International Journal of Molecular Sciences

27 pages, 6703 KiB

Open AccessArticle

Development of a Bispecific Antibody-Based Platform for Retargeting of Capsid Modified AAV Vectors

by Juliane Kuklik, Stefan Michelfelder, Felix Schiele, Sebastian Kreuz, Thorsten Lamla, Philipp Müller and John E. Park

Int. J. Mol. Sci. 2021, 22(15), 8355; https://doi.org/10.3390/ijms22158355 - 3 Aug 2021

Cited by 6 | Viewed by 5641

Abstract

A major limiting factor for systemically delivered gene therapies is the lack of novel tissue specific AAV (Adeno-associated virus) derived vectors. Bispecific antibodies can be used to redirect AAVs to specific target receptors. Here, we demonstrate that the insertion of a short linear [...] Read more.

A major limiting factor for systemically delivered gene therapies is the lack of novel tissue specific AAV (Adeno-associated virus) derived vectors. Bispecific antibodies can be used to redirect AAVs to specific target receptors. Here, we demonstrate that the insertion of a short linear epitope “2E3” derived from human proprotein-convertase subtilisin/kexin type 9 (PCSK9) into different surface loops of the VP capsid proteins can be used for AAV de-targeting from its natural receptor(s), combined with a bispecific antibody-mediated retargeting. We chose to target a set of distinct disease relevant membrane proteins—fibroblast activation protein (FAP), which is upregulated on activated fibroblasts within the tumor stroma and in fibrotic tissues, as well as programmed death-ligand 1 (PD-L1), which is strongly upregulated in many cancers. Upon incubation with a bispecific antibody recognizing the 2E3 epitope and FAP or PD-L1, the bispecific antibody/rAAV complex was able to selectively transduce receptor positive cells. In summary, we developed a novel, rationally designed vector retargeting platform that can target AAVs to a new set of cellular receptors in a modular fashion. This versatile platform may serve as a valuable tool to investigate the role of disease relevant cell types and basis for novel gene therapy approaches. Full article

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

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21 pages, 2408 KiB

Open AccessArticle

Activation of Local and Systemic Defence Responses by Flg22 Is Dependent on Daytime and Ethylene in Intact Tomato Plants

by Zalán Czékus, András Kukri, Kamirán Áron Hamow, Gabriella Szalai, Irma Tari, Attila Ördög and Péter Poór

Int. J. Mol. Sci. 2021, 22(15), 8354; https://doi.org/10.3390/ijms22158354 - 3 Aug 2021

Cited by 17 | Viewed by 4069

Abstract

The first line of plant defence responses against pathogens can be induced by the bacterial flg22 and can be dependent on various external and internal factors. Here, we firstly studied the effects of daytime and ethylene (ET) using Never ripe (Nr) [...] Read more.

The first line of plant defence responses against pathogens can be induced by the bacterial flg22 and can be dependent on various external and internal factors. Here, we firstly studied the effects of daytime and ethylene (ET) using Never ripe (Nr) mutants in the local and systemic defence responses of intact tomato plants after flg22 treatments. Flg22 was applied in the afternoon and at night and rapid reactions were detected. The production of hydrogen peroxide and nitric oxide was induced by flg22 locally, while superoxide was induced systemically, in wild type plants in the light period, but all remained lower at night and in Nr leaves. Flg22 elevated, locally, the ET, jasmonic acid (JA) and salicylic acid (SA) levels in the light period; these levels did not change significantly at night. Expression of Pathogenesis-related 1 (PR1), Ethylene response factor 1 (ERF1) and Defensin (DEF) showed also daytime- and ET-dependent changes. Enhanced ERF1 and DEF expression and stomatal closure were also observable in systemic leaves of wild type plants in the light. These data demonstrate that early biotic signalling in flg22-treated leaves and distal ones is an ET-dependent process and it is also determined by the time of day and inhibited in the early night phase. Full article

(This article belongs to the Special Issue Plant Innate Immunity 4.0)

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40 pages, 2164 KiB

Open AccessReview

Molecular Drivers of Developmental Arrest in the Human Preimplantation Embryo: A Systematic Review and Critical Analysis Leading to Mapping Future Research

by Konstantinos Sfakianoudis, Evangelos Maziotis, Eleni Karantzali, Georgia Kokkini, Sokratis Grigoriadis, Amelia Pantou, Polina Giannelou, Konstantina Petroutsou, Christina Markomichali, Maria Fakiridou, Michael Koutsilieris, Byron Asimakopoulos, Konstantinos Pantos and Mara Simopoulou

Int. J. Mol. Sci. 2021, 22(15), 8353; https://doi.org/10.3390/ijms22158353 - 3 Aug 2021

Cited by 20 | Viewed by 5740

Abstract

Developmental arrest of the preimplantation embryo is a multifactorial condition, characterized by lack of cellular division for at least 24 hours, hindering the in vitro fertilization cycle outcome. This systematic review aims to present the molecular drivers of developmental arrest, focusing on embryonic [...] Read more.

Developmental arrest of the preimplantation embryo is a multifactorial condition, characterized by lack of cellular division for at least 24 hours, hindering the in vitro fertilization cycle outcome. This systematic review aims to present the molecular drivers of developmental arrest, focusing on embryonic and parental factors. A systematic search in PubMed/Medline, Embase and Cochrane-Central-Database was performed in January 2021. A total of 76 studies were included. The identified embryonic factors associated with arrest included gene variations, mitochondrial DNA copy number, methylation patterns, chromosomal abnormalities, metabolic profile and morphological features. Parental factors included, gene variation, protein expression levels and infertility etiology. A valuable conclusion emerging through critical analysis indicated that genetic origins of developmental arrest analyzed from the perspective of parental infertility etiology and the embryo itself, share common ground. This is a unique and long-overdue contribution to literature that for the first time presents an all-inclusive methodological report on the molecular drivers leading to preimplantation embryos’ arrested development. The variety and heterogeneity of developmental arrest drivers, along with their inevitable intertwining relationships does not allow for prioritization on the factors playing a more definitive role in arrested development. This systematic review provides the basis for further research in the field. Full article

