International Journal of Molecular Sciences

19 pages, 5016 KiB

Open AccessArticle

The Discovery of Highly Potent THP Derivatives as OCTN2 Inhibitors: From Structure-Based Virtual Screening to In Vivo Biological Activity

by Francesca Di Cristo, Anna Calarco, Filomena Anna Digilio, Maria Stefania Sinicropi, Camillo Rosano, Umberto Galderisi, Mariarosa Anna Beatrice Melone, Carmela Saturnino and Gianfranco Peluso

Int. J. Mol. Sci. 2020, 21(19), 7431; https://doi.org/10.3390/ijms21197431 - 8 Oct 2020

Cited by 8 | Viewed by 3093

Abstract

A mismatch between β-oxidation and the tricarboxylic acid cycle (TCA) cycle flux in mitochondria produces an accumulation of lipid metabolic intermediates, resulting in both blunted metabolic flexibility and decreased glucose utilization in the affected cells. The ability of the cell to switch to [...] Read more.

A mismatch between β-oxidation and the tricarboxylic acid cycle (TCA) cycle flux in mitochondria produces an accumulation of lipid metabolic intermediates, resulting in both blunted metabolic flexibility and decreased glucose utilization in the affected cells. The ability of the cell to switch to glucose as an energy substrate can be restored by reducing the reliance of the cell on fatty acid oxidation. The inhibition of the carnitine system, limiting the carnitine shuttle to the oxidation of lipids in the mitochondria, allows cells to develop a high plasticity to metabolic rewiring with a decrease in fatty acid oxidation and a parallel increase in glucose oxidation. We found that 3-(2,2,2-trimethylhydrazine)propionate (THP), which is able to reduce cellular carnitine levels by blocking both carnitine biosynthesis and the cell membrane carnitine/organic cation transporter (OCTN2), was reported to improve mitochondrial dysfunction in several diseases, such as Huntington’s disease (HD). Here, new THP-derived carnitine-lowering agents (TCL), characterized by a high affinity for the OCTN2 with a minimal effect on carnitine synthesis, were developed, and their biological activities were evaluated in both in vitro and in vivo HD models. Certain compounds showed promising biological activities: reducing protein aggregates in HD cells, ameliorating motility defects, and increasing the lifespan of HD Drosophila melanogaster. Full article

(This article belongs to the Section Molecular Biology)

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Graphical abstract

Graphical abstract
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(A) Binding modes of l-carnitine (white sticks) THP (yellow sticks) resulting from docking simulations. Residues involved in protein‒ligand interactions are drawn as balls and sticks; the receptor hOCTN2 is reported in tan ribbons; (B) structural modifications carried out on THP; (C) synthesis of TCL 4a–t. (a) HCOH 37%, HCOOH, 60 °C, 16 h; (b) for 3a-j and 3l-t: amine, DCC, HOBT, Et3N, CH2Cl2, or THF, RT, 24 h, for 3k: 4-nitroaniline, PCl3, pyridine, 3 h, 40 °C; (c) CH3I, acetone, 18 h, RT. Abbreviations: 3-(2,2,2-trimethylhydrazine)propionate, THP; THP structurally related compounds, TCL; hydroxymethylene, HCOH; formic acid, HCOOH; N,N’- dicyclohexylcarbodiimide, DCC; hydroxybenzotriazole, HOBT; N,N-diethylethanamine, Et3N; dichloromethane, CH2Cl2; tetrahydrofurane, THF; room temperature, RT; phosphorus trichloride, iodomethane, PCl3. Full article ">Figure 2
(a,b) Western blot analysis of hOCTN2 expression in STHdhQ7/7, STHdhQ111/111, and transfected STHdhQ111/111 (STHdhQ111/111/hOCTN2). §§ p < 0.01 vs. STHdhQ7/7. (c) The total accumulation of carnitine (10 µM), THP, or TCL (50 μM) in STHdhQ111/111 cells with or without the heterologous expression of hOCTN2. (d) The efficiency of the transport of drugs by hOCTN2 in STHdhQ111/111 cells with the heterologous expression of hOCTN2. (e) Accumulation at 37 °C or at 4 °C of carnitine, THP, or TCL in STHdhQ111/111 cells with or without the heterologous expression of hOCTN2. (f) Carnitine uptake in OCTN2-transfected STHdhQ111/111 cells in the presence of 50 μM THP, and the newly synthesized compound. The bars represent the mean ± standard deviation (n = 3). Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CTL. Full article ">Figure 3
(a,b) STHdhQ111/111 treated with 50 μM of THP or selected TCL; (c,d) Bioenergetics studies of STHdhQ111/111 cultured in the presence of pyruvate as a specific energetic substrate. The percent capacity to use pyruvate for respiration as well as the percent dependence on pyruvate was calculated as described in the Materials and Methods <a href="#sec4dot9-ijms-21-07431" class="html-sec">Section 4.9</a>. Each experiment was independently repeated at least three times, in separate 24-well plates with each treatment in 4–12 replicate wells. Full article ">Figure 4
The binding modes of the different ligands tested, resulting from docking simulations. The receptor hOCTN2 is depicted by tan ribbons, and all the ligands are sticks. The residues involved in protein‒ligand interactions are drawn as shapes and sticks. (a,b) The binding mode for 4i (pink sticks) and 4j (light blue sticks); 4k is in green in (c); (d) molecule 4t is shown in orange. Full article ">Figure 5
Representative western blot of mHtt aggregates in the STHdhQ111/111 cell line incubated for 72 h in the presence of of 50 μM 4i, 4j, 4k, or 4t. Densitometric quantification was performed on three different experiments, and the results are expressed as the mean of the values obtained (mean ± SD). ** p < 0.01 and *** p < 0.001 versus control. Full article ">Figure 6
The effects of THP-derived molecules 4i, 4j, 4k, and 4t on fly lifespan and motor dysfunction. (a–d) Lifespan curves of transgenic UAS-Q128HD-FL/Elav-GAL4 flies fed with diets supplemented with the tested molecules. For each molecule, the comparison of age-dependent survival curves was done both with respect to the solvent alone and to THP. All treated groups had increased lifespans compared to both the control group and the THP group. (a) A longer extension in survival with respect to THP was observed in response to 4i, (p < 0.001), with a 44.95% increase in the mean lifespan. (b) A dramatic comparable increase was obtained after treatment with 4j (p < 0.001), with an increase in the mean lifespan of 25.3%. (c) A moderate but highly significant increase in survival was observed in response to 4k (p < 0.001; mean lifespan +16.96%). (d) A minor increase in survival, although highly significant, was observed in response to 4t (p < 0.01; mean lifespan +9.18%). n = 100 flies. (e) The climbing ability of UAS-Q128HD-FL/Elav-GAL4 transgenic flies fed with different media supplemented with one of four different derived-THP molecules, with THP, or with vehicle alone for control, was evaluated. All the tested THP-derived molecules improved the climbing ability of transgenic flies. The data are given as the mean ± SD. n = 60. p < 0.001 compared with THP for 4j, 4i, 4k, and 4t. Full article ">

16 pages, 1153 KiB

Open AccessReview

Helicobacter pylori Virulence Factor Cytotoxin-Associated Gene A (CagA)-Mediated Gastric Pathogenicity

by Shamshul Ansari and Yoshio Yamaoka

Int. J. Mol. Sci. 2020, 21(19), 7430; https://doi.org/10.3390/ijms21197430 - 8 Oct 2020

Cited by 63 | Viewed by 7460

Abstract

Helicobacter pylori causes persistent infection in the gastric epithelium of more than half of the world’s population, leading to the development of severe complications such as peptic ulcer diseases, gastric cancer, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Several virulence factors, including cytotoxin-associated [...] Read more.

Helicobacter pylori causes persistent infection in the gastric epithelium of more than half of the world’s population, leading to the development of severe complications such as peptic ulcer diseases, gastric cancer, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Several virulence factors, including cytotoxin-associated gene A (CagA), which is translocated into the gastric epithelium via the type 4 secretory system (T4SS), have been indicated to play a vital role in disease development. Although infection with strains harboring the East Asian type of CagA possessing the EPIYA-A, -B, and -D sequences has been found to potentiate cell proliferation and disease pathogenicity, the exact mechanism of CagA involvement in disease severity still remains to be elucidated. Therefore, we discuss the possible role of CagA in gastric pathogenicity. Full article

(This article belongs to the Special Issue Microbial Virulence Factors 2.0)

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17 pages, 3604 KiB

Open AccessArticle

Vitamin E Is Superior to Vitamin C in Delaying Seedling Senescence and Improving Resistance in Arabidopsis Deficient in Macro-Elements

by Zhong-Wei Zhang, Xin-Yue Yang, Xiao-Jian Zheng, Yu-Fan Fu, Ting Lan, Xiao-Yan Tang, Chang-Quan Wang, Guang-Deng Chen, Jian Zeng and Shu Yuan

Int. J. Mol. Sci. 2020, 21(19), 7429; https://doi.org/10.3390/ijms21197429 - 8 Oct 2020

Cited by 7 | Viewed by 3042

Abstract

Nitrogen (N), phosphorus (P), and potassium (K) are three essential macro-elements for plant growth and development. Used to improve yield in agricultural production, the excessive use of chemical fertilizers often leads to increased production costs and ecological environmental pollution. Vitamins C and E [...] Read more.

Nitrogen (N), phosphorus (P), and potassium (K) are three essential macro-elements for plant growth and development. Used to improve yield in agricultural production, the excessive use of chemical fertilizers often leads to increased production costs and ecological environmental pollution. Vitamins C and E are antioxidants that play an important role in alleviating abiotic stress. However, there are few studies on alleviating oxidative stress caused by macro-element deficiency. Here, we used Arabidopsis vitamin E synthesis-deficient mutant vte4 and vitamin C synthesis-deficient mutant vtc1 on which exogenous vitamin E and vitamin C, respectively, were applied at the bolting stage. In the deficiency of macro-elements, the Arabidopsis chlorophyll content decreased, malondialdehyde (MDA) content and relative electric conductivity increased, and reactive oxygen species (ROS) accumulated. The mutants vtc1 and vte4 are more severely stressed than the wild-type plants. Adding exogenous vitamin E was found to better alleviate stress than adding vitamin C. Vitamin C barely affected and vitamin E significantly inhibited the synthesis of ethylene (ETH) and jasmonic acid (JA) genes, thereby reducing the accumulation of ETH and JA that alleviated the senescence caused by macro-element deficiency at the later stage of bolting in Arabidopsis. A deficiency of macro-elements also reduced the yield and germination rate of the seeds, which were more apparent in vtc1 and vte4, and adding exogenous vitamin C and vitamin E, respectively, could restore them. This study reported, for the first time, that vitamin E is better than vitamin C in delaying seedling senescence caused by macro-element deficiency in Arabidopsis. Full article

(This article belongs to the Special Issue Regulation of Physiological and Morphological Responses to Plant Nutrient Deficiencies)

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Figure 1
Leaf phenotypes and chlorophyll contents of Arabidopsis thaliana at the 35th day (A) and the 45th day (B) after germination. CK, DN, DP, DK represent normal, deficiency of nitrogen, deficiency of phosphorus, and deficiency of potassium treatments, respectively. vtc1 and vte4 are vitamin C and vitamin E synthetic deletion mutants, respectively. WT+VC and WT+VE represent treatments of 5 mM exogenous vitamin C and vitamin E, respectively, to the wild-type (WT) plants. Bar = 1 cm. FW, fresh weight. The data represent average values ± SEM (n = 3). Different small letters show significant differences (p < 0.05). Full article ">Figure 2
Seed pod phenotypes and chlorophyll contents of Arabidopsis thaliana at the 35th day (A) and the 45th day (B) after germination. CK, DN, DP, DK represent normal, deficiency of nitrogen, deficiency of phosphorus, and deficiency of potassium treatments, respectively. vtc1 and vte4 are vitamin C and vitamin E synthetic deletion mutants, respectively. WT+VC and WT+VE represent treatments of 5 mM exogenous vitamin C and vitamin E, respectively, to the wild-type (WT) plants. Bar = 1 cm. FW, fresh weight. The data represent average values ± SEM (n = 3). Different small letters show significant differences (p < 0.05). Full article ">Figure 3
Malondialdehyde (MDA) content (A) and relative electric conductivity (B) of 45-day-old seedlings (20 days of nutrient deficiency treatments). CK, DN, DP, DK represent normal, deficiency of nitrogen, deficiency of phosphorus, and deficiency of potassium treatments, respectively. vtc1 and vte4 are vitamin C and vitamin E synthetic deletion mutants, respectively. WT+VC and WT+VE represent treatments of 5 mM exogenous vitamin C and vitamin E, respectively, to the wild-type (WT) plants. FW, fresh weight. The data represent average values ± SEM (n = 3). Different small letters show significant differences (p < 0.05). Full article ">Figure 4
O2− (A) and H2O2 (B) accumulation levels in 45-day-old seedlings (20 days of nutrient deficiency treatments). Histochemical assays for superoxide anion radicals (O2−) and H2O2 were performed by nitro-blue tetrazolium (NBT) and 3,3’-diamino-benzidine (DAB) staining, respectively. Quantitative data are presented below the staining images. CK, DN, DP, DK represent normal, deficiency of nitrogen, deficiency of phosphorus, and deficiency of potassium treatments, respectively. vtc1 and vte4 are vitamin C and vitamin E synthetic deletion mutants, respectively. WT+VC and WT+VE represent treatments of 5 mM exogenous vitamin C and vitamin E, respectively, to the wild-type (WT) plants. FW, fresh weight. The data represent average values ± SEM (n = 3). Different small letters show significant differences (p < 0.05). Full article ">Figure 5
Relative expression of vitamin C synthetic genes VTC1 (A) and VTC2 (B) and vitamin E synthetic genes VTE4 (C) and GGR (D) in 45-day-old seedlings (20 days of nutrient deficiency treatments). CK, DN, DP, DK represent normal, deficiency of nitrogen, deficiency of phosphorus, and deficiency of potassium treatments, respectively. vtc1 and vte4 are vitamin C and vitamin E synthetic deletion mutants, respectively. WT+VC and WT+VE represent treatments of 5 mM exogenous vitamin C and vitamin E, respectively, to the wild-type (WT) plants. VTC1, Vitamin C defective 1; VTC2, Vitamin C defective 2; VTE4, Vitamin E defective 4; GGR, Geranylgeranyl Reductase. The specific gene expression levels are represented as the percentages relatively to ACTIN7 expression levels. The expression level of WT in the control sample (CK) was normalized into “1.0”. The data represent average values ± SEM (n = 3). Different small letters show significant differences (p < 0.05). Full article ">Figure 6
Relative expression of ethylene synthetic genes ACS2 (A) and ACO1 (B) and ethylene-signaling genes EIN3 (C) and ERF1 (D) and ethylene contents (E) in 45-day-old seedlings. ACS2, 1-Aminocyclopropane-1 -Carboxylic acid Synthase 2; ACO1, 1-Aminocyclopropane-1-Carboxylate Oxidase 1; EIN3, Ethylene Insensitive 3; ERF1, Ethylene Response Factor 1. The expression level of WT in the control sample (CK) was normalized into “1.0”. The data represent average values ± SEM (n = 3). Different small letters show significant differences (p < 0.05). Full article ">Figure 7
Relative expression of jasmonic acid synthetic gene COI1 (A) and jasmonic acid-signaling gene PDF1.2 (B) and jasmonic acid contents (C) in 45-day-old seedlings (20 days of nutrient deficiency treatments). COL1, CONSTANS-Like 1; PDF1.2, Plant Defensin 1.2. The expression level of WT in the control sample (CK) was normalized into “1.0”. The data represent average values ± SEM (n = 3). Different small letters show significant differences (p < 0.05). Full article ">Figure 8
Yield per plant (A), 1000 seed weight (B), pod number per plant (C), length of pod (D), and seed germination rate (E) of Arabidopsis plants after macro-element deficiency treatments. CK, DN, DP, DK represent normal, deficiency of nitrogen, deficiency of phosphorus, and deficiency of potassium treatments, respectively. vtc1 and vte4 are vitamin C and vitamin E synthetic deletion mutants, respectively. WT+VC and WT+VE represent treatments of 5 mM exogenous vitamin C and vitamin E, respectively, to the wild-type (WT) plants. The data represent average values ± SEM (n = 3). Different small letters show significant differences (p < 0.05). Full article ">

