We found that dendrimers cause chronic inflammation which may cause adverse changes in the skin, ultimately– leading to apoptosis in the case of dendrimers in lower concentrations or necrosis at higher concentrations (especially 3rd generation dendrimers). In addition, the inflammatory path induced by the tested compounds was caused by damage in the mitochondria, which we observed as a significant decrease in the mitochondrial membrane potential.ConclusionThe results of our study showed that PAMAM dendrimers can cause disorders of cell proliferation and differentiation and may be the cause of cell cycle deregulation and chronic adverse inflammation.

Gluconeogenesis is essential for trypanosome development in the tsetse fly vector.

In the glucose-free environment that is the midgut of the tsetse fly vector, the procyclic form of Trypanosoma brucei primarily uses proline to feed its central carbon and energy metabolism. In these conditions, the parasite needs to produce glucose 6-phosphate (G6P) through gluconeogenesis from metabolism of non-glycolytic carbon source(s). We showed here that two phosphoenolpyruvate-producing enzymes, PEP carboxykinase (PEPCK) and pyruvate phosphate dikinase (PPDK) have a redundant function for the essential gluconeogenesis from proline. Indeed, incorporation of 13C-enriched proline into G6P was abolished in the PEPCK/PPDK null double mutant (Δppdk/Δpepck), but not in the single Δppdk and Δpepck mutant cell lines. The procyclic trypanosome also uses the glycerol conversion pathway to feed gluconeogenesis, since the death of the Δppdk/Δpepck double null mutant in glucose-free conditions is only observed after RNAi-mediated down-regulation of the expression of the glycerol kinase, the first enzyme of the glycerol conversion pathways. Deletion of the gene encoding fructose-1,6-bisphosphatase (Δfbpase), a key gluconeogenic enzyme irreversibly producing fructose 6-phosphate from fructose 1,6-bisphosphate, considerably reduced, but not abolished, incorporation of 13C-enriched proline into G6P. In addition, the Δfbpase cell line is viable in glucose-free conditions, suggesting that an alternative pathway can be used for G6P production in vitro. However, FBPase is essential in vivo, as shown by the incapacity of the Δfbpase null mutant to colonise the fly vector salivary glands, while the parental phenotype is restored in the Δfbpase rescued cell line re-expressing FBPase. The essential role of FBPase for the development of T. brucei in the tsetse was confirmed by taking advantage of an in vitro differentiation assay based on the RNA-binding protein 6 over-expression, in which the procyclic forms differentiate into epimastigote forms but not into mammalian-infective metacyclic parasites. In total, morphology, immunofluorescence and cytometry analyses showed that the differentiation of the epimastigote stages into the metacyclic forms is abolished in the Δfbpase mutant.

Quantitative Proteomic Analysis in Candida albicans Using SILAC-Based Mass Spectrometry.

Stable isotope labelling by amino acids in cell culture (SILAC) in conjunction with MS analysis is a sensitive and reliable technique for quantifying relative differences in protein abundance and posttranslational modifications between cell populations. We develop and utilise SILAC-MS workflows for quantitative proteomics in the fungal pathogen Candida albicans. Arginine metabolism provides important cues for escaping host defences during pathogenesis, which limits the use of auxotrophs in Candida research. Our strategy eliminates the need for engineering arginine auxotrophs for SILAC experiments and allows the use of ARG4 as selectable marker during strain construction. Cells that are auxotrophic for lysine are successfully labelled with both lysine and arginine stable isotopes. We find that prototrophic C. albicans preferentially uses exogenous arginine and down-regulates internal production, which allow it to achieve high incorporation rates. However, similar to other yeast, C. albicans is able to metabolise heavy arginine to heavy proline, which compromised the accuracy of protein quantification. A computational method is developed to correct for the incorporation of heavy proline. In addition, we utilise the developed SILAC labelling in C. albicans for the global quantitative proteomic analysis of a strain expressing a phosphatase-dead mutant Cdc14PD .

Novel Wine Yeast for Improved Utilisation of Proline during Fermentation

Proline is the predominant amino acid in grape juice, but it is poorly assimilated by wine yeast under the anaerobic conditions typical of most fermentations. Exploiting the abundance of this naturally occurring nitrogen source to overcome the need for nitrogen supplementation and/or the risk of stuck or sluggish fermentations would be most beneficial. This study describes the isolation and evaluation of a novel wine yeast isolate, Q7, obtained through ethyl methanesulfonate (EMS) mutagenesis. The utilisation of proline by the EMS isolate was markedly higher than by the QA23 wild type strain, with approximately 700 and 300 mg/L more consumed under aerobic and self-anaerobic fermentation conditions, respectively, in the presence of preferred nitrogen sources. Higher intracellular proline contents in the wild type strain implied a lesser rate of proline catabolism or incorporation by this strain, but with higher cell viability after freezing treatment. The expression of key genes (PUT1, PUT2, PUT3, PUT4, GAP1 and URE2) involved in proline degradation, transport and repression were compared between the parent strain and the isolate, revealing key differences. The application of these strains for efficient conduct for nitrogen-limited fermentations is a possibility.

Inactivation of Beclin-1-dependent autophagy promotes ursolic acid-induced apoptosis in hypertrophic scar fibroblasts.

A hypertrophic scar (HS) is caused by abnormal proliferation of dermal fibroblasts. Thus, promoting hypertrophic scar fibroblast (HSFB) apoptosis is an effective strategy for HS therapy. Ursolic acid (UA) has been widely used as an inducer of apoptosis in diverse cancers. However, whether UA plays an inhibitory role in HS formation is still unknown. In our study, UA was used to treat HSFBs and the cell viability, apoptosis, and collagen synthesis were determined by a Cell Counting Kit 8 assay, flow cytometry, and an H3 -proline incorporation assay, respectively. Autophagy activity was detected by LC3 immunoblotting and electron microscopy, and siRNAs targeting Beclin-1 were used to inhibit autophagy. Western blotting was performed to investigate the molecular changes in HSFBs after various treatments. We found that UA inhibited collagen synthesis and induced cell apoptosis in HSFBs, evidenced by the deregulated expression of Bim, Bcl-2 and Cyto C. Furthermore, we demonstrated that UA induced autophagy and inactivation of autophagy promoted UA-induced apoptosis and collagen synthesis inhibition in HSFBs. Molecular investigation indicated that UA-induced autophagy through upregulation of Beclin-1 and knockdown of Beclin-1 prevent UA-induced autophagy. Overexpression of Bcl-2 prevents UA-induced autophagy, Beclin-1 upregulation, apoptosis and collagen synthesis inhibition in HSFBs. Collectively, our study demonstrated that UA is a novel agent for inhibiting HS formation by promoting apoptosis, especially in combination with an autophagy inhibitor. Our results provide strong evidence of the application of UA in clinical HS treatment.