(This article belongs to the Special Issue Novel Molecular Mechanisms and Pathophysiology of Human Embryos)

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15 pages, 3845 KiB

Open AccessArticle

Mechanistic and Predictive QSAR Analysis of Diverse Molecules to Capture Salient and Hidden Pharmacophores for Anti-Thrombotic Activity

by Magdi E. A. Zaki, Sami A. Al-Hussain, Vijay H. Masand, Manoj K. Sabnani and Abdul Samad

Int. J. Mol. Sci. 2021, 22(15), 8352; https://doi.org/10.3390/ijms22158352 - 3 Aug 2021

Cited by 5 | Viewed by 2483

Abstract

Thrombosis is a life-threatening disease with a high mortality rate in many countries. Even though anti-thrombotic drugs are available, their serious side effects compel the search for safer drugs. In search of a safer anti-thrombotic drug, Quantitative Structure-Activity Relationship (QSAR) could be useful [...] Read more.

Thrombosis is a life-threatening disease with a high mortality rate in many countries. Even though anti-thrombotic drugs are available, their serious side effects compel the search for safer drugs. In search of a safer anti-thrombotic drug, Quantitative Structure-Activity Relationship (QSAR) could be useful to identify crucial pharmacophoric features. The present work is based on a larger data set comprising 1121 diverse compounds to develop a QSAR model having a balance of acceptable predictive ability (Predictive QSAR) and mechanistic interpretation (Mechanistic QSAR). The developed six parametric model fulfils the recommended values for internal and external validation along with Y-randomization parameters such as R²_tr = 0.831, Q²_LMO = 0.828, R²_ex = 0.783. The present analysis reveals that anti-thrombotic activity is found to be correlated with concealed structural traits such as positively charged ring carbon atoms, specific combination of aromatic Nitrogen and sp2-hybridized carbon atoms, etc. Thus, the model captured reported as well as novel pharmacophoric features. The results of QSAR analysis are further vindicated by reported crystal structures of compounds with factor Xa. The analysis led to the identification of useful novel pharmacophoric features, which could be used for future optimization of lead compounds. Full article

(This article belongs to the Special Issue Drug Design and Virtual Screening)

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13 pages, 28055 KiB

Open AccessArticle

Extracellular Vesicles as Biological Indicators and Potential Sources of Autologous Therapeutics in Osteoarthritis

by Xin Zhang, Janet L. Huebner and Virginia Byers Kraus

Int. J. Mol. Sci. 2021, 22(15), 8351; https://doi.org/10.3390/ijms22158351 - 3 Aug 2021

Cited by 15 | Viewed by 2797

Abstract

Along with cytokines, extracellular vesicles (EVs) released by immune cells in the joint contribute to osteoarthritis (OA) pathogenesis. By high-resolution flow cytometry, we characterized 18 surface markers and 4 proinflammatory cytokines carried by EVs of various sizes in plasma and synovial fluid (SF) [...] Read more.

Along with cytokines, extracellular vesicles (EVs) released by immune cells in the joint contribute to osteoarthritis (OA) pathogenesis. By high-resolution flow cytometry, we characterized 18 surface markers and 4 proinflammatory cytokines carried by EVs of various sizes in plasma and synovial fluid (SF) from individuals with knee OA, with a primary focus on immune cells that play a major role in OA pathogenesis. By multiplex immunoassay, we also measured concentrations of cytokines within (endo) and outside (exo) EVs. EVs carrying HLA-DR, -DP and -DQ were the most enriched subpopulations in SF relative to plasma (25–50-fold higher depending on size), suggesting a major contribution to the SF EV pool from infiltrating immune cells in OA joints. In contrast, the CD34⁺ medium and small EVs, reflecting hematopoietic stem cells, progenitor cells, and endothelial cells, were the most significantly enriched subpopulations in plasma relative to SF (7.3- and 7.7-fold higher). Ratios of EVs derived from neutrophils and lymphocytes were highly correlated between SF and plasma, indicating that plasma EVs could reflect OA severity and serve as systemic biomarkers of OA joint pathogenesis. Select subsets of plasma EVs might also provide next generation autologous biological products for intra-articular therapy of OA joints. Full article

(This article belongs to the Special Issue Extracellular Vesicles in Inflammation)