18 pages, 1909 KiB

Open AccessArticle

The Paralogue of the Intrinsically Disordered Nuclear Protein 1 Has a Nuclear Localization Sequence that Binds to Human Importin α3

by José L. Neira, Bruno Rizzuti, Ana Jiménez-Alesanco, Olga Abián, Adrián Velázquez-Campoy and Juan L. Iovanna

Int. J. Mol. Sci. 2020, 21(19), 7428; https://doi.org/10.3390/ijms21197428 - 8 Oct 2020

Cited by 7 | Viewed by 2536

Abstract

Numerous carrier proteins intervene in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin α, with several human isoforms; among them, importin α3 (Impα3) features a particularly high flexibility. The protein NUPR1L is an intrinsically disordered [...] Read more.

Numerous carrier proteins intervene in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin α, with several human isoforms; among them, importin α3 (Impα3) features a particularly high flexibility. The protein NUPR1L is an intrinsically disordered protein (IDP), evolved as a paralogue of nuclear protein 1 (NUPR1), which is involved in chromatin remodeling and DNA repair. It is predicted that NUPR1L has a nuclear localization sequence (NLS) from residues Arg51 to Gln74, in order to allow for nuclear translocation. We studied in this work the ability of intact NUPR1L to bind Impα3 and its depleted species, ∆Impα3, without the importin binding domain (IBB), using fluorescence, isothermal titration calorimetry (ITC), circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular docking techniques. Furthermore, the binding of the peptide matching the isolated NLS region of NUPR1L (NLS-NUPR1L) was also studied using the same methods. Our results show that NUPR1L was bound to Imp α3 with a low micromolar affinity (~5 μM). Furthermore, a similar affinity value was observed for the binding of NLS-NUPR1L. These findings indicate that the NLS region, which was unfolded in isolation in solution, was essentially responsible for the binding of NUPR1L to both importin species. This result was also confirmed by our in silico modeling. The binding reaction of NLS-NUPR1L to ∆Impα3 showed a larger affinity (i.e., lower dissociation constant) compared with that of Impα3, confirming that the IBB could act as an auto-inhibition region of Impα3. Taken together, our findings pinpoint the theoretical predictions of the NLS region in NUPR1L and, more importantly, suggest that this IDP relies on an importin for its nuclear translocation. Full article

(This article belongs to the Special Issue Intrinsically Disordered Proteins (IDPs): From Physical Chemistry to Pathogenic Mechanisms)

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Figure 1
Binding of intact nuclear protein 1 NUPR1L to ∆Impα3 monitored by spectroscopic techniques: (A) Fluorescence spectrum obtained by excitation at 295 nm of the complex between ∆Impα3 and intact NUPR1L, and addition spectrum obtained by the sum of the spectra of both isolated macromolecules. (B) Far-UV circular dichroism (CD) spectrum of the complex between ∆Impα3 and NUPR1L and the addition spectrum obtained by the sum of the spectra of both isolated macromolecules. (C) Thermal denaturations of ∆Impα3 in the presence and absence of NUPR1L followed by the changes in ellipticity at 222 nm. All experiments were carried out in phosphate buffer (50 mM, pH 7.0). Full article ">Figure 2
Interaction of intact NUPR1L with both importin species as measured by fluorescence: (A) Titration curve monitoring the changes of Impα3 fluorescence at 330 nm in the presence of NUPR1L, after excitation at 280 nm. (B) Titration curve monitoring the changes of ∆Impα3 fluorescence at 330 nm in the presence of NUPR1L, after excitation at 280 nm. All experiments were carried out in phosphate buffer (50 mM, pH 7.0). Full article ">Figure 3
Conformational features of isolated nuclear localization sequence (NLS)-NUPR1L in solution: (A) Far-UV CD spectrum of NLS-NUPR1L at 298 K in phosphate buffer (50 mM, pH 7.0). (B) 1D-1H-nuclear magnetic resonance (NMR) spectrum of isolated NLS-NUPR1L at 283 K and pH 7.2 (50 mM, Tris buffer). (C) NOE (Nuclear Overhuaser effect) diagram of isolated NLS-NUPR1L at 283 K: NOEs are classified into strong, medium, or weak, as represented by the height of the bar underneath the sequence; signal intensity was judged by visual inspection from the NOESY (Nuclear Overhuaser effect spectrosocopy) experiments. The corresponding Hα NOEs with the Hδ of the following proline residue are indicated by an open bar in the row corresponding to the sequential αN contacts. The dotted lines indicate NOE contacts that could not be unambiguously assigned owing to signal overlap. The numbering of residues corresponds to that of the sequence of intact NUPR1L. The symbols αN, βN, γN, and NN correspond to the sequential contacts (that is, for instance, the αN corresponds to the αN (i,i + 1) contacts). Full article ">Figure 4
Binding of NLS-NUNPR1L to both importin species: (A) Titration curve monitoring the changes of Impα3 fluorescence at 340 nm in the presence of NLS-NUPR1L, after excitation at 280 nm. (B) Titration curve monitoring the changes of ∆Impα3 fluorescence at 340 nm in the presence of NLS-NUPR1L, after excitation at 280 nm. All experiments were carried out in phosphate buffer (50 mM, pH 7.0). (C) Calorimetric binding isotherms (ligand normalized heat effect per injection as a function of the ligand/protein molar ratio) for the interaction of NLS-NUPR1L with Impα3 (left) and ∆Impα3 (right) are shown, with the thermogram (raw thermal power data as a function of time) at the top of each panel. Binding parameters were estimated by non-linear least squares regression data analysis of the interaction isotherms applying a single ligand binding site model implemented in Origin 7.0. (OriginLab, Northampton, MA, USA). Full article ">Figure 5
Binding energy of 8-residue-long fragments of NLS-NUPR1L peptide to importin: Affinity of each fragment is shown in correspondence of the two residues at the centre of each 8-residue sequence. (Inset) Difference in the binding energy between the docking pose with the highest affinity found at increasing exhaustiveness in the search versus the best pose found at any exhaustiveness value, for three 8-residue-long fragments that together span the whole sequence of the twenty-residue-long NLS-NUPR1L: (light grey) fragment RTRREQAL, (dark grey) RTNWPAPG, and (black) GHERKVAQ. Full article ">Figure 6
Predicted docking poses for NUPR1L peptide on importin. (A) Bound conformation of the capped fragment PAPGGHER: backbone (–N–Cα–C– atoms) representation of the best five docking poses on ∆Impα3. (B) Most favorable binding pose of the same fragment (cyan), compared with the crystallographic conformation [<a href="#B37-ijms-21-07428" class="html-bibr">37</a>] of the NLS of the Epstein–Barr virus EBNA-LP protein (purple). For clarity, H atoms and backbone O atoms are omitted. The tryptophan residues (orange) in the major NLS-binding site of importin are labeled. All images were created with PyMol [<a href="#B38-ijms-21-07428" class="html-bibr">38</a>]. Full article ">

14 pages, 971 KiB

Open AccessArticle

Circulating miR-99a-5p Expression in Plasma: A Potential Biomarker for Early Diagnosis of Breast Cancer

by Iris Garrido-Cano, Vera Constâncio, Anna Adam-Artigues, Ana Lameirinhas, Soraya Simón, Belen Ortega, María Teresa Martínez, Cristina Hernando, Begoña Bermejo, Ana Lluch, Paula Lopes, Rui Henrique, Carmen Jerónimo, Juan Miguel Cejalvo and Pilar Eroles

Int. J. Mol. Sci. 2020, 21(19), 7427; https://doi.org/10.3390/ijms21197427 - 8 Oct 2020

Cited by 28 | Viewed by 3579

Abstract

MicroRNAs have emerged as new diagnostic and therapeutic biomarkers for breast cancer. Herein, we analysed miR-99a-5p expression levels in primary tumours and plasma of breast cancer patients to evaluate its usefulness as a minimally invasive diagnostic biomarker. MiR-99a-5p expression levels were determined by [...] Read more.

MicroRNAs have emerged as new diagnostic and therapeutic biomarkers for breast cancer. Herein, we analysed miR-99a-5p expression levels in primary tumours and plasma of breast cancer patients to evaluate its usefulness as a minimally invasive diagnostic biomarker. MiR-99a-5p expression levels were determined by quantitative real-time PCR in three independent cohorts of patients: (I) Discovery cohort: breast cancer tissues (n = 103) and healthy breast tissues (n = 26); (II) Testing cohort: plasma samples from 105 patients and 98 healthy donors; (III) Validation cohort: plasma samples from 89 patients and 85 healthy donors. Our results demonstrated that miR-99a-5p was significantly downregulated in breast cancer tissues compared to healthy breast tissues. Conversely, miR-99a-5p levels were significantly higher in breast cancer patients than in healthy controls in plasma samples from both testing and validation cohorts, and ROC curve analysis revealed that miR-99a-5p has good diagnostic potential even to detect early breast cancer. In conclusion, miR-99a-5p’s deregulated expression distinguished healthy patients from breast cancer patients in two different types of samples (tissues and plasma). Interestingly, expression levels in plasma were significantly lower in healthy controls than in early-stage breast cancer patients. Our findings suggest circulating miR-99a-5p as a novel promising non-invasive biomarker for breast cancer detection. Full article

(This article belongs to the Section Biochemistry)

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Study design to develop a novel miRNA biomarker. Full article ">Figure 2
(A) MiR-99a expression levels in breast cancer tissues from Cohort #1. Differential miR-99a expression levels in 103 breast cancer tissues were compared with 26 normal breast tissues. Red horizontal line: median with interquartile range. Mann–Whitney U, **** p < 0.0001. (B) Receiver-operating characteristic (ROC) curve analysis for miR-99a expression levels in breast cancer tissue samples. (C) TCGA data for the expression of miR-99a-5p in normal solid tissue (n = 52) and breast primary tumour (n = 782). Expression is represented as reads per million miRNA mapped. Horizontal line: median with interquartile range. Mann–Whitney U, **** p < 0.0001. Full article ">Figure 3
(A) Circulating miR-99a levels in cohort #2. Differential miR-99a levels in 105 plasma of breast cancer patients were compared with those of 98 healthy controls. Expression levels were significantly lower in healthy controls than in breast cancer patients. Red horizontal line: median with interquartile range. Mann–Whitney U, **** p < 0.0001. (B) Receiver-operating characteristic (ROC) curve analysis for circulating miR-99a levels in cohort #2. Full article ">Figure 4
(A) Circulating miR-99a levels in cohort #3. Differential miR-99a levels in plasma of 89 breast cancer patients were compared with those of 85 healthy controls. Expression levels were significantly lower in healthy controls than in breast cancer patients. Red horizontal line: median with interquartile range. Mann–Whitney U, **** p < 0.0001. (B) Receiver-operating characteristic (ROC) curve analysis for circulating miR-99a levels in cohort #3. Full article ">Figure 5
(A) Expression of miR-99a in early-stage breast cancer plasma. Distribution of circulating miR-99a levels in 125 plasma of early-stage breast cancer patients (stage I and II) and 193 healthy controls. Expression levels were significantly lower in healthy controls than in early-stage breast cancer patients. Horizontal line: median with interquartile range. Mann–Whitney U, **** p < 0.0001. (B) Receiver-operating characteristic (ROC) curve analysis for circulating miR-99a levels in early-stage breast cancer patients. Full article ">

12 pages, 2915 KiB

Open AccessArticle

Discovery of Novel Fetal Hemoglobin Inducers through Small Chemical Library Screening

by Giulia Breveglieri, Salvatore Pacifico, Cristina Zuccato, Lucia Carmela Cosenza, Shaiq Sultan, Elisabetta D’Aversa, Roberto Gambari, Delia Preti, Claudio Trapella, Remo Guerrini and Monica Borgatti

Int. J. Mol. Sci. 2020, 21(19), 7426; https://doi.org/10.3390/ijms21197426 - 8 Oct 2020

Cited by 1 | Viewed by 2853

Abstract

The screening of chemical libraries based on cellular biosensors is a useful approach to identify new hits for novel therapeutic targets involved in rare genetic pathologies, such as β-thalassemia and sickle cell disease. In particular, pharmacologically mediated stimulation of human γ-globin [...] Read more.

The screening of chemical libraries based on cellular biosensors is a useful approach to identify new hits for novel therapeutic targets involved in rare genetic pathologies, such as β-thalassemia and sickle cell disease. In particular, pharmacologically mediated stimulation of human γ-globin gene expression, and increase of fetal hemoglobin (HbF) production, have been suggested as potential therapeutic strategies for these hemoglobinopathies. In this article, we screened a small chemical library, constituted of 150 compounds, using the cellular biosensor K562.GR, carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and β-globin gene promoters, respectively. Then the identified compounds were analyzed as HbF inducers on primary cell cultures, obtained from β-thalassemia patients, confirming their activity as HbF inducers, and suggesting these molecules as lead compounds for further chemical and biological investigations. Full article

(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)

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15 pages, 2417 KiB

Open AccessArticle

Phosphoglycerate Mutase 1 Prevents Neuronal Death from Ischemic Damage by Reducing Neuroinflammation in the Rabbit Spinal Cord

by Hyo Young Jung, Hyun Jung Kwon, Woosuk Kim, Kyu Ri Hahn, Seung Myung Moon, Yeo Sung Yoon, Dae Won Kim and In Koo Hwang

Int. J. Mol. Sci. 2020, 21(19), 7425; https://doi.org/10.3390/ijms21197425 - 8 Oct 2020

Cited by 10 | Viewed by 2928

Abstract

Phosphoglycerate mutase 1 (PGAM1) is a glycolytic enzyme that increases glycolytic flux in the brain. In the present study, we examined the effects of PGAM1 in conditions of oxidative stress and ischemic damage in motor neuron-like (NSC34) cells and the rabbit spinal cord. [...] Read more.

Phosphoglycerate mutase 1 (PGAM1) is a glycolytic enzyme that increases glycolytic flux in the brain. In the present study, we examined the effects of PGAM1 in conditions of oxidative stress and ischemic damage in motor neuron-like (NSC34) cells and the rabbit spinal cord. A Tat-PGAM1 fusion protein was prepared to allow easy crossing of the blood-brain barrier, and Control-PGAM1 was synthesized without the Tat peptide protein transduction domain. Intracellular delivery of Tat-PGAM1, not Control-PGAM1, was achieved in a time- and concentration-dependent manner. Immunofluorescent staining confirmed the intracellular expression of Tat-PGAM1 in NSC34 cells. Tat-PGAM1, but not Control-PGAM1, significantly alleviated H₂O₂-induced oxidative stress, neuronal death, mitogen-activated protein kinase, and apoptosis-inducing factor expression in NSC34 cells. After ischemia induction in the spinal cord, Tat-PGAM1 treatment significantly improved ischemia-induced neurological impairments and ameliorated neuronal cell death in the ventral horn of the spinal cord 72 h after ischemia. Tat-PGAM1 treatment significantly mitigated the ischemia-induced increase in malondialdehyde and 8-iso-prostaglandin F2α production in the spinal cord. In addition, Tat-PGAM1, but not Control-PGAM1, significantly decreased microglial activation and secretion of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α induced by ischemia in the ventral horn of the spinal cord. These results suggest that Tat-PGAM1 can be used as a therapeutic agent to reduce spinal cord ischemia-induced neuronal damage by lowering the oxidative stress, microglial activation, and secretion of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α. Full article

(This article belongs to the Section Molecular Neurobiology)

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26 pages, 2997 KiB

Open AccessReview

T Cell Activation Machinery: Form and Function in Natural and Engineered Immune Receptors

by Nicholas J. Chandler, Melissa J. Call and Matthew E. Call

Int. J. Mol. Sci. 2020, 21(19), 7424; https://doi.org/10.3390/ijms21197424 - 8 Oct 2020

Cited by 10 | Viewed by 8519

Abstract

The impressive success of chimeric antigen receptor (CAR)-T cell therapies in treating advanced B-cell malignancies has spurred a frenzy of activity aimed at developing CAR-T therapies for other cancers, particularly solid tumors, and optimizing engineered T cells for maximum clinical benefit in many [...] Read more.

The impressive success of chimeric antigen receptor (CAR)-T cell therapies in treating advanced B-cell malignancies has spurred a frenzy of activity aimed at developing CAR-T therapies for other cancers, particularly solid tumors, and optimizing engineered T cells for maximum clinical benefit in many different disease contexts. A rapidly growing body of design work is examining every modular component of traditional single-chain CARs as well as expanding out into many new and innovative engineered immunoreceptor designs that depart from this template. New approaches to immune cell and receptor engineering are being reported with rapidly increasing frequency, and many recent high-quality reviews (including one in this special issue) provide comprehensive coverage of the history and current state of the art in CAR-T and related cellular immunotherapies. In this review, we step back to examine our current understanding of the structure-function relationships in natural and engineered lymphocyte-activating receptors, with an eye towards evaluating how well the current-generation CAR designs recapitulate the most desirable features of their natural counterparts. We identify key areas that we believe are under-studied and therefore represent opportunities to further improve our grasp of form and function in natural and engineered receptors and to rationally design better therapeutics. Full article

(This article belongs to the Special Issue Recent Advances in T Cell Immunity)

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T cell activation following TCR recognition of stimulatory pMHC requires sensitivity enhancing co-receptor engagement of MHC (CD4 or CD8αβ) as well as co-stimulatory signals from constitutively expressed CD28 and several TCR induced co-stimulatory molecules (4-1BB depicted here). Yellow boxes represent ITAMs, green boxes represent non-ITAM stimulatory motifs. (A) Co-receptors CD4/CD8αβ engage MHC, dramatically increasing TCR sensitivity. (B) Positively charged tails interact with negatively charged lipid head groups. (C) Stalk cysteines facilitate interchain disulfide crosslinking. (D) Homo/hetero-typic TM interactions are vital to immunoreceptor assembly and function. Protein data bank (PDB) codes of structures shown in this figure: CD8αβ 2ATP, CD4/pMHC/TCRαβ 3TOE, TCR 6XJR (TCRαβ from 3TOE aligned against TCRαβ chains in 6XJR using pymol, 3TOE TCRαβ chains not shown), CD28 1YJD, 4-1BB/4-1BBL 6CPR. Full article ">Figure 2
2nd Generation CAR constructs: the native receptor sequences commonly incorporated and the benefits and liabilities of those domains with regard to CAR function. Structure of the scFv domain is from PDB code 3H3B. Full article ">Figure 3
Novel design approaches to improving CAR function. 3rd Gen CARs use two or more co-stimulatory tails to improve signalling outcomes (OX40, ICOS, CD3ε also used). KIR-CARs and NKG2D CARs leverage native TM interactions to constitutively recruit endogenous signaling modules DAP12 and DAP10, respectively. TCR-CAR uses a CD3ε-scFv fusion to harness the high number of ITAMs and regulatory sequences within all six CD3 tails. PDB codes of structures shown are: scFv: 3H3B, NKG2D ECD: 1MPU, TCR: 6XJR. Full article ">

5 pages, 186 KiB

Open AccessEditorial

Wheat and Barley: Acclimatization to Abiotic and Biotic Stress

by Tomasz Hura

Int. J. Mol. Sci. 2020, 21(19), 7423; https://doi.org/10.3390/ijms21197423 - 8 Oct 2020

Cited by 15 | Viewed by 3366

Abstract

Twelve articles (ten research papers and two reviews) included in the Special Issue entitled “Wheat and Barley: Acclimatization to Abiotic and Biotic Stress” are summed up here to present the latest research on the molecular background of adaptation to environmental stresses in two [...] Read more.

Twelve articles (ten research papers and two reviews) included in the Special Issue entitled “Wheat and Barley: Acclimatization to Abiotic and Biotic Stress” are summed up here to present the latest research on the molecular background of adaptation to environmental stresses in two cereal species. Crucial research results were presented and discussed, as they may be of importance in breeding aimed at increasing wheat and barley tolerance to abiotic and biotic stresses. Full article

(This article belongs to the Special Issue Wheat and Barley: Acclimatization to Abiotic and Biotic Stress)

22 pages, 3309 KiB

Open AccessArticle

Comprehensive Analysis of Antibodies Induced by Vaccination with 4 Kinds of Avian Influenza H5N1 Pre-Pandemic Vaccines

by Nobuko Ohshima, Yoshitaka Iba, Ritsuko Kubota-Koketsu, Ayami Yamasaki, Keiko Majima, Gene Kurosawa, Daisuke Hirano, Shunji Yoshida, Mototaka Sugiura, Yoshizo Asano, Yoshinobu Okuno and Yoshikazu Kurosawa

Int. J. Mol. Sci. 2020, 21(19), 7422; https://doi.org/10.3390/ijms21197422 - 8 Oct 2020

Cited by 2 | Viewed by 2383

Abstract

Four kinds of avian-derived H5N1 influenza virus, A/Vietnam/1194/2004 (Clade 1), A/Indonesia/5/2005 (Clade 2.1), A/Qinghai/1A/2005 (Clade 2.2), and A/Anhui/1/2005 (Clade 2.3), have been stocked in Japan for use as pre-pandemic vaccines. When a pandemic occurs, these viruses would be used as vaccines in the [...] Read more.

Four kinds of avian-derived H5N1 influenza virus, A/Vietnam/1194/2004 (Clade 1), A/Indonesia/5/2005 (Clade 2.1), A/Qinghai/1A/2005 (Clade 2.2), and A/Anhui/1/2005 (Clade 2.3), have been stocked in Japan for use as pre-pandemic vaccines. When a pandemic occurs, these viruses would be used as vaccines in the hope of inducing immunity against the pandemic virus. We analyzed the specificity of antibodies (Abs) produced by B lymphocytes present in the blood after immunization with these vaccines. Eighteen volunteers took part in this project. After libraries of Ab-encoding sequences were constructed using blood from subjects vaccinated with these viruses, a large number of clones that encoded Abs that bound to the virus particles used as vaccines were isolated. These clones were classified into two groups according to the hemagglutination inhibition (HI) activity of the encoded Abs. While two-thirds of the clones were HI positive, the encoded Abs exhibited only restricted strain specificity. On the other hand, half of the HI-negative clones encoded Abs that bound not only to the H5N1 virus but also to the H1N1 virus; with a few exceptions, these Abs appeared to be encoded by memory B cells present before vaccination. The HI-negative clones included those encoding broadly cross-reactive Abs, some of which were encoded by non-V_H1-69 germline genes. However, although this work shows that various kinds of anti-H5N1 Abs are encoded by volunteers vaccinated with pre-pandemic vaccines, broad cross-reactivity was seen only in a minority of clones, raising concern regarding the utility of these H5N1 vaccine viruses for the prevention of H5N1 pandemics. Full article

(This article belongs to the Section Molecular Microbiology)

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19 pages, 13043 KiB

Open AccessArticle

Intraarticular Administration Effect of Hydrogen Sulfide on an In Vivo Rat Model of Osteoarthritis

by Carlos Vaamonde-García, Elena F. Burguera, Ángela Vela-Anero, Tamara Hermida-Gómez, Purificación Filgueira-Fernández, Jennifer A. Fernández-Rodríguez, Rosa Meijide-Faílde and Francisco J. Blanco

Int. J. Mol. Sci. 2020, 21(19), 7421; https://doi.org/10.3390/ijms21197421 - 8 Oct 2020

Cited by 17 | Viewed by 4876

Abstract

Osteoarthritis (OA) is the most common articular chronic disease. However, its current treatment is limited and mostly symptomatic. Hydrogen sulfide (H₂S) is an endogenous gas with recognized physiological activities. The purpose here was to evaluate the effects of the intraarticular administration [...] Read more.

Osteoarthritis (OA) is the most common articular chronic disease. However, its current treatment is limited and mostly symptomatic. Hydrogen sulfide (H₂S) is an endogenous gas with recognized physiological activities. The purpose here was to evaluate the effects of the intraarticular administration of a slow-releasing H₂S compound (GYY-4137) on an OA experimental model. OA was induced in Wistar rats by the transection of medial collateral ligament and the removal of the medial meniscus of the left joint. The animals were randomized into three groups: non-treated and intraarticularly injected with saline or GYY-4137. Joint destabilization induced articular thickening (≈5% increment), the loss of joint mobility and flexion (≈12-degree angle), and increased levels of pain (≈1.5 points on a scale of 0 to 3). Animals treated with GYY-4137 presented improved motor function of the joint, as well as lower pain levels (≈75% recovery). We also observed that cartilage deterioration was attenuated in the GYY-4137 group (≈30% compared with the saline group). Likewise, these animals showed a reduced presence of pro-inflammatory mediators (cyclooxygenase-2, inducible nitric oxide synthase, and metalloproteinase-13) and lower oxidative damage in the cartilage. The increment of the nuclear factor-erythroid 2-related factor 2 (Nrf-2) levels and Nrf-2-regulated gene expression (≈30%) in the GYY-4137 group seem to be underlying its chondroprotective effects. Our results suggest the beneficial impact of the intraarticular administration of H₂S on experimental OA, showing a reduced cartilage destruction and oxidative damage, and supporting the use of slow H₂S-producing molecules as a complementary treatment in OA. Full article

(This article belongs to the Special Issue Redox Signaling and Oxidative Stress in Bone Health and Disease)