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Plasma and SF EVs from OA participants carry surface markers from the major hematopoietic cell subsets indicating their cell origins. EVs from plasma and SF of OA participants were profiled with the indicated surface markers by high-resolution multicolor flow cytometry. (A,B) The graphs present the representative color dot plots of all tested surface markers in gated large (LEVs), medium (MEVs) and small (SEVs) EVs from the matched plasma (A) and SF (B) of one OA participant. (C) The table summarizes the tested surface markers and their major expressing cells in human. HSCs: hematopoietic stem cells; ASCs: adipose stem cells; MSCs, mesenchymal stem cells; NK cells: natural killer cells; APCs: antigen presenting cells (including monocytes, macrophages and dendritic cells); HLA-ABC: HLA-A, HLA-B and HLA-C; HLA-DRDPDQ: HLA-DR, -DP and -DQ. Full article ">Figure 2
Compared to plasma, multiple immune cell-related EVs are enriched in synovial fluid (SF). EVs from both matched and unmatched plasma and SF of OA participants were profiled with the indicated surface markers by high-resolution multicolor flow cytometry. (A) Comparisons between the matched plasma and SF EVs (n = 16 pairs) were performed using Wilcoxon matched-pairs signed rank test with desired FDR q < 0.05. Volcano plots were generated using −Log 10 (q value) and Log 2 (Fold Changes of iMFI of the individual surface marker in LEVs, MEVs, and SEVs from SF vs Plasma). A positive fold change reflects a higher level of SF EVs relative to plasma EVs; a negative fold change reflects a lower level of SF EVs relative to plasma EVs). The graphs representing a summary of iMFI of each surface marker in gated LEVs, MEVs, or SEVs in all participants are presented in <a href="#app1-ijms-22-08351" class="html-app">Figure S1</a>. (B) These graphs plot the correlation of fold changes (SF ratio to plasma) of iMFI of each surface marker in gated LEVs, MEVs, or SEVs in matched (n = 16 SF-plasma pairs, x axis) and unmatched (n = 32 SF, n = 30 plasma, y axis) SF and plasma EVs. Full article ">Figure 3
The amount of several immune cell-related EVs and ratio of neutrophil-EVs to lymphocyte-EVs was positively correlated between plasma and SF. EVs from the matched plasma and SF of OA participants (n = 16) were profiled with the indicated surface markers by high-resolution multicolor flow cytometry. Spearman correlation was used for assessing correlations between the matched plasma and SF for the iMFI of individual surface markers and the iMFI ratio of CD15+ neutrophil-related EVs to lymphocyte (CD8+ and CD4+ T cell, CD19+ B cell, and CD56+ NK cell)-related EVs in gated LEVs, MEVs and SEVs. The heat maps were generated using the Spearman correlation coefficient r value with * p < 0.05 and ** p < 0.01. Full article ">Figure 4
Exo-EV and endo-EV cytokines generally correlated in plasma and SF. (A,B) The concentrations of exo-EV and endo-EV cytokines in plasma and SF of OA participants were measured by multiplex immunoassay. The floating bars (min to max with line at mean) represent the differential concentrations between exo-EV and endo-EV cytokines in plasma (A), n = 46) and SF (B), n = 48). Comparisons between the concentrations of the matched exo-EV and endo-EV cytokines were performed using Wilcoxon matched-pairs signed rank test with desired FDR q < 0.05, and the results are indicated as q value **** < 0.0001. (C,D) EVs from the matched plasma and SF of OA participants (n = 8) were profiled for the indicated intra-vesicle cytokines by high-resolution multicolor flow cytometry. The graphics are representative color dot plots of all tested intra-vesicle cytokines in gated LEVs, MEVs, and SEVs from the matched plasma (C) and SF (D) of one OA participant. (E) The floating bars represent a summary of iMFI (min to max with line at mean) of the tested endo-EV cytokine in gated LEVs, MEVs, or SEVs. Comparisons between the matched plasma and SF EVs (n = 8 each group) were performed using Wilcoxon matched-pairs signed rank test with significant results defined by FDR q < 0.05, asterisks indicate the q value as * < 0.05. (F) Spearman correlation was used for assessing correlations between the concentration of each exo-EV and endo-EV cytokine in plasma (n = 46) and SF (n = 48). The heat maps were generated using the correlation coefficient r value with * p < 0.05, *** p < 0.001, and **** p < 0.0001. (G) Spearman correlation was used for assessing correlations between the matched plasma and SF (n = 8 each group) for the concentration of each exo-EV and endo-EV cytokine. The heat maps were generated using the correlation coefficient r value with * p < 0.05 and ** p < 0.01. Full article ">Figure 5
Graphic summary of results. (A) Multiple immune cell-derived EV subpopulations were enriched in SF compared with plasma; the pro-inflammatory phenotype of SF EVs was supported by their pro-inflammatory cytokine cargo. (B) HSC-, progenitor cell-, and endothelial cell-associated CD34+ EV populations were enriched in plasma relative to SF. (C) Ratios of neutrophil-EVs to lymphocyte-EVs were positively correlated between plasma and SF. (D) EVs related to several types of stem cells, progenitor cells, neutrophils and B cells, and endo-EV pro-inflammatory cytokines IL-6 and TNF-α were highly correlated between SF and plasma. Graph was created with BioRender.com. Full article ">

21 pages, 5989 KiB

Open AccessArticle

Cardiolipin-Containing Lipid Membranes Attract the Bacterial Cell Division Protein DivIVA

by Naďa Labajová, Natalia Baranova, Miroslav Jurásek, Robert Vácha, Martin Loose and Imrich Barák

Int. J. Mol. Sci. 2021, 22(15), 8350; https://doi.org/10.3390/ijms22158350 - 3 Aug 2021

Cited by 5 | Viewed by 3943

Abstract

DivIVA is a protein initially identified as a spatial regulator of cell division in the model organism Bacillus subtilis, but its homologues are present in many other Gram-positive bacteria, including Clostridia species. Besides its role as topological regulator of the Min system [...] Read more.

DivIVA is a protein initially identified as a spatial regulator of cell division in the model organism Bacillus subtilis, but its homologues are present in many other Gram-positive bacteria, including Clostridia species. Besides its role as topological regulator of the Min system during bacterial cell division, DivIVA is involved in chromosome segregation during sporulation, genetic competence, and cell wall synthesis. DivIVA localizes to regions of high membrane curvature, such as the cell poles and cell division site, where it recruits distinct binding partners. Previously, it was suggested that negative curvature sensing is the main mechanism by which DivIVA binds to these specific regions. Here, we show that Clostridioides difficile DivIVA binds preferably to membranes containing negatively charged phospholipids, especially cardiolipin. Strikingly, we observed that upon binding, DivIVA modifies the lipid distribution and induces changes to lipid bilayers containing cardiolipin. Our observations indicate that DivIVA might play a more complex and so far unknown active role during the formation of the cell division septal membrane. Full article

(This article belongs to the Collection Feature Papers in Molecular Microbiology)