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Graphical abstract

Graphical abstract
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Macroscopic and clinical evaluation. Macroscopic evaluation of the animals from the experimental groups (under non-treatment (C); intraarticular injection of saline (CI) or H2S donor (SI)) was performed. Firstly, the weight (a) and articular diameter (b) were monitored. Evaluation of the pain levels (c) was performed with an arbitrary scale, as previously indicated. Finally, the angle of joint flexion (d) and extension (e) over the course of the model were assessed by a protractor. Values are mean ± SEM (n = 6 independent animals for each condition). * p ≤ 0.05 vs. day 0; $ p ≤ 0.05 vs. day 7; & p ≤ 0.05 vs. the respective day in the control group; # p ≤ 0.05 vs. the respective day in the CI group. Full article ">Figure 2
Analysis of the articular motor function with the Rotarod performance test. Animals from the three experimental groups were forced to move in the rotating cylinder for 300 s. The number of falls (a) during this period was monitored, as well as the time remaining on the rotarod (b). Values are mean ± SEM (n = 6 independent animals for each condition). * p ≤ 0.05 vs. day 0; $ p ≤ 0.05 vs. day 7; & p ≤ 0.05 vs. the respective day in the C group; # p ≤ 0.05 vs. the respective day in the CI group. C, non-treated; CI, control injection; SI, H2S donor injection. Full article ">Figure 3
Histological analysis of articular cartilage. Cartilage lesions were evaluated by the semi-quantitative modified osteoarthritis Research Society International (OARSI) score, as previously indicated in the Materials and Methods section. (a) Representative images of the joint sections stained with Safranin-O-fast green from each group of the study, showing the cartilage of the medial compartment from the tibial plateau (MTP) and femoral condyle (MFC) in the right knee (sham surgery) and left knee (OA surgery). Arrows indicate areas with a loss of cartilage matrix and ★ indicates cartilage with the loss of Safranin staining (indicator of proteoglycan content). Analysis of the semi-quantitative score of the pathological alterations in the cartilage from MFC (b) and MTP (c). Values are mean ± SEM (n = 3 independent animals for each condition). * p ≤ 0.05 vs. the respective sham-operated joint; & p ≤ 0.05 vs. the C group; # p ≤ 0.05 vs. the CI group. Control, non-treated; CI, control injection; SI, H2S donor injection. Scale bar = 500 µm. Full article ">Figure 4
Histological analysis of synovial tissue. Pathological responses in the synovial tissue were evaluated by a semi-quantitative Krenn score, as previously indicated in the Materials and Methods section. (a) Representative images of synovial sections stained with hematoxylin and eosin from each group of the study, showing synovium in the medial compartment of the right knee (sham surgery) and left knee (OA surgery). Analysis of the semi-quantitative score of the following pathological alterations in the synovial tissue: number of lining cell layers (b), proliferation of the subintima tissue (c), and infiltration of inflammatory cells (d). (e) The sum up of the pathological changes in the tissue is shown. Values are mean ± SEM (n = 3 independent animals for each condition). * p ≤ 0.05 vs. the respective sham-operated joint. Control, non-treated; CI, control injection; SI, H2S donor injection. Scale bar = 50 µm. Full article ">Figure 5
Presence of oxidative damage markers and antioxidant response in the articular cartilage. (a) Representative images of 8-hydroxy-2′-deoxyguanosine (8-oxo-dG), 4-hydroxy-2-nonenal (4-HNE), and nuclear factor erythroid-derived 2-like 2 (Nrf-2) immunohistochemistry in the cartilage from each group of the study. Magnification (2x) of the images are shown on its bottom-left corner. Quantitative analysis of 8-oxo-dG (b), 4-HNE (c), and Nrf-2 (d)-positive cells. Values are mean ± SEM (n = 3 independent animals for each condition). & p ≤ 0.05 vs. the C group; # p ≤ 0.05 vs. the CI group. Control, non-treated; CI, control injection; SI, H2S donor injection. Scale bar = 20 µm. Full article ">Figure 6
Presence of pro-catabolic mediators in the articular cartilage. (a) Representative images of metalloproteinase-13 (MMP-13), inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX-2) immunohistochemistry in the cartilage from each group of study. Magnifications (2x) of the images are shown on their bottom-left corners. Quantitative analysis of MMP-13 (b), iNOS (c), and COX-2 (d)-positive cells. Values are mean ± SEM (n = 3 independent animals for each condition). & p ≤ 0.05 vs. the C group; # p ≤ 0.05 vs. the CI group. Control, non-treated; CI, control injection; SI, H2S donor injection. Scale bar = 50 µm. Full article ">Figure 7
Measurement of the gene expression of antioxidant and pro-inflammatory markers. Relative expression levels of genes involved in antioxidant response (a) (nuclear factor erythroid-derived 2-like 2 (Nfe2l2), nicotinamide adenine dinucleotide phosphate hydrogen (NADPH):quinone oxidoreductase (Nqo1), and heme oxygenase-1 (Hmox1)) and in pro-inflammatory signaling (b) (interleukin-1β (Il1b), tumor necrosis factor-α (Tnf), cytokine-induced neutrophil chemoattractant-1 (Cxcl1), cyclooxygenase 2 (Ptgse)) were analyzed in blood samples from each group of the study, as previously indicated. Values are mean ± SEM (n = 3 independent animals for each condition). * p ≤ 0.05 vs. the CI group. CI, control injection; SI, H2S donor injection. Full article ">

20 pages, 5481 KiB

Open AccessArticle

Prognostic Role of Survivin and Macrophage Infiltration Quantified on Protein and mRNA Level in Molecular Subtypes Determined by RT-qPCR of KRT5, KRT20, and ERBB2 in Muscle-Invasive Bladder Cancer Treated by Adjuvant Chemotherapy

by Thorsten H. Ecke, Adisch Kiani, Thorsten Schlomm, Frank Friedersdorff, Anja Rabien, Klaus Jung, Ergin Kilic, Peter Boström, Minna Tervahartiala, Pekka Taimen, Jan Gleichenhagen, Georg Johnen, Thomas Brüning, Stefan Koch, Jenny Roggisch and Ralph M. Wirtz

Int. J. Mol. Sci. 2020, 21(19), 7420; https://doi.org/10.3390/ijms21197420 - 8 Oct 2020

Cited by 2 | Viewed by 3339

Abstract

Objectives: Bladder cancer is a heterogeneous malignancy. Therefore, it is difficult to find single predictive markers. Moreover, most studies focus on either the immunohistochemical or molecular assessment of tumor tissues by next-generation sequencing (NGS) or PCR, while a combination of immunohistochemistry (IHC) and [...] Read more.

Objectives: Bladder cancer is a heterogeneous malignancy. Therefore, it is difficult to find single predictive markers. Moreover, most studies focus on either the immunohistochemical or molecular assessment of tumor tissues by next-generation sequencing (NGS) or PCR, while a combination of immunohistochemistry (IHC) and PCR for tumor marker assessment might have the strongest impact to predict outcome and select optimal therapies in real-world application. We investigated the role of proliferation survivin/BIRC5 and macrophage infiltration (CD68, MAC387, CLEVER-1) on the basis of molecular subtypes of bladder cancer (KRT5, KRT20, ERBB2) to predict outcomes of adjuvant treated muscle-invasive bladder cancer patients with regard to progression-free survival (PFS) and disease-specific survival (DSS). Materials and Methods: We used tissue microarrays (TMA) from n = 50 patients (38 males, 12 female) with muscle-invasive bladder cancer. All patients had been treated with radical cystectomy followed by adjuvant triple chemotherapy. Median follow-up time was 60.5 months. CD68, CLEVER-1, MAC387, and survivin protein were detected by immunostaining and subsequent visual inspection. BIRC5, KRT5, KRT20, ERBB2, and CD68 mRNAs were detected by standardized RT-qPCR after tissue dot RNA extraction using a novel stamp technology. All these markers were evaluated in three different centers of excellence. Results: Nuclear staining rather than cytoplasmic staining of survivin predicted DSS as a single marker with high levels of survivin being associated with better PFS and DSS upon adjuvant chemotherapy (p = 0.0138 and p = 0.001, respectively). These results were validated by the quantitation of BIRC5 mRNA by PCR (p = 0.0004 and p = 0.0508, respectively). Interestingly, nuclear staining of survivin protein was positively associated with BIRC5 mRNA, while cytoplasmic staining was inversely related, indicating that the translocation of survivin protein into the nucleus occurred at a discrete, higher level of its mRNA. Combining survivin/BIRC5 levels based on molecular subtype being assessed by KRT20 expression improved the predictive value, with tumors having low survivin/BIRC5 and KRT20 mRNA levels having the best survival (75% vs. 20% vs. 10% 5-year DSS, p = 0.0005), and these values were independent of grading, node status, and tumor stage in multivariate analysis (p = 0.0167). Macrophage infiltration dominated in basal tumors and was inversely related with the luminal subtype marker gene expression. The presence of macrophages in survivin-positive or ERBB2-positive tumors was associated with worse DSS. Conclusions: For muscle-invasive bladder cancer patients, the proliferative activity as determined by the nuclear staining of survivin or RT-qPCR on the basis of molecular subtype characteristics outperforms single marker detections and single technology approaches. Infiltration by macrophages detected by IHC or PCR is associated with worse outcome in defined subsets of tumors. The limitations of this study are the retrospective nature and the limited number of patients. However, the number of molecular markers has been restricted and based on predefined assumptions, which resulted in the dissection of muscle-invasive disease into tumor–biological axes of high prognostic relevance, which warrant further investigation and validation. Full article

(This article belongs to the Special Issue Diagnostic, Prognostic and Predictive Biological Markers in Bladder Cancer – Illumination of a Vision 2.0)

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19 pages, 620 KiB

Open AccessReview

Recent Advances in Drosophila Models of Charcot-Marie-Tooth Disease

by Fukiko Kitani-Morii and Yu-ichi Noto

Int. J. Mol. Sci. 2020, 21(19), 7419; https://doi.org/10.3390/ijms21197419 - 8 Oct 2020

Cited by 8 | Viewed by 8866

Abstract

Charcot-Marie-Tooth disease (CMT) is one of the most common inherited peripheral neuropathies. CMT patients typically show slowly progressive muscle weakness and sensory loss in a distal dominant pattern in childhood. The diagnosis of CMT is based on clinical symptoms, electrophysiological examinations, and genetic [...] Read more.

Charcot-Marie-Tooth disease (CMT) is one of the most common inherited peripheral neuropathies. CMT patients typically show slowly progressive muscle weakness and sensory loss in a distal dominant pattern in childhood. The diagnosis of CMT is based on clinical symptoms, electrophysiological examinations, and genetic testing. Advances in genetic testing technology have revealed the genetic heterogeneity of CMT; more than 100 genes containing the disease causative mutations have been identified. Because a single genetic alteration in CMT leads to progressive neurodegeneration, studies of CMT patients and their respective models revealed the genotype-phenotype relationships of targeted genes. Conventionally, rodents and cell lines have often been used to study the pathogenesis of CMT. Recently, Drosophila has also attracted attention as a CMT model. In this review, we outline the clinical characteristics of CMT, describe the advantages and disadvantages of using Drosophila in CMT studies, and introduce recent advances in CMT research that successfully applied the use of Drosophila, in areas such as molecules associated with mitochondria, endosomes/lysosomes, transfer RNA, axonal transport, and glucose metabolism. Full article

(This article belongs to the Special Issue Role of Drosophila in Human Disease Research 2.0)

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27 pages, 9780 KiB

Open AccessArticle

A Comparative Transcriptome Analysis, Conserved Regulatory Elements and Associated Transcription Factors Related to Accumulation of Fusariotoxins in Grain of Rye (Secale cereale L.) Hybrids

by Khalid Mahmood, Jihad Orabi, Peter Skov Kristensen, Pernille Sarup, Lise Nistrup Jørgensen and Ahmed Jahoor

Int. J. Mol. Sci. 2020, 21(19), 7418; https://doi.org/10.3390/ijms21197418 - 8 Oct 2020

Cited by 4 | Viewed by 3236

Abstract

Detoxification of fusariotoxin is a type V Fusarium head blight (FHB) resistance and is considered a component of type II resistance, which is related to the spread of infection within spikes. Understanding this type of resistance is vital for FHB resistance, but to [...] Read more.

Detoxification of fusariotoxin is a type V Fusarium head blight (FHB) resistance and is considered a component of type II resistance, which is related to the spread of infection within spikes. Understanding this type of resistance is vital for FHB resistance, but to date, nothing is known about candidate genes that confer this resistance in rye due to scarce genomic resources. In this study, we generated a transcriptomic resource. The molecular response was mined through a comprehensive transcriptomic analysis of two rye hybrids differing in the build-up of fusariotoxin contents in grain upon pathogen infection. Gene mining identified candidate genes and pathways contributing to the detoxification of fusariotoxins in rye. Moreover, we found cis regulatory elements in the promoters of identified genes and linked them to transcription factors. In the fusariotoxin analysis, we found that grain from the Nordic seed rye hybrid “Helltop” accumulated 4 times higher concentrations of deoxynivalenol (DON), 9 times higher nivalenol (NIV), and 28 times higher of zearalenone (ZEN) than that of the hybrid “DH372” after artificial inoculation under field conditions. In the transcriptome analysis, we identified 6675 and 5151 differentially expressed genes (DEGs) in DH372 and Helltop, respectively, compared to non-inoculated control plants. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that DEGs were associated with glycolysis and the mechanistic target of rapamycin (mTOR) signaling pathway in Helltop, whereas carbon fixation in photosynthesis organisms were represented in DH372. The gene ontology (GO) enrichment and gene set enrichment analysis (GSEA) of DEGs lead to identification of the metabolic and biosynthetic processes of peptides and amides in DH372, whereas photosynthesis, negative regulation of catalytic activity, and protein-chromophore linkage were the significant pathways in Helltop. In the process of gene mining, we found four genes that were known to be involved in FHB resistance in wheat and that were differentially expressed after infection only in DH372 but not in Helltop. Based on our results, we assume that DH372 employed a specific response to pathogen infection that led to detoxification of fusariotoxin and prevented their accumulation in grain. Our results indicate that DH372 might resist the accumulation of fusariotoxin through activation of the glycolysis and drug metabolism via cytochrome P450. The identified genes in DH372 might be regulated by the WRKY family transcription factors as associated cis regulatory elements found in the in silico analysis. The results of this study will help rye breeders to develop strategies against type V FHB. Full article

(This article belongs to the Section Molecular Plant Sciences)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Graphical representation of the experimental setup and data analysis: this setup covers the fusariotoxin analysis, RNAseq experimental setup, RNA extraction, and subsequently next generation sequencing (NGS) data analysis; 5 DAI represents 5 days after inoculation. Full article ">Figure 2
Distribution and annotations of differentially expressed genes (DEGs) of both hybrids after inoculation with Fusarium spp. through assigning enzyme classes and KEGG (Kyoto Encyclopedia of Genes and Genome) pathways: (A) the Venn diagram illustrates the number of differentially expressed genes in both hybrids and divides them into three categories. (B) The major classes of enzymes of DEGs associated to exclusive DH372, exclusive Helltop, and common category and (C) the top 12 most highly represented KEGG pathways belonging to each category are shown. Analysis was performed using the OmicsBox and the KEGG database. Full article ">Figure 3
Gene enrichment analysis of differentially expressed genes (DEGs): this enrichment analysis was carried out using the upregulated genes after inoculation and belong to three different categories, i.e., (A) upregulated genes exclusive to DH372, (B) upregulated genes exclusive to Helltop, and (C) upregulated genes common in both hybrids. The top 15 significant gene-enriched gene ontology (GO) terms are shown. The test set represents the DEGs that were upregulated after inoculation, and the reference set is the total number of genes in rye known to be involved in that specific function. Full article ">Figure 4
Gene enrichment analysis of differentially expressed genes (DEGs): this enrichment analysis was carried out using the downregulated genes after inoculation in both hybrids and belong to three different categories, i.e., (A) downregulated genes exclusive to DH372, (B) downregulated genes exclusive to Helltop, and (C) downregulated genes common in both hybrids. The top 15 significant gene-enriched gene ontology (GO) terms are shown. The test set represents the DEGs that were upregulated after inoculation, and the reference set is the total number of genes in rye known to be involved in that specific function. Full article ">Figure 5
Gene set enrichment analysis (GSEA) of differentially expressed genes in both hybrids: (A) GSEA of DH372 and (B) GSEA of Helltop. The GSEA revealed that genes were recruited into different pathways in both hybrids after Fusarium spp. inoculation. The GSEA is presented in the form of a word cloud. Full article ">Figure 6
Dissection and mining of differentially expressed genes (DEGs): (A) the overall comparison of all DEGs was divided into three different categories known as exclusively responding genes, common responding genes, and contrasting responding genes categories. The exclusively responsive genes are the ones that were differentially expressed in one hybrid but absent in other. The common responding genes are the ones that exhibited similar differential expression pattern in both hybrids, whereas contrasting responding genes are the one that exhibited opposite response. (B) Comparison of cytochrome P450s in both hybrids and their division into three categories as mentioned above and (C) comparison of glycosyltransferases (GTs) in both hybrids and their division into the categories: the pentagons, stars, and circle encompass the total number of genes present in different comparisons. Venn diagrams were plotted using online tools Venny 2.0 (<a href="http://bioinfogp.cnb.csic.es/tools/venny/" target="_blank">http://bioinfogp.cnb.csic.es/tools/venny/</a>). Full article ">Figure 7
Gene expression patterns of wheat orthologs during different developmental stages and under various stresses: (A,B) these seven wheat orthologs belong to common responding genes, and (C,D) these four genes belong to exclusively responding genes in DH372. Developmental stage-specific expression patterns are shown in A and C. Our selected genes tend to show higher expression during anthesis. “HIGH,” “MEDIUM,” and “LOW” expressions were based on gene expression data found in GENEVESTIGATOR (<a href="http://www.genevestigator.com" target="_blank">http://www.genevestigator.com</a>). Heat map of expression of selected genes in response to various stresses (B,D) using Genevestigator perturbation tool: the relative expression of the genes was represented in a log2 ratio, and significant changes in expression were filtered out based on p < 0.001. Expression of genes was strongly induced in response to biotic stresses including Fusarium spp. Full article ">

24 pages, 5532 KiB

Open AccessArticle

Structural and Computational Insights into a Blebbistatin-Bound Myosin•ADP Complex with Characteristics of an ADP-Release Conformation along the Two-Step Myosin Power Stoke

by Wiebke Ewert, Peter Franz, Georgios Tsiavaliaris and Matthias Preller

Int. J. Mol. Sci. 2020, 21(19), 7417; https://doi.org/10.3390/ijms21197417 - 8 Oct 2020

Cited by 3 | Viewed by 4149

Abstract

The motor protein myosin drives a wide range of cellular and muscular functions by generating directed movement and force, fueled through adenosine triphosphate (ATP) hydrolysis. Release of the hydrolysis product adenosine diphosphate (ADP) is a fundamental and regulatory process during force production. However, [...] Read more.

The motor protein myosin drives a wide range of cellular and muscular functions by generating directed movement and force, fueled through adenosine triphosphate (ATP) hydrolysis. Release of the hydrolysis product adenosine diphosphate (ADP) is a fundamental and regulatory process during force production. However, details about the molecular mechanism accompanying ADP release are scarce due to the lack of representative structures. Here we solved a novel blebbistatin-bound myosin conformation with critical structural elements in positions between the myosin pre-power stroke and rigor states. ADP in this structure is repositioned towards the surface by the phosphate-sensing P-loop, and stabilized in a partially unbound conformation via a salt-bridge between Arg131 and Glu187. A 5 Å rotation separates the mechanical converter in this conformation from the rigor position. The crystallized myosin structure thus resembles a conformation towards the end of the two-step power stroke, associated with ADP release. Computationally reconstructing ADP release from myosin by means of molecular dynamics simulations further supported the existence of an equivalent conformation along the power stroke that shows the same major characteristics in the myosin motor domain as the resolved blebbistatin-bound myosin-II·ADP crystal structure, and identified a communication hub centered on Arg232 that mediates chemomechanical energy transduction. Full article

(This article belongs to the Section Molecular Biophysics)

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15 pages, 4074 KiB

Open AccessArticle

PPAR-α Deletion Attenuates Cisplatin Nephrotoxicity by Modulating Renal Organic Transporters MATE-1 and OCT-2

by Leandro Ceotto Freitas-Lima, Alexandre Budu, Adriano Cleis Arruda, Mauro Sérgio Perilhão, Jonatan Barrera-Chimal, Ronaldo Carvalho Araujo and Gabriel Rufino Estrela

Int. J. Mol. Sci. 2020, 21(19), 7416; https://doi.org/10.3390/ijms21197416 - 8 Oct 2020

Cited by 29 | Viewed by 3383

Abstract

Cisplatin is a chemotherapy drug widely used in the treatment of solid tumors. However, nephrotoxicity has been reported in about one-third of patients undergoing cisplatin therapy. Proximal tubules are the main target of cisplatin toxicity and cellular uptake; elimination of this drug can [...] Read more.

Cisplatin is a chemotherapy drug widely used in the treatment of solid tumors. However, nephrotoxicity has been reported in about one-third of patients undergoing cisplatin therapy. Proximal tubules are the main target of cisplatin toxicity and cellular uptake; elimination of this drug can modulate renal damage. Organic transporters play an important role in the transport of cisplatin into the kidney and organic cations transporter 2 (OCT-2) has been shown to be one of the most important transporters to play this role. On the other hand, multidrug and toxin extrusion 1 (MATE-1) transporter is the main protein that mediates the extrusion of cisplatin into the urine. Cisplatin nephrotoxicity has been shown to be enhanced by increased OCT-2 and/or reduced MATE-1 activity. Peroxisome proliferator-activated receptor alpha (PPAR-α) is the transcription factor which controls lipid metabolism and glucose homeostasis; it is highly expressed in the kidneys and interacts with both MATE-1 and OCT-2. Considering the above, we treated wild-type and PPAR-α knockout mice with cisplatin in order to evaluate the severity of nephrotoxicity. Cisplatin induced renal dysfunction, renal inflammation, apoptosis and tubular injury in wild-type mice, whereas PPAR-α deletion protected against these alterations. Moreover, we observed that cisplatin induced down-regulation of organic transporters MATE-1 and OCT-2 and that PPAR-α deletion restored the expression of these transporters. In addition, PPAR-α knockout mice at basal state showed increased MATE-1 expression and reduced OCT-2 levels. Here, we show for the first time that PPAR-α deletion protects against cisplatin nephrotoxicity and that this protection is via modulation of the organic transporters MATE-1 and OCT-2. Full article

(This article belongs to the Special Issue Physiology, Biochemistry, and Pharmacology of Transporters for Organic Cations)

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PPAR-α deletion attenuates cisplatin-induced increased pro-inflammatory cytokines and apoptosis-related genes. Cisplatin treatment (CP) increased mRNA levels of pro-inflammatory cytokines, (A) TNF-α, (B) IL-1β and (C) IL-6 in renal tissue; PPAR-α knockout mice (CP PPARKO) prevented this increase. Apoptosis-related genes (D) TNFR-2 and (E) Bax/Bcl-2 ratio were also increased by cisplatin (CP) and PPAR-α deletion (CP PPARKO) avoided this increase. n = 5–6 per group. One-way ANOVA followed by post hoc Tukey’s test. * p < 0.05 compared to the VEH group. # p < 0.05; ## p < 0.01 compared to the CP group. Full article ">Figure 2
PPAR-α deletion attenuates tubular injury and apoptosis induced by cisplatin after 96 h. Representative photomicrography of H&E staining. (A) CP treatment increases tubular injury while PPAR-α deletion attenuates it. (B) Immunofluorescence was performed to assess apoptosis. CP increases cleaved caspase-3 staining and CP PPARKO reverses this increase. In arrows is indicated tubules with the tubular lumen obstructed by the tubular casts and cell detachment from the tubular basement membrane. G to indicate glomeruli and a T for examples of tubules with normal structure, no cell detachment and free tubular lumen. n = 5 per group. Scale bar = 100 µm. One-way ANOVA followed by post hoc Tukey’s test. ** p < 0.01, **** p < 0.0001. compared to the VEH group. ## p < 0.01, #### p < 0.0001; compared to the CP group. Full article ">Figure 3
PPAR-α knockout mice mitigate the decreased mRNA and protein expression by immunofluorescence of MATE-1. Ninety-six hours after cisplatin treatment (CP) downregulates (A) mRNA and (B) protein levels of MATE-1. PPAR-α knockout mice (CP PPARKO) prevented this downregulation. G to indicate glomeruli and a T to indicate tubules. n = 5 per group. One-way ANOVA followed by post hoc Tukey´s test. Scale bar = 100 µm. * p < 0.05, *** p < 0.001 compared to the VEH group. # p < 0.05, ### p < 0.001 compared to the CP group. Full article ">Figure 4
PPAR-α ablation attenuates downregulation of mRNA and protein expression by immunofluorescence of organic cations transporter 2 (OCT-2). Ninety-six hours after cisplatin treatment (CP) downregulates (A) mRNA and (B) protein (levels of OCT-2. PPAR-α knockout mice (CP PPARKO) attenuated this downregulation. G to indicate glomeruli and a T to indicate tubules. n = 5 per group. One-way ANOVA followed by post hoc Tukey´s test. Scale bar = 100 µm. * p < 0.05, *** p < 0.001 compared to the VEH group. # p < 0.05, ## p < 0.01; compared to the CP group. Full article ">Figure 5
PPAR-α absence enhances protein expression by immunofluorescence of multidrug and toxin extrusion 1 (MATE-1). (A) No differences between WT and PPARKO mice were found in MATE-1 mRNA levels. (B) However, PPAR-α knockout mice enhanced MATE-1 protein levels. G to indicate glomeruli and a T to indicate tubules. n = 5 per group. Scale bar = 100 µm. Two-tailed Student´s t-test. * p < 0.05, compared to the WT group. Full article ">Figure 6
PPAR-α absence decreases mRNA and protein expression by immunofluorescence of organic cations transporter 2 (OCT-2). PPAR-α knockout mice presented reduced (A) mRNA and (B) protein levels of renal OCT-2. G to indicate glomeruli and a T to indicate tubules. n = 5 per group. Two-tailed Student´s t-test. Scale bar = 100 µm. * p < 0.05, *** p < 0.001 compared to the WT group. Full article ">

26 pages, 2228 KiB

Open AccessArticle

Serum-Based Proteomics Profiling in Adult Patients with Cystic Fibrosis

by Hicham Benabdelkamel, Hanadi Alamri, Meshail Okla, Afshan Masood, Mai Abdel Jabar, Ibrahim O. Alanazi, Assim A. Alfadda, Imran Nizami, Majed Dasouki and Anas M. Abdel Rahman

Int. J. Mol. Sci. 2020, 21(19), 7415; https://doi.org/10.3390/ijms21197415 - 8 Oct 2020

Cited by 13 | Viewed by 3631

Abstract

Cystic fibrosis (CF), the most common lethal autosomal recessive disorder among Caucasians, is caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel gene. Despite significant advances in the management of CF patients, novel disease-related biomarkers and therapies must be identified. [...] Read more.

Cystic fibrosis (CF), the most common lethal autosomal recessive disorder among Caucasians, is caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel gene. Despite significant advances in the management of CF patients, novel disease-related biomarkers and therapies must be identified. We performed serum proteomics profiling in CF patients (n = 28) and healthy subjects (n = 10) using the 2D-DIGE MALDI-TOF proteomic approach. Out of a total of 198 proteins identified, 134 showed a statistically significant difference in abundance and a 1.5-fold change (ANOVA, p < 0.05), including 80 proteins with increased abundance and 54 proteins with decreased abundance in CF patients. A multiple reaction monitoring-mass spectrometry analysis of six differentially expressed proteins identified by a proteomic approach (DIGE-MALD-MS) showed a significant increase in C3 and CP proteins and a decrease in APOA1, Complement C1, Hp, and RBP4proteins compared with healthy controls. Fifteen proteins were identified as potential biomarkers for CF diagnosis. An ingenuity pathway analysis of the differentially regulated proteins indicates that the central nodes dysregulated in CF subjects involve pro-inflammatory cytokines, ERK1/2, and P38 MAPK, which are primarily involved in catalytic activities and metabolic processes. The involved canonical pathways include those related to FXR/RXR, LXR/RXR, acute phase response, IL12, nitric oxide, and reactive oxygen species in macrophages. Our data support the current efforts toward augmenting protease inhibitors in patients with CF. Perturbations in lipid and vitamin metabolism frequently observed in CF patients may be partly due to abnormalities in their transport mechanism. Full article

(This article belongs to the Special Issue Biomarkers in Rare Diseases)

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Representative fluorescent protein profiles of 2D-DIGE containing a control sample labeled with Cy3 (A), a CF sample labeled with Cy5, (B) a pooled internal control labeled with Cy2, (C), and a merged 2D-DIGE comparison Cy3/Cy5 (D). Full article ">Figure 2
Multiple reaction monitoring (MRM)mass spectrometry for validating the study findings. The MRM method based on signature peptides was developed to validate the expression of six proteins found in the proteomics approach (DIGE-MALD-MS). The expression of these six proteins in CF patients was expressed in fold changes compared with the healthy controls (Ctrl). The statistical significance was evaluated using an unpaired t-test (n = 10), in which * represents p < 0.05, and **** represents p < 0.0001. Full article ">Figure 3
Statistical analysis of proteomics expression for the CF patients compared with the healthy control subjects. A volcano plot between the Ctrl and CF groups shows the significantly dysregulated proteins (42 down-regulated and 22 up-regulated in the CF group), with cutoffs of 2 and 0.05 for the fold change (x-axes) and t-test, respectively (A). An orthogonal PLS-DA score plot with eight components indicates a significant separation between the study groups (Q2: 0.805, and R2: 0.971) for 1000 permutations due to the proteomics dysregulations (B). Full article ">Figure 4
Biomarker statistical evaluation. An ROC exploratory analysis was generated by the PLS-DA model with many latent variable 2 and with increased sensitivity (x-axis) and specificity (y-axis), in which the area under the curve is at least 0.959 with a minimum combination of five variables (A). The features are ranked based on the selected frequency using the PLS-DA model, in which the color change from green to red indicates their relative expression of low to high, respectively (B); represents isoforms of the same protein found in different spots of the gel. Representative ROC curves for apolipoprotein A-I, which is down-regulated in CF (C), and protein-glutamine gamma-glutamyltransferase 6, which is up-regulated in CF (D). Full article ">Figure 5
Schematic representation of the most significant IPA networks involving the proteins that were differentially regulated between the CF and control states. The IPA analysis found that the functional interaction networks pathway with the highest score (33) was related to “metabolic diseases, neurological diseases, and disease organismal injury and abnormalities.” This pathway incorporated pro-inflammatory cytokines, ERK1/2, and P38 MAPK as central nodes that were deregulated in CF samples. The nodes in green and red correspond to down-regulated and up-regulated proteins, respectively. The colorless nodes were proposed by the IPA and suggest potential targets that are functionally coordinated with the differentially abundant proteins (A). The solid lines indicate direct molecular interactions, and the dashed lines represent indirect interactions. The diagram shows the top six canonical pathways ranked by the P-values obtained from the IPA (B). Full article ">

25 pages, 5362 KiB

Open AccessArticle

Investigating the Transition of Pre-Symptomatic to Symptomatic Huntington’s Disease Status Based on Omics Data

by Christiana C. Christodoulou, Margarita Zachariou, Marios Tomazou, Evangelos Karatzas, Christiana A. Demetriou, Eleni Zamba-Papanicolaou and George M. Spyrou

Int. J. Mol. Sci. 2020, 21(19), 7414; https://doi.org/10.3390/ijms21197414 - 8 Oct 2020

Cited by 23 | Viewed by 4062

Abstract

Huntington’s disease is a rare neurodegenerative disease caused by a cytosine–adenine–guanine (CAG) trinucleotide expansion in the Huntingtin (HTT) gene. Although Huntington’s disease (HD) is well studied, the pathophysiological mechanisms, genes and metabolites involved in HD remain poorly understood. Systems bioinformatics can [...] Read more.