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DivIVACd binds better to SUVs containing CL and the N-terminal domain is essential for its binding. (A) A scheme of the sedimentation assay. DivIVACd was mixed with solutions of SUVs that did not contain cardiolipin (in yellow) or contained SUVs with different cardiolipin concentrations (CL is in red). All SUV mixtures were prepared with the buffer containing 30% sucrose to enhance their sedimentation. The SUVs were pelleted by centrifugation and DivIVACd amounts were quantified. The DivIVACd illustration is based on the structure of the full-length DivIVABs tetramer [<a href="#B38-ijms-22-08350" class="html-bibr">38</a>]. The sedimentation assay with N-terminally truncated Δ60-DivIVACd was performed similarly. (B) Box and whisker plots of the percentages of DivIVACd identified in the pellets under each of the tested conditions. Sedimentation assays were usually performed in triplets and in 3 independent experiments with full-length DivIVACd samples. Sedimentation assays with Δ60-DivIVACd were performed in triplets two times. Selected significant p-values from two-tailed unpaired t-tests are depicted above the plot; the threshold for statistical significance was taken to be p < 0.05. Stars indicate significantly different values, where * corresponds to p-value ≤ 0.05, ** to p ≤ 0.01, *** to p ≤ 0.001 and **** to p ≤ 0.0001. All p-values and percentages of DivIVACd or Δ60-DivIVACd in supernatants and pellets are listed in <a href="#app1-ijms-22-08350" class="html-app">Tables S4–S6</a>. (C) An SDS-PAGE analysis of a representative sedimentation assay. The compositions of the individual SUVs tested are given above the gel. S refers to the soluble fraction; P, to the pellet. Full article ">Figure 2
QCM-D measurements confirm that DivIVACd binding depends on the lipid composition. (A) Simplified scheme of the QCM-D experiment. Full-length DivIVACd (violet) was layered over the SLB on the QCM-D sensor chip. While the sedimentation assay investigated the binding of DivIVACd to vesicles of different lipid composition, this experiment examined the ability of DivIVACd to bind to different lipid bilayers. The DivIVACd illustration is based on the structure of the full-length DivIVABs tetramer [<a href="#B38-ijms-22-08350" class="html-bibr">38</a>]. The illustration was adapted from biolinscientific.com. (B–E) Results from measurements using supported lipid bilayers (SLBs) containing different cardiolipin concentrations. Layers were formed on a silicon dioxide-coated surface of a quartz crystal from a suspension of liposomes. DivIVACd was added to the SLB stepwise, starting with 0.25 µM after t = 1 min, followed by 0.5, 1.0, 2.0, and 3.0 µM DivIVACd, as indicated on the top margin of each plot, and the frequency (Δf) and dissipation (ΔD) changes were measured. The plots show Δf (in blue) and ΔD (in red) of the third, fifth, seventh, and ninth overtones (lower overtones are in lighter shades, the highest overtone is the darkest shade) normalized to the baseline obtained after SLB formation. (F) A summary plot of the normalized Δf7 versus DivIVACd concentration shows a clear preference of DivIVACd for the SLB with CL. The Δf7 curve is reversed, meaning that the ascending curve indicates increased mass absorption. The series were fit non-linearly to the Hill equation. Full article ">Figure 3
Changes to SLBs introduced by DivIVACd visualized using fluorescently labelled cardiolipin. (A) Representative image of SLB composed of 69.9% PC, 30%, PG and 0.1% TF-CL (L1*, the asterisk designates the presence of fluorescent TopFluor® Cardiolipin) before the addition of DivIVACd. Most of the fluorescent signal was homogenously distributed, with only few brighter foci of TF-CL visible. (B) The addition of 2 µM DivIVACd to L1* caused changes to the fluorescence distribution, resulting in TF-CL clustering. However, we did not observe clusters with areas larger than 4 µm2. (C) SLB with 64.9% PC, 30% PG, 5% CL, and 0.1% TF-CL (L4*) before DivIVACd addition. (D) Adding DivIVACd to the chamber with an L4* SLB changed the lipid distribution and morphology of the bilayer even more visibly and larger clusters or foci can be observed (4% larger than 4 µm2). The scale bars represent 10 µm. The bar charts show the distribution of fluorescent areas from several images analysed using the built-in “Analyse Particle” plugin for automatic particle counting in Fiji. The number of particles (n) detected and measured is given at the top of each chart. Full article ">Figure 4
DivIVACd binding also affects the lipid distribution in SLBs containing cardiolipin and labelled phosphatidylethanolamine. (A) The fluorescence in SLBs composed of 69.9% PC, 30% PG, and 0.1% Rh-PE (L1**) without DivIVACd was evenly distributed (a double asterisk indicates the presence of fluorescent phosphatidylethanolamine in the lipid mixture designation). (B) In L1** samples, the fluorescence distribution remained unchanged after 2 µM DivIVACd was added. (C) SLB containing 64.9% PC, 30% PG, 5% CL, and 0.1% Rh-PE (L4**) was also examined before the addition of DivIVACd. (D) The addition of 2 µM DivIVACd to SLBs containing 5% CL caused changes in the lipid distribution and possibly morphological changes to the bilayer. Larger clusters formed, of which some measured several micrometres (5% larger than 4 µm2). (E) Details of the L4** bilayer changes observed in the presence of DivIVACd. The scale bars represent 10 μm in (A–E) and 5 μm in the magnified views. Full article ">Figure 5
Simulations of the N-terminal domain of DivIVACd binding to the membrane. (A) A comparison of the N-terminal domain models of C. difficile DivIVACd, S. coelicolor DivIVASc, and B. subtilis DivIVABs. The left view of each structure shows the electrostatic potential of the domain surface (the range is ±1 kT/e, negative is red, positive is blue). The right view shows the domain tertiary structure. The upper views show the side of the domain and the lower views show the view down the membrane-interacting end. (B) On the left are structures of the lipids used in the simulations. Coloured circles around lipid head-groups, with marked negative charges, are used to denote different lipid types in the density maps. On the right side is the DivIVACd N-terminal domain with three orthogonal axes defined by vectors a, b, and c, which were used to describe the domain orientation with respect to the membrane. Angle α represents the tilt of the domain long axis, while angles β and γ capture the orientation of the domain sides. (C–E) Results from molecular dynamics simulations using 100% PC (L2), (C) 65% + 30% PG + 5% CL (L4), and (D) 55% + 30% PG + 15% CL (L6) bilayers. Each panel shows results from two independent simulation trajectories t1 and t2. Snapshots of the last frames of each simulation are shown on the left side; the molecular surface domain is shown coloured based on its electrostatic potential; the membrane lipids and phosphates are in grey and orange; water is omitted. The middle panels contain histograms showing the distribution of domain orientations with respect to the membrane, where cos(α) = 1 corresponds to a perpendicular orientation. The right-hand side shows the densities of the lipid head-groups around the domain tip. Full article ">Figure 6
Model of DivIVACd membrane binding. DivIVACd preferentially binds to membrane areas with an increased cardiolipin concentration. Bound DivIVACd attracts more DivIVACd molecules and cardiolipin to these areas. The bilayer morphology is heavily distorted, and curvature develops. This, in turn, causes more cardiolipin to sort towards these areas and enhance DivIVACd binding. The DivIVACd illustration (violet) is based on the structure of the full-length DivIVABs tetramer [<a href="#B38-ijms-22-08350" class="html-bibr">38</a>]. This illustration is simplified, however, as the protein forms higher order structures, and the shape and rigidity of these structures are likely to play a role in bilayer shaping. Cardiolipin (CL) is highlighted in red, other lipids are in yellow. Full article ">