Huntington’s disease is a rare neurodegenerative disease caused by a cytosine–adenine–guanine (CAG) trinucleotide expansion in the Huntingtin (HTT) gene. Although Huntington’s disease (HD) is well studied, the pathophysiological mechanisms, genes and metabolites involved in HD remain poorly understood. Systems bioinformatics can reveal synergistic relationships among different omics levels and enables the integration of biological data. It allows for the overall understanding of biological mechanisms, pathways, genes and metabolites involved in HD. The purpose of this study was to identify the differentially expressed genes (DEGs), pathways and metabolites as well as observe how these biological terms differ between the pre-symptomatic and symptomatic HD stages. A publicly available dataset from the Gene Expression Omnibus (GEO) was analyzed to obtain the DEGs for each HD stage, and gene co-expression networks were obtained for each HD stage. Network rewiring, highlights the nodes that change most their connectivity with their neighbors and infers their possible implication in the transition between different states. The CACNA1I gene was the mostly highly rewired node among pre-symptomatic and symptomatic HD network. Furthermore, we identified AF198444 to be common between the rewired genes and DEGs of symptomatic HD. CNTN6, DEK, LTN1, MST4, ZFYVE16, CEP135, DCAKD, MAP4K3, NUPL1 and RBM15 between the DEGs of pre-symptomatic and DEGs of symptomatic HD and CACNA1I, DNAJB14, EPS8L3, HSDL2, SNRPD3, SOX12, ACLY, ATF2, BAG5, ERBB4, FOCAD, GRAMD1C, LIN7C, MIR22, MTHFR, NABP1, NRG2, OTC, PRAMEF12, SLC30A10, STAG2 and Y16709 between the rewired genes and DEGs of pre-symptomatic HD. The proteins encoded by these genes are involved in various biological pathways such as phosphatidylinositol-4,5-bisphosphate 3-kinase activity, cAMP response element-binding protein binding, protein tyrosine kinase activity, voltage-gated calcium channel activity, ubiquitin protein ligase activity, adenosine triphosphate (ATP) binding, and protein serine/threonine kinase. Additionally, prominent molecular pathways for each HD stage were then obtained, and metabolites related to each pathway for both disease stages were identified. The transforming growth factor beta (TGF-β) signaling (pre-symptomatic and symptomatic stages of the disease), calcium (Ca²⁺) signaling (pre-symptomatic), dopaminergic synapse pathway (symptomatic HD patients) and Hippo signaling (pre-symptomatic) pathways were identified. The in silico metabolites we identified include Ca²⁺, inositol 1,4,5-trisphosphate, sphingosine 1-phosphate, dopamine, homovanillate and L-tyrosine. The genes, pathways and metabolites identified for each HD stage can provide a better understanding of the mechanisms that become altered in each disease stage. Our results can guide the development of therapies that may target the altered genes and metabolites of the perturbed pathways, leading to an improvement in clinical symptoms and hopefully a delay in the age of onset. Full article

(This article belongs to the Special Issue Systems Bioinformatics: How Networks and Systems Biology Boost Precision/Personalized Medicine)

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18 pages, 3284 KiB

Open AccessReview

Pericyte-Endothelial Interactions in the Retinal Microvasculature

by Hu Huang

Int. J. Mol. Sci. 2020, 21(19), 7413; https://doi.org/10.3390/ijms21197413 - 8 Oct 2020

Cited by 116 | Viewed by 10190

Abstract

Retinal microvasculature is crucial for the visual function of the neural retina. Pericytes and endothelial cells (ECs) are the two main cellular constituents in the retinal microvessels. Formation, maturation, and stabilization of the micro-vasculatures require pericyte-endothelial interactions, which are perturbed in many retinal [...] Read more.

Retinal microvasculature is crucial for the visual function of the neural retina. Pericytes and endothelial cells (ECs) are the two main cellular constituents in the retinal microvessels. Formation, maturation, and stabilization of the micro-vasculatures require pericyte-endothelial interactions, which are perturbed in many retinal vascular disorders, such as retinopathy of prematurity, retinal vein occlusion, and diabetic retinopathy. Understanding the cellular and molecular mechanisms of pericyte-endothelial interaction and perturbation can facilitate the design of therapeutic intervention for the prevention and treatment of retinal vascular disorders. Pericyte-endothelial interactions are indispensable for the integrity and functionality of retinal neurovascular unit (NVU), including vascular cells, retinal neurons, and glial cells. The essential autocrine and paracrine signaling pathways, such as Vascular endothelial growth factor (VEGF), Platelet-derived growth factor subunit B (PDGFB), Notch, Angipointein, Norrin, and Transforming growth factor-beta (TGF-β), have been well characterized for the regulation of pericyte-endothelial interactions in the neo-vessel formation processes (vasculogenesis and angiogenesis) during embryonic development. They also play a vital role in stabilizing and remodeling mature vasculature under pathological conditions. Awry signals, aberrant metabolisms, and pathological conditions, such as oxidative stress and inflammation, can disrupt the communication between pericytes and endothelial cells, thereby resulting in the breakdown of the blood-retinal barrier (BRB) and other microangiopathies. The emerging evidence supports extracellular exosomes’ roles in the (mis)communications between the two cell types. This review summarizes the essential knowledge and updates about new advancements in pericyte-EC interaction and communication, emphasizing the retinal microvasculature. Full article

(This article belongs to the Special Issue Recent Advancements in Research and Therapy of Ocular Neovascular Diseases)

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Retinal microvasculature in the adult mouse retina. The retinas were dissected from the adult mice and processed with clarity method. The cleared retinas were performed with the immunofluorescence staining of anti-PECAM1/CD31 primary antibody and Alexa Fluor 594 secondary antibody (red). The three-dimensional (3D) architectures of retinal vasculatures are visualized with low (A) and high magnification (B). One points to superficial vascular plexus at the nerve fiber layer. Two points to intermediate vascular plexus at the inner plexiform form layer. Three points to deep vascular plexus at the outer plexiform layer. The outer inner nuclear layer and the inner nuclear layer were stained with DAPI. The DAPI signals in Panel B were shown with pseudocolor (grey). Full article ">Figure 2
Pericytes-endothelial cell interactions and abnormalities in the retinal microvasculature. (A) Overview of an entire retinal microvasculature made from the adult mouse retina with the trypsin digestion method. The circles divided the vascular network into the periphery, middle, and central zones. (B) The high magnitude of retinal vasculature indicates the close interactions of pericytes and endothelial cells in the normal blood vessel walls. Red arrows point to the nuclei of endothelial cells. Green arrows point to the nuclei of pericytes. (C) The retinal vasculatures are made from the CXCR5 knockout mouse, which was subjected to ischemia-reperfusion injury, leading to a substantial loss of endothelial cells (ECs) and pericytes. Arrows point to the acellular capillary. This image is excerpted from a previous publication with the journal’s permission [<a href="#B22-ijms-21-07413" class="html-bibr">22</a>]. Full article ">Figure 3
The proposed regulation of PlGF and TGF-β in early diabetic retinopathy. Diabetes upregulates both PlGF and TGF-β in endothelial cells. PlGF can promote early diabetic retinopathy through the activation of VEGFR1 and Erk1/2 signaling, and the expression of downstream target genes, such as G6pdh (pentose phosphate pathway), Prdx3 and 6 (antioxidants), as well as the tight and adhesion junction genes (Cadh5, ZO1, and occludin). The transcription factor(s) that regulate downstream genes’ expressions are to be identified (question maker). Increased TGF-β by diabetes can protect the retina from diabetic injury in the early disease phase through the activation of TGFBR2/ALK5 signaling, which regulates the nuclear translocation of Smad2/3 and then activates the transcription of downstream target genes, such as Egf2, Edn2, and Pcam1. TGF-β can regulate PlGF through the unknown factor (question maker). Arrow lines: stimulation. Blunt line: inhibition. Full article ">Figure 4
The features of vascular organoids derived from human-induced pluripotent stem cells. (A–C) The vascular organoids are differentiated from human induced pluripotent stem cells (iPSCs). The 10-micron cryosections are prepared and stained with the endothelial cell (ECs) marker CD31 (or PECAM1, A, red) and the pericyte marker PDGFRb (B, green). The nuclei were stained with DAPI (blue). (C) The merged image shows the co-localization of the two markers in the blood vessels. (D–E) the enlarged images of the boxed areas in panels A–C. Note that the integral vascular networks of organoids vasculatures with vessel lumens, ECs, and pericytes in the blood vessels. Human organoids can be an excellent model to study EC-pericyte interaction and other human vascular diseases’ pathophysiology. Full article ">Figure 5
Co-localization analysis of pericytes and endothelial cells in the organoid vasculature. Part of the organoid vasculature (1/6) is used for analysis with the ImageJ software and EzColocalization plugin [<a href="#B96-ijms-21-07413" class="html-bibr">96</a>]. (A,B) The heat maps show the localization of PDGFRB and CD31 staining signals in the vessels. The scale bars indicate the signal intensity from low to high. (C) The scatterplot shows the relationship between the signal intensity for PDGFRB-Alex fluor 488 and CD31-Cy 5 channels. Note that (1) the numbers of data represent the vessel segments identified by the software, and (2) data with similar signal intensity for each channel indicate colocalization versus anti-colocalization by the differences in signal intensity. (D) The metric matrix for the threshold overlap score (TOS) linear median values. The X-axis and Y-axis values are the top percentile (FT) of threshold pixels for signal intensity. The black color is not informative. The red color indicates co-localization. Full article ">

19 pages, 2167 KiB

Open AccessReview

p38 MAPK Pathway in the Heart: New Insights in Health and Disease

by Rafael Romero-Becerra, Ayelén M. Santamans, Cintia Folgueira and Guadalupe Sabio

Int. J. Mol. Sci. 2020, 21(19), 7412; https://doi.org/10.3390/ijms21197412 - 8 Oct 2020

Cited by 89 | Viewed by 8014

Abstract

The p38 mitogen-activated kinase (MAPK) family controls cell adaptation to stress stimuli. p38 function has been studied in depth in relation to cardiac development and function. The first isoform demonstrated to play an important role in cardiac development was p38α; however, all p38 [...] Read more.

The p38 mitogen-activated kinase (MAPK) family controls cell adaptation to stress stimuli. p38 function has been studied in depth in relation to cardiac development and function. The first isoform demonstrated to play an important role in cardiac development was p38α; however, all p38 family members are now known to collaborate in different aspects of cardiomyocyte differentiation and growth. p38 family members have been proposed to have protective and deleterious actions in the stressed myocardium, with the outcome of their action in part dependent on the model system under study and the identity of the activated p38 family member. Most studies to date have been performed with inhibitors that are not isoform-specific, and, consequently, knowledge remains very limited about how the different p38s control cardiac physiology and respond to cardiac stress. In this review, we summarize the current understanding of the role of the p38 pathway in cardiac physiology and discuss recent advances in the field. Full article

(This article belongs to the Special Issue P38 Signaling Pathway)

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p38 in cardiovascular regeneration. (A) p38α blocks cardiovascular regeneration by inhibiting the expression of genes involved in cardiomyocyte proliferation and regeneration, such as Ect2, Crp1, ki67, cdc2, cyclin A, and p27, and reducing Rb phosphorylation to block cell-cycle progression. FGF1 stimulation has the opposite effect. (B) p38γ activates differentiation and myogenesis in satellite cells by phosphorylating MyoD and activating proliferation. p38α prevents p38γ activation in satellite cells, blocking regeneration. ↑ increase, ↓ decrease. Full article ">Figure 2
Dual role of p38 activation during preconditioning and ischemia–reperfusion injury. Activation of p38β during preconditioning triggers pro-survival signaling pathways, whereas decreased p38α activation during the ischemic episode leads to cardioprotection. On the other hand, ROS-induced p38α activation during the ischemic insult triggers HIF1-α stabilization; increases (↑) fibrosis, arrhythmias, and inflammation; and disrupts mitochondrial homeostasis. SB203580 administration during preconditioning increases myocardial injury, whereas administration during ischemia–reperfusion improves cardiac outcome. Indirect p38 downregulators, such as gamboge, statins, and antioxidants, seem to have beneficial effects when administered during or after the ischemia. Further research is needed to determine the precise reciprocity of ROS–p38 regulation. ↑ increase. Full article ">Figure 3
p38 in heart failure and cardiac arrhythmia. p38 activation participates in the development of heart failure and cardiac arrhythmia through three main mechanisms: (A) Increased cardiac fibrosis by induction of TNF-α and IL-6 in cardiomyocytes, differentiation of fibroblasts, and induction of TGF-β in cardiac myofibroblasts; (B) Reduced cardiac contractility due to dephosphorylation of a-tropomyosin and troponin I via PP2C-α/PP2C-β; (C) Promotion of cardiac arrhythmias due to reduced expression and activity of SERCA2 (Atp2a2), increased expression of NCX1 (Ncx1) and Cx43 (Cnx43), and altered Cx43 phosphorylation, inducing cardiac contractile dysfunction and altered action potential propagation. ↑ increase, ↓ decrease. Full article ">

37 pages, 4518 KiB

Open AccessReview

Amphiphilic Aminoglycosides as Medicinal Agents

by Clément Dezanet, Julie Kempf, Marie-Paule Mingeot-Leclercq and Jean-Luc Décout

Int. J. Mol. Sci. 2020, 21(19), 7411; https://doi.org/10.3390/ijms21197411 - 8 Oct 2020

Cited by 15 | Viewed by 4211

Abstract

The conjugation of hydrophobic group(s) to the polycationic hydrophilic core of the antibiotic drugs aminoglycosides (AGs), targeting ribosomal RNA, has led to the development of amphiphilic aminoglycosides (AAGs). These drugs exhibit numerous biological effects, including good antibacterial effects against susceptible and multidrug-resistant bacteria [...] Read more.

The conjugation of hydrophobic group(s) to the polycationic hydrophilic core of the antibiotic drugs aminoglycosides (AGs), targeting ribosomal RNA, has led to the development of amphiphilic aminoglycosides (AAGs). These drugs exhibit numerous biological effects, including good antibacterial effects against susceptible and multidrug-resistant bacteria due to the targeting of bacterial membranes. In the first part of this review, we summarize our work in identifying and developing broad-spectrum antibacterial AAGs that constitute a new class of antibiotic agents acting on bacterial membranes. The target-shift strongly improves antibiotic activity against bacterial strains that are resistant to the parent AG drugs and to antibiotic drugs of other classes, and renders the emergence of resistant Pseudomonas aeruginosa strains highly difficult. Structure–activity and structure–eukaryotic cytotoxicity relationships, specificity and barriers that need to be crossed in their development as antibacterial agents are delineated, with a focus on their targets in membranes, lipopolysaccharides (LPS) and cardiolipin (CL), and the corresponding mode of action against Gram-negative bacteria. At the end of the first part, we summarize the other recent advances in the field of antibacterial AAGs, mainly published since 2016, with an emphasis on the emerging AAGs which are made of an AG core conjugated to an adjuvant or an antibiotic drug of another class (antibiotic hybrids). In the second part, we briefly illustrate other biological and biochemical effects of AAGs, i.e., their antifungal activity, their use as delivery vehicles of nucleic acids, of short peptide (polyamide) nucleic acids (PNAs) and of drugs, as well as their ability to cleave DNA at abasic sites and to inhibit the functioning of connexin hemichannels. Finally, we discuss some aspects of structure–activity relationships in order to explain and improve the target selectivity of AAGs. Full article

(This article belongs to the Special Issue Structure–Activity Studies of Antibacterials and Potentiators of Antibiotics)

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16 pages, 11529 KiB

Open AccessArticle

Self-Replication of Prion Protein Fragment 89-230 Amyloid Fibrils Accelerated by Prion Protein Fragment 107-143 Aggregates

by Tomas Sneideris, Mantas Ziaunys, Brett K.-Y. Chu, Rita P.-Y. Chen and Vytautas Smirnovas

Int. J. Mol. Sci. 2020, 21(19), 7410; https://doi.org/10.3390/ijms21197410 - 8 Oct 2020

Cited by 4 | Viewed by 3250

Abstract

Prion protein amyloid aggregates are associated with infectious neurodegenerative diseases, known as transmissible spongiform encephalopathies. Self-replication of amyloid structures by refolding of native protein molecules is the probable mechanism of disease transmission. Amyloid fibril formation and self-replication can be affected by many different [...] Read more.

Prion protein amyloid aggregates are associated with infectious neurodegenerative diseases, known as transmissible spongiform encephalopathies. Self-replication of amyloid structures by refolding of native protein molecules is the probable mechanism of disease transmission. Amyloid fibril formation and self-replication can be affected by many different factors, including other amyloid proteins and peptides. Mouse prion protein fragments 107-143 (PrP(107-143)) and 89-230 (PrP(89-230)) can form amyloid fibrils.

β

-sheet core in PrP(89-230) amyloid fibrils is limited to residues ∼160–220 with unstructured N-terminus. We employed chemical kinetics tools, atomic force microscopy and Fourier-transform infrared spectroscopy, to investigate the effects of mouse prion protein fragment 107-143 fibrils on the aggregation of PrP(89-230). The data suggest that amyloid aggregates of a short prion-derived peptide are not able to seed PrP(89-230) aggregation; however, they accelerate the self-replication of PrP(89-230) amyloid fibrils. We conclude that PrP(107-143) fibrils could facilitate the self-replication of PrP(89-230) amyloid fibrils in several possible ways, and that this process deserves more attention as it may play an important role in amyloid propagation. Full article

(This article belongs to the Special Issue Amyloid Hetero-Aggregation)

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14 pages, 627 KiB

Open AccessReview

Genetic Aspects of Inflammation and Immune Response in Stroke

by Dejan Nikolic, Milena Jankovic, Bojana Petrovic and Ivana Novakovic

Int. J. Mol. Sci. 2020, 21(19), 7409; https://doi.org/10.3390/ijms21197409 - 8 Oct 2020

Cited by 22 | Viewed by 4979

Abstract

Genetic determinants play important role in the complex processes of inflammation and immune response in stroke and could be studied in different ways. Inflammation and immunomodulation are associated with repair processes in ischemic stroke, and together with the concept of preconditioning are promising [...] Read more.

Genetic determinants play important role in the complex processes of inflammation and immune response in stroke and could be studied in different ways. Inflammation and immunomodulation are associated with repair processes in ischemic stroke, and together with the concept of preconditioning are promising modes of stroke treatment. One of the important aspects to be considered in the recovery of patients after the stroke is a genetic predisposition, which has been studied extensively. Polymorphisms in a number of candidate genes, such as IL-6, BDNF, COX2, CYPC19, and GPIIIa could be associated with stroke outcome and recovery. Recent GWAS studies pointed to the variant in genesPATJ and LOC as new genetic markers of long term outcome. Epigenetic regulation of immune response in stroke is also important, with mechanisms of histone modifications, DNA methylation, and activity of non-coding RNAs. These complex processes are changing from acute phase over the repair to establishing homeostasis or to provoke exaggerated reaction and death. Pharmacogenetics and pharmacogenomics of stroke cures might also be evaluated in the context of immuno-inflammation and brain plasticity. Potential novel genetic treatment modalities are challenged but still in the early phase of the investigation. Full article

(This article belongs to the Special Issue Immunoinflammatory Background of Neuronal Damage in Stroke)

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18 pages, 3478 KiB

Open AccessArticle

Rose Bengal Crosslinking to Stabilize Collagen Sheets and Generate Modulated Collagen Laminates

by Stefanie Eckes, Joy Braun, Julia S. Wack, Ulrike Ritz, Daniela Nickel and Katja Schmitz

Int. J. Mol. Sci. 2020, 21(19), 7408; https://doi.org/10.3390/ijms21197408 - 8 Oct 2020

Cited by 6 | Viewed by 3651

Abstract

For medical application, easily accessible biomaterials with tailored properties are desirable. Collagen type I represents a biomaterial of choice for regenerative medicine and tissue engineering. Here, we present a simple method to modify the properties of collagen and to generate collagen laminates. We [...] Read more.

For medical application, easily accessible biomaterials with tailored properties are desirable. Collagen type I represents a biomaterial of choice for regenerative medicine and tissue engineering. Here, we present a simple method to modify the properties of collagen and to generate collagen laminates. We selected three commercially available collagen sheets with different thicknesses and densities and examined the effect of rose bengal and green light collagen crosslinking (RGX) on properties such as microstructure, swelling degree, mechanical stability, cell compatibility and drug release. The highest impact of RGX was measured for Atelocollagen, for which the swelling degree was reduced from 630% (w/w) to 520% (w/w) and thickness measured under force application increased from 0.014 mm to 0.455 mm, indicating a significant increase in mechanical stability. Microstructural analysis revealed that the sponge-like structure was replaced by a fibrous structure. While the initial burst effect during vancomycin release was not influenced by crosslinking, RGX increased cell proliferation on sheets of Atelocollagen and on Collagen Solutions. We furthermore demonstrate that RGX can be used to covalently attach different sheets to create materials with combined properties, making the modification and combination of readily available sheets with RGX an attractive approach for clinical application. Full article

(This article belongs to the Special Issue Optimization of Biomaterials for Reconstructive and Regenerative Medicine)

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15 pages, 8399 KiB

Open AccessArticle

LINC00885 a Novel Oncogenic Long Non-Coding RNA Associated with Early Stage Breast Cancer Progression

by Martin C. Abba, Romina Canzoneri, Agustina Gurruchaga, Jaeho Lee, Pradeep Tatineni, Hyunsuk Kil, Ezequiel Lacunza and C. Marcelo Aldaz

Int. J. Mol. Sci. 2020, 21(19), 7407; https://doi.org/10.3390/ijms21197407 - 8 Oct 2020

Cited by 15 | Viewed by 3121

Abstract

Long intergenic non-protein coding RNA 885 (LINC00885) was identified as significantly upregulated in breast ductal carcinoma in situ (DCIS). The aim of this study was to characterize the phenotypic effects and signaling pathways modulated by LINC00885 in non-invasive and invasive breast [...] Read more.

Long intergenic non-protein coding RNA 885 (LINC00885) was identified as significantly upregulated in breast ductal carcinoma in situ (DCIS). The aim of this study was to characterize the phenotypic effects and signaling pathways modulated by LINC00885 in non-invasive and invasive breast cancer models. We determined that LINC00885 induces premalignant phenotypic changes by increasing cell proliferation, motility, migration and altering 3D growth in normal and DCIS breast cell lines. Transcriptomic studies (RNA-seq) identified the main signaling pathways modulated by LINC00885, which include bioprocesses related to TP53 signaling pathway and proliferative signatures such as activation of EREG, EGFR and FOXM1 pathways. LINC00885 silencing in breast cancer lines overexpressing this lncRNA leads to downregulation of proliferation related transcripts such as EREG, CMYC, CCND1 and to significant decrease in cell migration and motility. TCGA-BRCA data analyses show an association between high LINC00885 expression and worse overall survival in patients with primary invasive breast carcinomas (p = 0.024), suggesting that the pro-tumorigenic effects of LINC00885 overexpression persist post-invasion. We conclude that LINC00885 behaves as a positive regulator of cell growth both in normal and DCIS breast cells possibly operating as a ceRNA and representing a novel oncogenic lncRNA associated with early stage breast cancer progression. Full article

(This article belongs to the Special Issue Epithelial Cells and Cancer: Victims, Villains or Heroes?)

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37 pages, 974 KiB

Open AccessReview

Therapeutic Potential of Endothelial Colony-Forming Cells in Ischemic Disease: Strategies to Improve their Regenerative Efficacy

by Pawan Faris, Sharon Negri, Angelica Perna, Vittorio Rosti, Germano Guerra and Francesco Moccia

Int. J. Mol. Sci. 2020, 21(19), 7406; https://doi.org/10.3390/ijms21197406 - 7 Oct 2020

Cited by 33 | Viewed by 4605

Abstract

Cardiovascular disease (CVD) comprises a range of major clinical cardiac and circulatory diseases, which produce immense health and economic burdens worldwide. Currently, vascular regenerative surgery represents the most employed therapeutic option to treat ischemic disorders, even though not all the patients are amenable [...] Read more.

Cardiovascular disease (CVD) comprises a range of major clinical cardiac and circulatory diseases, which produce immense health and economic burdens worldwide. Currently, vascular regenerative surgery represents the most employed therapeutic option to treat ischemic disorders, even though not all the patients are amenable to surgical revascularization. Therefore, more efficient therapeutic approaches are urgently required to promote neovascularization. Therapeutic angiogenesis represents an emerging strategy that aims at reconstructing the damaged vascular network by stimulating local angiogenesis and/or promoting de novo blood vessel formation according to a process known as vasculogenesis. In turn, circulating endothelial colony-forming cells (ECFCs) represent truly endothelial precursors, which display high clonogenic potential and have the documented ability to originate de novo blood vessels in vivo. Therefore, ECFCs are regarded as the most promising cellular candidate to promote therapeutic angiogenesis in patients suffering from CVD. The current briefly summarizes the available information about the origin and characterization of ECFCs and then widely illustrates the preclinical studies that assessed their regenerative efficacy in a variety of ischemic disorders, including acute myocardial infarction, peripheral artery disease, ischemic brain disease, and retinopathy. Then, we describe the most common pharmacological, genetic, and epigenetic strategies employed to enhance the vasoreparative potential of autologous ECFCs by manipulating crucial pro-angiogenic signaling pathways, e.g., extracellular-signal regulated kinase/Akt, phosphoinositide 3-kinase, and Ca²⁺ signaling. We conclude by discussing the possibility of targeting circulating ECFCs to rescue their dysfunctional phenotype and promote neovascularization in the presence of CVD. Full article

(This article belongs to the Special Issue Endothelial Progenitor Cells in Health and Disease)

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13 pages, 1963 KiB

Open AccessArticle

Effect of Vesicle Size on the Cytolysis of Cell-Penetrating Peptides (CPPs)

by Kazutami Sakamoto, Takeshi Kitano, Haruka Kuwahara, Megumi Tedani, Kenichi Aburai, Shiroh Futaki, Masahiko Abe, Hideki Sakai, Hiroyasu Ohtaka and Yuji Yamashita

Int. J. Mol. Sci. 2020, 21(19), 7405; https://doi.org/10.3390/ijms21197405 - 7 Oct 2020

Cited by 8 | Viewed by 3309

Abstract

A specific series of peptides, called a cell-penetrating peptide (CPP), is known to be free to directly permeate through cell membranes into the cytosol (cytolysis); hence, this CPP would be a potent carrier for a drug delivery system (DDS). Previously, we proposed the [...] Read more.