16 pages, 4467 KiB

Open AccessArticle

Quercetin Administration Suppresses the Cytokine Storm in Myeloid and Plasmacytoid Dendritic Cells

by Giulio Verna, Marina Liso, Elisabetta Cavalcanti, Giusy Bianco, Veronica Di Sarno, Angelo Santino, Pietro Campiglia and Marcello Chieppa

Int. J. Mol. Sci. 2021, 22(15), 8349; https://doi.org/10.3390/ijms22158349 - 3 Aug 2021

Cited by 14 | Viewed by 2981

Abstract

Dendritic cells (DCs) can be divided by lineage into myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs). They both are present in mucosal tissues and regulate the immune response by secreting chemokines and cytokines. Inflammatory bowel diseases (IBDs) are characterized by a [...] Read more.

Dendritic cells (DCs) can be divided by lineage into myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs). They both are present in mucosal tissues and regulate the immune response by secreting chemokines and cytokines. Inflammatory bowel diseases (IBDs) are characterized by a leaky intestinal barrier and the consequent translocation of bacterial lipopolysaccharide (LPS) to the basolateral side. This results in DCs activation, but the response of pDCs is still poorly characterized. In the present study, we compared mDCs and pDCs responses to LPS administration. We present a broad panel of DCs secreted factors, including cytokines, chemokines, and growth factors. Our recent studies demonstrated the anti-inflammatory effects of quercetin administration, but to date, there is no evidence about quercetin’s effects on pDCs. The results of the present study demonstrate that pDCs can respond to LPS and that quercetin exposure modulates soluble factors release through the same molecular pathway used by mDCs (Slpi, Hmox1, and AP-1). Full article

(This article belongs to the Special Issue Role of Dendritic Cells in Inflammation 2.0)

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Graphical abstract
Full article ">Figure 1
Representative density plots for dendritic cells (DCs) found in Peyer’s patches (PP) from wild-type (WT) (A) and Winnie mice (B), and in mesenteric lymphnodes (MLNs) from WT (D) and Winnie (E); 20,000 cells were acquired per each condition. Graphs represent percentages of the different populations of DCs in PP (C) and MLNs (F) in WT and Winnie mice; bars represent mean relative expression ± SEM (n = 4) for each genotype. * p < 0.05 **** p < 0.0001. Full article ">Figure 2
Representative density plots for CD11c, B220, and Ly6C staining in mDCs (A), myeloid dendritic cells (mDCs) stimulated with lipopolysaccharide (LPS) for 24 h (B), plasmacytoid dendritic cells (pDCs) (C), pDCs stimulated with LPS for 24 h (D). Bar plots for mean ± SEM (n = 4) showing the percentages of CD11c+B220+Ly6C+ cells (E), CD11c+CD80+ cells (F), and major histocompatibility complex II (MHCII) MFI (mean fluorescence intensity) (G) in control and after LPS stimulation for 24 h. Bar plots representing the mean ± SEM (n = 6) of toll-like receptor 4 (Tlr4) gene expression relative to control mDCs (H). * p < 0.05 *** p < 0.001 **** p < 0.0001. Full article ">Figure 3
Bar plots expressing the mean ± SEM (n = 4) for MHCII MFI and CD80 expression (A,B) and 7-Aminoactinomycin D (7-AAD) vitality staining (C) of mDCs and pDCs in baseline conditions and after stimulation with LPS and/or quercetin. *** p < 0.001 **** p < 0.0001. Full article ">Figure 4
Scatter plots expressing the mean ± SEM (n = 5) for secreted cytokines of mDCs and pDCs in baseline conditions and after stimulation with LPS and/or quercetin. * p < 0.05 ** p < 0.005 *** p < 0.001 **** p < 0.0001; $ p < 0.05 $$ p < 0.005 $$$ p < 0.001 $$$$ p < 0.0001; * LPS vs. quercetin + LPS; $ mDCs vs. pDCs. Full article ">Figure 5
Scatter plots expressing the mean ± SEM (n = 5) for secreted chemokines of mDCs and pDCs in baseline conditions and after stimulation with LPS and/or quercetin. * p < 0.05 *** p < 0.001 **** p < 0.0001; $$$ p < 0.001 $$$$ p < 0.0001; * LPS vs. quercetin + LPS; $ mDCs vs. pDCs. Full article ">Figure 6
Scatter plots expressing the mean ± SEM (n = 5) for secreted growth factors of mDCs and pDCs in baseline conditions and after stimulation with LPS and/or quercetin. **** p < 0.0001; $ p < 0.05 $$$$ p < 0.0001; * LPS vs. quercetin + LPS; $ mDCs vs. pDCs. Full article ">Figure 7
Molecular pathways activated in mDCs and pDCs 6 h after LPS stimulation and/or quercetin administration. Bar plots representing the mean ± SEM (n = 6) of each gene expression relative to control mDCs. * p < 0.05 ** p < 0.005 **** p < 0.0001; $$ p < 0.005 $$$ p < 0.001 $$$$ p < 0.0001; * LPS vs. quercetin + LPS; $ mDCs vs. pDCs. Full article ">

12 pages, 4279 KiB

Open AccessArticle

Preparation and Size Control of Efficient and Safe Nanopesticides by Anodic Aluminum Oxide Templates-Assisted Method

by Chunxin Wang, Bo Cui, Yan Wang, Mengjie Wang, Zhanghua Zeng, Fei Gao, Changjiao Sun, Liang Guo, Xiang Zhao and Haixin Cui

Int. J. Mol. Sci. 2021, 22(15), 8348; https://doi.org/10.3390/ijms22158348 - 3 Aug 2021

Cited by 9 | Viewed by 2296

Abstract

Efficient and safe nanopesticides play an important role in pest control due to enhancing target efficiency and reducing undesirable side effects, which has become a hot spot in pesticide formulation research. However, the preparation methods of nanopesticides are facing critical challenges including low [...] Read more.

Efficient and safe nanopesticides play an important role in pest control due to enhancing target efficiency and reducing undesirable side effects, which has become a hot spot in pesticide formulation research. However, the preparation methods of nanopesticides are facing critical challenges including low productivity, uneven particle size and batch differences. Here, we successfully developed a novel, versatile and tunable strategy for preparing buprofezin nanoparticles with tunable size via anodic aluminum oxide (AAO) template-assisted method, which exhibited better reproducibility and homogeneity comparing with the traditional method. The storage stability of nanoparticles at different temperatures was evaluated, and the release properties were also determined to evaluate the performance of nanoparticles. Moreover, the present method is further demonstrated to be easily applicable for insoluble drugs and be extended for the study of the physicochemical properties of drug particles with different sizes. Full article

(This article belongs to the Special Issue Multifunctional Nanomaterials: Synthesis, Properties and Applications)

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20 pages, 4067 KiB

Open AccessReview

Importance of Surface Topography in Both Biological Activity and Catalysis of Nanomaterials: Can Catalysis by Design Guide Safe by Design?

by Mary Gulumian, Charlene Andraos, Antreas Afantitis, Tomasz Puzyn and Neil J. Coville

Int. J. Mol. Sci. 2021, 22(15), 8347; https://doi.org/10.3390/ijms22158347 - 3 Aug 2021

Cited by 15 | Viewed by 3840

Abstract

It is acknowledged that the physicochemical properties of nanomaterials (NMs) have an impact on their toxicity and, eventually, their pathogenicity. These properties may include the NMs’ surface chemical composition, size, shape, surface charge, surface area, and surface coating with ligands (which can carry [...] Read more.

It is acknowledged that the physicochemical properties of nanomaterials (NMs) have an impact on their toxicity and, eventually, their pathogenicity. These properties may include the NMs’ surface chemical composition, size, shape, surface charge, surface area, and surface coating with ligands (which can carry different functional groups as well as proteins). Nanotopography, defined as the specific surface features at the nanoscopic scale, is not widely acknowledged as an important physicochemical property. It is known that the size and shape of NMs determine their nanotopography which, in turn, determines their surface area and their active sites. Nanotopography may also influence the extent of dissolution of NMs and their ability to adsorb atoms and molecules such as proteins. Consequently, the surface atoms (due to their nanotopography) can influence the orientation of proteins as well as their denaturation. However, although it is of great importance, the role of surface topography (nanotopography) in nanotoxicity is not much considered. Many of the issues that relate to nanotopography have much in common with the fundamental principles underlying classic catalysis. Although these were developed over many decades, there have been recent important and remarkable improvements in the development and study of catalysts. These have been brought about by new techniques that have allowed for study at the nanoscopic scale. Furthermore, the issue of quantum confinement by nanosized particles is now seen as an important issue in studying nanoparticles (NPs). In catalysis, the manipulation of a surface to create active surface sites that enhance interactions with external molecules and atoms has much in common with the interaction of NP surfaces with proteins, viruses, and bacteria with the same active surface sites of NMs. By reviewing the role that surface nanotopography plays in defining many of the NMs’ surface properties, it reveals the need for its consideration as an important physicochemical property in descriptive and predictive toxicology. Through the manipulation of surface topography, and by using principles developed in catalysis, it may also be possible to make safe-by-design NMs with a reduction of the surface properties which contribute to their toxicity. Full article

(This article belongs to the Special Issue Advances in Cheminformatics and Nanoinformatics: From Materials Modelling to Precision Toxicology, Medicine and Agriculture)

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Shape-dependent synthesized nanoclusters. Reprinted with permission from Reference [<a href="#B5-ijms-22-08347" class="html-bibr">5</a>]. Copyright 2020 Springer Nature. Full article ">Figure 2
Side view scheme of the selected (100), (110), and (111) slabs of a copper(II) oxide (CuO) cube. Adapted with permission from Reference [<a href="#B8-ijms-22-08347" class="html-bibr">8</a>]. Copyright 2021 Elsevier. Full article ">Figure 3
Simple block model of defects on a single-crystal surface. Reprinted with permission from Reference [<a href="#B9-ijms-22-08347" class="html-bibr">9</a>]. Copyright 2021 American Chemical Society. Full article ">Figure 4
A Schematic representation of the quantum confinement effect: (a) The bandgap (or HOMO–LUMO gap) of the semiconductor nanocrystal increases with decreasing size, while discrete energy levels arise at the band-edges; (b) Five colloidal dispersions of cadmium–selenium quantum dots (CdSe QDs) under UV excitation ranging from 6 nm (red) to 2 nm (blue). Adapted with permission from refs. [<a href="#B17-ijms-22-08347" class="html-bibr">17</a>,<a href="#B18-ijms-22-08347" class="html-bibr">18</a>]. Copyright 2021 Springer Link (<a href="http://creativecommons.org/licenses/by/4.0/" target="_blank">http://creativecommons.org/licenses/by/4.0/</a>; accessed on 7 June 2021). Full article ">Figure 5
TEM image of a carbon nano-onion (a) and carbon dot (b). Image courtesy from Neil J. Coville. Full article ">Figure 6
Nanotopography and biological activities of NMs. Full article ">Figure 7
The role of nanotopography in both the catalytic and biological activities of NMs. Full article ">

51 pages, 997 KiB

Open AccessReview

Role of Virus-Induced Host Cell Epigenetic Changes in Cancer

by Valeria Pietropaolo, Carla Prezioso and Ugo Moens

Int. J. Mol. Sci. 2021, 22(15), 8346; https://doi.org/10.3390/ijms22158346 - 3 Aug 2021

Cited by 37 | Viewed by 5829

Abstract

The tumor viruses human T-lymphotropic virus 1 (HTLV-1), hepatitis C virus (HCV), Merkel cell polyomavirus (MCPyV), high-risk human papillomaviruses (HR-HPVs), Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpes virus (KSHV) and hepatitis B virus (HBV) account for approximately 15% of all human cancers. Although the [...] Read more.