A specific series of peptides, called a cell-penetrating peptide (CPP), is known to be free to directly permeate through cell membranes into the cytosol (cytolysis); hence, this CPP would be a potent carrier for a drug delivery system (DDS). Previously, we proposed the mechanism of cytolysis as a temporal and local phase transfer of membrane lipid caused by positive membrane curvature generation. Moreover, we showed how to control the CPP cytolysis. Here, we investigate the phospholipid vesicle’s size effect on CPP cytolysis because this is the most straightforward way to control membrane curvature. Contrary to our expectation, we found that the smaller the vesicle diameter (meaning a higher membrane curvature), the more cytolysis was suppressed. Such controversial findings led us to seek the reason for the unexpected results, and we ended up finding out that the mobility of membrane lipids as a liquid crystal is the key to cytolysis. As a result, we could explain the cause of cytolysis suppression by reducing the vesicle size (because of the restriction of lipid mobility); osmotic pressure reduction to enhance positive curvature generation works as long as the membrane is mobile enough to modulate the local structure. Taking all the revealed vital factors and their effects as a tool, we will further explore how to control CPP cytolysis for developing a DDS system combined with appropriate cargo selection to be tagged with CPPs. Full article

(This article belongs to the Special Issue Assembly Superstructures in Chemistry)

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Figure 1
Confirmation of the vesicle formation for 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC). (a) Freeze-fractured transmission electron microscope (FF-TEM) image of small unilamellar vesicle (SUV); (b) FF-TEM image of large unilamellar vesicle (LUV); (c) optical microscope image of giant unilamellar vesicle (GUV). Full article ">Figure 2
Size distribution analysis for egg yolk phosphatidylcholine (EPC)-vesicles by dynamic light scattering (DLS). Full article ">Figure 3
Effect of vesicle size with osmotic pressure change for FITC–R8 cytolysis to EPC vesicles. L/P = 1000, 37 °C, 10 min, n = 3; cytolysis amount of FITC–R8/outer membrane lipids (μmol/mol). Full article ">Figure 4
Effect of osmotic pressure for FITC–R8 cytolysis vs. EPC vesicle size. L/P = 1000, 37 °C, 10 min, n = 3; cytolysis amount of FITC–R8 (CPP)/vesicle (number of CPP molecule/each vesicle). Full article ">Figure 5
Influence of temperature on cytolysis of FITC–R8 to SOPC vesicle (SOPC; L/P = 1000, 10 min, n = 3). Full article ">Figure 6
DSC profiles of SOPC vesicles. Full article ">Figure 7
Gel to liquid crystal (LC) phase transition enthalpy (ΔH) for SOPC vesicles. Full article ">Figure 8
Effect of osmotic pressure on the DSC peak profiles of SOPC vesicles. DSC peaks are recorded for a repeated freeze-and-thaw process (5–8 times). Osmotic pressures (mOsm) are 112 for hypertonic, 56 for isotonic, and 14 for hypotonic. Arrow (a) indicates the diminution of the trace of the ripple phase, and arrow (b) shows the incremental height of the phase transition peak. Full article ">

25 pages, 11077 KiB

Open AccessArticle

Conserved and Opposite Transcriptome Patterns during Germination in Hordeum vulgare and Arabidopsis thaliana

by Yanqiao Zhu, Oliver Berkowitz, Jennifer Selinski, Andreas Hartmann, Reena Narsai, Yan Wang, Peisheng Mao and James Whelan

Int. J. Mol. Sci. 2020, 21(19), 7404; https://doi.org/10.3390/ijms21197404 - 7 Oct 2020

Cited by 7 | Viewed by 3260

Abstract

Seed germination is a critical process for completion of the plant life cycle and for global food production. Comparing the germination transcriptomes of barley (Hordeum vulgare) to Arabidopsis thaliana revealed the overall pattern was conserved in terms of functional gene ontology; [...] Read more.

Seed germination is a critical process for completion of the plant life cycle and for global food production. Comparing the germination transcriptomes of barley (Hordeum vulgare) to Arabidopsis thaliana revealed the overall pattern was conserved in terms of functional gene ontology; however, many oppositely responsive orthologous genes were identified. Conserved processes included a set of approximately 6000 genes that peaked early in germination and were enriched in processes associated with RNA metabolism, e.g., pentatricopeptide repeat (PPR)-containing proteins. Comparison of orthologous genes revealed more than 3000 orthogroups containing almost 4000 genes that displayed similar expression patterns including functions associated with mitochondrial tricarboxylic acid (TCA) cycle, carbohydrate and RNA/DNA metabolism, autophagy, protein modifications, and organellar function. Biochemical and proteomic analyses indicated mitochondrial biogenesis occurred early in germination, but detailed analyses revealed the timing involved in mitochondrial biogenesis may vary between species. More than 1800 orthogroups representing 2000 genes displayed opposite patterns in transcript abundance, representing functions of energy (carbohydrate) metabolism, photosynthesis, protein synthesis and degradation, and gene regulation. Differences in expression of basic-leucine zippers (bZIPs) and Apetala 2 (AP2)/ethylene-responsive element binding proteins (EREBPs) point to differences in regulatory processes at a high level, which provide opportunities to modify processes in order to enhance grain quality, germination, and storage as needed for different uses. Full article

(This article belongs to the Special Issue Plant Respiration)

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12 pages, 4065 KiB

Open AccessArticle

N-Butylidenephthalide Inhibits the Phenotypic Switch of VSMCs through Activation of AMPK and Prevents Stenosis in an Arteriovenous Fistula Rat Model

by Hsin-Han Yang, Yue-Xuan Xu, Jie-Yi Chen, Horng-Jyh Harn and Tzyy-Wen Chiou

Int. J. Mol. Sci. 2020, 21(19), 7403; https://doi.org/10.3390/ijms21197403 - 7 Oct 2020

Cited by 6 | Viewed by 2588

Abstract

The phenotypic switch of vascular smooth muscle cells (VSMCs) plays a pivotal role in the development of vascular disorders, such as atherosclerosis, stenosis and restenosis, after vascular intervention. In our previous study, n-butylidenephthalide (BP) was reported to have anti-proliferating and apoptotic effects on [...] Read more.

The phenotypic switch of vascular smooth muscle cells (VSMCs) plays a pivotal role in the development of vascular disorders, such as atherosclerosis, stenosis and restenosis, after vascular intervention. In our previous study, n-butylidenephthalide (BP) was reported to have anti-proliferating and apoptotic effects on VSMCs. The purpose of the current study is to further investigate its role in platelet-derived growth factor (PDGF)-induced VSMC phenotypic modulation in an arteriovenous fistula model. In vitro, we observed that BP inhibited the PDGF-induced cytoskeleton reorganization of the VSMCs. The enhanced expression of vimentin and collagen, as well as the migration ability induced by PDGF, were also inhibited by BP. By cell cycle analysis, we found that BP inhibited the PDGF-induced VSMCs proliferation and arrested the VSMCs in the G0/G1 phase. In an arteriovenous fistula rat model, the formation of stenosis, which was coupled with a thrombus, and the expression of vimentin and collagen in VSMCs, were also inhibited by administration of BP, indicating that BP inhibited the PDGF-induced phenotypic switch and the migration of VSMCs. Besides, the inhibitory effects of BP on the phenotypic switch were found to accompany the activated 5’ AMP-activated protein kinase (AMPK) as well as the inhibited phosphorylation of mTOR. Knockdown of AMPK by gene silencing conflicted the effects of BP and further exacerbated the PDGF-induced VSMCs phenotypic switch, confirming the modulating effect that BP exerted on the VSMCs by this pathway. These findings suggest that BP may contribute to the vasculoprotective potential in vasculature. Full article

(This article belongs to the Section Biochemistry)

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Graphical abstract
Full article ">Figure 1
n-Butylidenephthalide (BP) inhibited PDGF-induced phenotypic switch. Measurements of morphology and protein expression in A7r5 treated with BP: (A) Representative images of PDGF-induced A7r5 in the absence or presence of BP (25 and 75 μg/mL) for 24 h. Scale bar = 50 μm; (B,C) Western blotting analysis of phenotypic marker expression. All data are presented as the means ± standard deviation of three independent experiments. * p < 0.05 and ** p < 0.01 versus the PDGF group. Full article ">Figure 2
BP inhibits migration and arrests the PDGF-induced A7r5 in the G0/G1 phase in a dose-dependent manner. (A) A wound-healing assay (upper panel, scale bar = 500 μm) and Oris system migrating assay (lower panel, scale bar = 500 μm) were performed since A7r5 was treated with PDGF (10 ng/mL) and BP (0, 25, 50, 75 and 100 µg/mL) for 24 h; (B) Cell cycle analysis through PI staining and following flow cytometry for the A7r5 after 24 h of PDGF (10 ng/mL) and BP treatment (0, 25 and 75 ug/mL); (C,D) Quantification of the Oris system migrating assay and cell cycle analysis. Data are expressed as the mean ± SEM. * p < 0.05, *** p < 0.001. Full article ">Figure 3
Histological staining of a venous limb of arteriovenous fistula (AVF) 30 days after establishment, and the comparison of lumen size among rats treated with different doses of BP. The thrombus and neointimal hyperplasia were indicated by a red arrow. (A) WT; (B) Sham; (C) 20 mg/kg-QD BP; (D) 50 mg/kg-QD BP; and (E) 50 mg/kg-BID BP. A significant reduction in thrombus and neointimal hyperplasia was observed in rats treated with BP. (F) Measurement of lumen size as reflected by the perimeter of the venous limb. Scale bar = 100 μm. Data are expressed as the mean ± SEM. n = 4 per group. * p < 0.05 versus sham. Full article ">Figure 4
Immunohistochemical detection (brown color) of a venous limb of AVF from rats treated with different doses of BP for 30 days. The expression of a phenotypic-specific marker (SMA, vimentin and collagen I) in an adjacent section of each group is indicated by a red arrow. Significant inhibition of phenotypic switch was observed in rats treated with BP compared to the control (sham) group. Scale bar = 100 μm. Full article ">Figure 5
BP inhibited the PDGF-induced phenotypic switch by activating AMPK. (A) Protein expression of AMPK and mTOR after treatment of PDGF and BP for 24 h; (B) Protein expression of AMPK, mTOR and phenotypic markers in PDGF-induced A7r5 treated without or with BP in the absence or presence of siAMPK; (C) Representative images of PDGF-induced A7r5 treated without or with BP in the absence or presence of siAMPK. Scale bar = 50 μm Full article ">

17 pages, 1946 KiB

Open AccessArticle

Antioxidant Amelioration of Riboflavin Transporter Deficiency in Motoneurons Derived from Patient-Specific Induced Pluripotent Stem Cells

by Chiara Marioli, Valentina Magliocca, Stefania Petrini, Alessia Niceforo, Rossella Borghi, Sara Petrillo, Piergiorgio La Rosa, Fiorella Colasuonno, Tiziana Persichini, Fiorella Piemonte, Keith Massey, Marco Tartaglia, Sandra Moreno, Enrico Bertini and Claudia Compagnucci

Int. J. Mol. Sci. 2020, 21(19), 7402; https://doi.org/10.3390/ijms21197402 - 7 Oct 2020

Cited by 13 | Viewed by 4771

Abstract

Mitochondrial dysfunction is a key element in the pathogenesis of neurodegenerative disorders, such as riboflavin transporter deficiency (RTD). This is a rare, childhood-onset disease characterized by motoneuron degeneration and caused by mutations in SLC52A2 and SLC52A3, encoding riboflavin (RF) transporters (RFVT2 and [...] Read more.

Mitochondrial dysfunction is a key element in the pathogenesis of neurodegenerative disorders, such as riboflavin transporter deficiency (RTD). This is a rare, childhood-onset disease characterized by motoneuron degeneration and caused by mutations in SLC52A2 and SLC52A3, encoding riboflavin (RF) transporters (RFVT2 and RFVT3, respectively), resulting in muscle weakness, ponto-bulbar paralysis and sensorineural deafness. Based on previous findings, which document the contribution of oxidative stress in RTD pathogenesis, we tested possible beneficial effects of several antioxidants (Vitamin C, Idebenone, Coenzyme Q₁₀ and EPI-743, either alone or in combination with RF) on the morphology and function of neurons derived from induced pluripotent stem cells (iPSCs) from two RTD patients. To identify possible improvement of the neuronal morphotype, neurite length was measured by confocal microscopy after β-III tubulin immunofluorescent staining. Neuronal function was evaluated by determining superoxide anion generation by MitoSOX assay and intracellular calcium (Ca²⁺) levels, using the Fluo-4 probe. Among the antioxidants tested, EPI-743 restored the redox status, improved neurite length and ameliorated intracellular calcium influx into RTD motoneurons. In conclusion, we suggest that antioxidant supplementation may have a role in RTD treatment. Full article

(This article belongs to the Special Issue hiPSC-Derived Cells as Models for Drug Discovery)

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Quantification of superoxide anion in riboflavin transporter deficiency (RTD) induced pluripotent stem cells (iPSCs) following treatment with antioxidants ascorbic acid (AA), CoQ10, Idebenone, and EPI-743, showing their effect on RTD iPSCs superoxide anion production. For each RTD cell line, P1 (A) and P2 (B), the arrows indicate the antioxidant concentration most effective in lowering the levels of superoxide anion. Experiments were conducted in triplicate and values expressed as mean ± standard error of the mean (SEM). According to Kruskal-Wallis tests * p < 0.05, *** p < 0.001, compared with controls’ group (Ctrl); # p < 0.05, ## p < 0.01 respect to untreated patients. Full article ">Figure 2
RTD iPSCs show increased lipid peroxidation. (A) Fluorescence micrographs of iPSCs labeled with BODIPY using the green (on the left, indicating oxidation of the butadienyl portion of the dye) and the red (on the middle) filter and then overlay of the red (nonoxidized) and green (oxidized) images (in the right column). Colocalization of oxidized and reduced BODIPY fluorescence appears in yellow. Bar = 100 μm. Control and RTD iPSCs were incubated for 45 min with BODIPY 581/591 C11. Treatment with EPI-743, but not other antioxidants, results in significantly reduced levels of oxidized lipids as shown by the shift of green to red fluorescent signal. (B) Bar graph reporting the quantitative analyses of the BODIPY experiments performed on control and RTD iPSCs. Values are expressed as mean ± SEM. According to Kruskal-Wallis test * p < 0.05 compared with controls iPSCs; # p < 0.05, ## p < 0.01, respect to untreated patients. Full article ">Figure 3
Analysis of neurite length following antioxidant treatment of MNs derived from RTD patient-derived iPSCs. Immunofluorescence images of β III tubulin (in red) show shorter neurites in RTD MNs compared to control cells. In both P1 and P2 RTD MNs, treatment with AA, CoQ10 and IDEB fails to cause significant changes. RF and EPI-743 ± RF causes improvement in neurite length for RTD MNs. Nuclei are counterstained with Hoechst (in blue). Scale bars = 50 μm. Data derived from four independent experiments, and values are expressed as mean ± SEM. According to Kruskal-Wallis test ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with control group (Ctrl); # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 with respect to untreated RTD patient MNs. Full article ">Figure 4
Intracellular Ca2+ flux in RTD motoneurons following EPI-743 treatment. (A) Confocal images showing changes in intracellular calcium flux in RTD and control MNs before (basal level) and after stimulation with 5 μM ionomycin. (B) Graphical representation of the mean fluorescence intensity over time of Control and RTD MNs following RF or EPI-743 treatment, showing an increase in intracellular Ca2+ following ionomycin supplementation (indicated by the black arrow) and decrease following addition of EGTA to the medium (30 s following ionomycin in all samples, as indicated by the grey arrow). Experiments were conducted in triplicate. Full article ">Figure 5
Schemae showing the morphological changes of the RTD MNs before and following treatment with EPI-743. The drawing depicts neurons with short neurites in RTD P1 and P2, but following EPI-743 treatment they extend longer neurites that, for RTD P2, are very similar to Ctrl MNs, while, for RTD P1 neurite length is improved but still not comparable to that of Ctrl MNs. N = Nucleus. n = neurite. Full article ">

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