The tumor viruses human T-lymphotropic virus 1 (HTLV-1), hepatitis C virus (HCV), Merkel cell polyomavirus (MCPyV), high-risk human papillomaviruses (HR-HPVs), Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpes virus (KSHV) and hepatitis B virus (HBV) account for approximately 15% of all human cancers. Although the oncoproteins of these tumor viruses display no sequence similarity to one another, they use the same mechanisms to convey cancer hallmarks on the infected cell. Perturbed gene expression is one of the underlying mechanisms to induce cancer hallmarks. Epigenetic processes, including DNA methylation, histone modification and chromatin remodeling, microRNA, long noncoding RNA, and circular RNA affect gene expression without introducing changes in the DNA sequence. Increasing evidence demonstrates that oncoviruses cause epigenetic modifications, which play a pivotal role in carcinogenesis. In this review, recent advances in the role of host cell epigenetic changes in virus-induced cancers are summarized. Full article

(This article belongs to the Special Issue Genetics and Epigenetics in Complex Diseases)

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15 pages, 2608 KiB

Open AccessArticle

Cigarette Smoke Condensate Exposure Induces Receptor for Advanced Glycation End-Products (RAGE)-Dependent Sterile Inflammation in Amniotic Epithelial Cells

by Helena Choltus, Régine Minet-Quinard, Corinne Belville, Julie Durif, Denis Gallot, Loic Blanchon and Vincent Sapin

Int. J. Mol. Sci. 2021, 22(15), 8345; https://doi.org/10.3390/ijms22158345 - 3 Aug 2021

Cited by 8 | Viewed by 4101

Abstract

Maternal smoking is a risk factor of preterm prelabor rupture of the fetal membranes (pPROM), which is responsible for 30% of preterm births worldwide. Cigarettes induce oxidative stress and inflammation, mechanisms both implicated in fetal membranes (FM) weakening. We hypothesized that the receptor [...] Read more.

Maternal smoking is a risk factor of preterm prelabor rupture of the fetal membranes (pPROM), which is responsible for 30% of preterm births worldwide. Cigarettes induce oxidative stress and inflammation, mechanisms both implicated in fetal membranes (FM) weakening. We hypothesized that the receptor for advanced glycation end-products (RAGE) and its ligands can result in cigarette-dependent inflammation. FM explants and amniotic epithelial cells (AECs) were treated with cigarette smoke condensate (CSC), combined or not with RAGE antagonist peptide (RAP), an inhibitor of RAGE. Cell suffering was evaluated by measuring lactate dehydrogenase (LDH) medium-release. Extracellular HMGB1 (a RAGE ligand) release by amnion and choriodecidua explants were checked by western blot. NF-κB pathway induction was determined by a luciferase gene reporter assay, and inflammation was evaluated by cytokine RT-qPCR and protein quantification. Gelatinase activity was assessed using a specific assay. CSC induced cell suffering and HMGB1 secretion only in the amnion, which is directly associated with a RAGE-dependent response. CSC also affected AECs by inducing inflammation (cytokine release and NFκB activation) and gelatinase activity through RAGE engagement, which was linked to an increase in extracellular matrix degradation. This RAGE dependent CSC-induced inflammation associated with an increase of gelatinase activity could explain a pathological FM weakening directly linked to pPROM. Full article

(This article belongs to the Collection Feature Papers in Molecular Pathology, Diagnostics, and Therapeutics)

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Graphical abstract

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Full article ">Figure 1
CSC treatment induces a cell danger response into amnion. (A) Cell toxicity was evaluated by measuring LDH release in a supernatant culture after a 24 h treatment with CSC in amnion and choriodecidua (n = 6). (B) HMGB1 release in a supernatant culture after a 48 h treatment with CSC was quantified by the western blot method in amnion and chori-odecidua; the results are reported in the histogram (n = 5). (C) Toxicity was evaluated by LDH release measurement in primary amniocytes (pAECs) cell-culture supernatants after a 48 h treatment with CSC (100 µg/mL) (n = 6). (D) HMGB1 nuclear toward cytosolic translocation was investigated by immunocytochemistry on pAECs after 7 h treatment with CSC (yellow staining; Alexa488). Nuclei were counterstained with Hoechst (red). Scales bars: 50 µM (magnification ×200). Negative control was realized without primary antibody hybridization. White arrows indicate the cytosolic cloud of HMGB1. Comparison with the control (DMSO) was realized by a Mann-Whitney t-test. *** means p < 0.001, and “ns” means “not significant”. Results are presented in Tukey boxes; means are indicated by “+”. Full article ">Figure 2
Amniotic epithelial cells expressed a functional RAGE axis. (A) RNA expressions of RAGE, TIRAP, Myd88, and Diaphanous-1 were detected by RT-PCR in pAECS. Negative controls were performed in the absence of a cDNA template. (B) The RAGE receptor and its adaptors, Diaphanous-1, MyD88, and TIRAP (green staining, Alexa488), were detected by immunocytochemistry on primary amniocytes (pAECs). Nuclei were counterstained with Hoechst (blue). Scales bars: 50 µM (magnification ×200). Negative control was realized without primary antibody hybridization. Full article ">Figure 3
Activation of RAGE axis by CSC-response in pAECs. (A) Quantification of RAGE and its signaling adaptors (Diapahnous-1, Myd88, and TIRAP) transcription by RT-qPCR following 48 h of CSC (100 µg/mL) +/− RAP (12.7 µg/mL) treatment of pAECs (n = 7). (B) NFκB luciferase reporter assay was performed after 48 h of CSC treatment (100 µg/mL), whether combined or not with RAP (12.7 µg/mL) (n = 4). (C) p65-NFκB relocalization was investigated by immunocytochemistry on pAECs, whether treated or not with CSC for 48 h (yellow staining; Alexa488). Nuclei were counterstained with Hoechst (red). Scales bars: 50 µM (magnification ×200). Negative controls were realized without primary antibody hybridization. White arrows indicate perinuclear/nuclear relocalization of the p65 protein. A comparison of conditions was realized by a Kruskal–Wallis one-way ANOVA test, followed by a Dunn’s post-test. ** means p < 0.01, *** means p < 0.001, and “ns” means “not significant”. Results are presented in Tukey boxes, and means are indicated by “+”. Full article ">Figure 4
RAGE is implicated in CSC-induced pro-inflammatory cytokine production in pAECs. (A) Quantification of cytokine mRNA expression by RT-qPCR following 48 h of CSC (100 µg/mL) +/− RAP (12.7 µg/mL) treatment of pAECs (n = 6). (B) Pro-inflammatory cytokine (IL8, IL6, IL1β) secretion was quantified by ELLA technology after 48 and 72 h (with 48 h re-treatment) of CSC treatment (n = 4). A comparison of conditions was realized by a Kruskal–Wallis one-way ANOVA test, followed by a Dunn’s post-test. * means p < 0.05, ** means p < 0.01, *** means p < 0.001, and “ns” means “not significant”. Cigarette Smoke Condensate Enhances Gelatinase Activity in pAECs through the RAGE Pathway. Full article ">Figure 5
CSC exposure stimulates gelatinase activity in pAECs through the RAGE pathway. (A) Transcription of MMP2 and MMP9 gelatinases were measured by RT-qPCR into pAECS after 48 h of CSC treatment (100 µg/mL), whether combined or not with RAP (12.7 µg/mL) (n = 8). (B) Gelatinase activity was studied by a zymography kit assay on pAECs cell media after 48 and 72 h (with 48 h re-treatment) of CSC treatment (n = 5). Statistical analysis was performed using a Kruskal–Wallis one-way ANOVA test, followed by a Dunn’s post-test. * means p < 0.05, ** means p < 0.01, *** means p < 0.001, and “ns” means “not significant”. Results are presented in Tukey boxes, and means are indicated by “+”. Black dots indicate values outside of the Tukey boxes. Full article ">Figure 6
Maternal tabagism model of negative consequences on amniotic cells by the induction of RAGE-dependent sterile inflammation and gelatinase activity. Sterile inflammation is a key phenomenon of FM weakening, not only in physiological rupture, but also in pPROM. Exposure to tobacco during pregnancy is a well-known risk factor of pPROM. We demonstrated here that CSC induces an in vitro HMGB1 release by the amnion, a well-known danger signal. Then, this alarmin, a major ligand of RAGE, can induce a pro-inflammatory response (NF-κB activation and cytokine production) through the RAGE pathway in amniotic epithelial cells, which suggests the essential role of RAGE in FM rupture and pPROM. smart.servier.com was used to create the figure. Full article ">

32 pages, 2286 KiB

Open AccessReview

Unraveling Axon Guidance during Axotomy and Regeneration

by Miguel E. Domínguez-Romero and Paula G. Slater

Int. J. Mol. Sci. 2021, 22(15), 8344; https://doi.org/10.3390/ijms22158344 - 3 Aug 2021

Cited by 17 | Viewed by 6374

Abstract

During neuronal development and regeneration axons extend a cytoskeletal-rich structure known as the growth cone, which detects and integrates signals to reach its final destination. The guidance cues “signals” bind their receptors, activating signaling cascades that result in the regulation of the growth [...] Read more.

During neuronal development and regeneration axons extend a cytoskeletal-rich structure known as the growth cone, which detects and integrates signals to reach its final destination. The guidance cues “signals” bind their receptors, activating signaling cascades that result in the regulation of the growth cone cytoskeleton, defining growth cone advance, pausing, turning, or collapse. Even though much is known about guidance cues and their isolated mechanisms during nervous system development, there is still a gap in the understanding of the crosstalk between them, and about what happens after nervous system injuries. After neuronal injuries in mammals, only axons in the peripheral nervous system are able to regenerate, while the ones from the central nervous system fail to do so. Therefore, untangling the guidance cues mechanisms, as well as their behavior and characterization after axotomy and regeneration, are of special interest for understanding and treating neuronal injuries. In this review, we present findings on growth cone guidance and canonical guidance cues mechanisms, followed by a description and comparison of growth cone pathfinding mechanisms after axotomy, in regenerative and non-regenerative animal models. Full article

(This article belongs to the Special Issue Role of Neuronal Guidance Cues in Inflammation and Vascular Biology)

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The growth cone. (A). Schematic representation of the axonal growth cone structure and cytoskeletal response to attractive and repulsive guidance cues. The axonal growth cone is composed of three principal domains: the central (C) domain (light blue), where the microtubules (cyan) coming from the axon shaft are splayed; the transition (T) zone (pink), where contractile F-actin arcs (red) are organized in a semi-circle structure separating the C domain from the peripheral (P) domain (gray), which is composed by filopodia (F-actin bundles, red) and lamellipodia (F-actin mesh, peach). In the upper right, an attractive gradient is represented, towards which the growth cone moves (green arrows). New lamellipodia (light green) and contact points are formed towards the leading edge, while in the opposite direction, F-actin is disassembled, and filopodia and lamellipodia retract (red arrows). (B). Schematic representation of F-actin dynamics in the growth cone. F-actin bundles polymerizing towards the leading edge of the growth cone (yellow arrow), generating growth cone protrusive forces, while the F-actin is subjected in turn to a retrograde movement towards the T zone, driven by myosin II (gray arrow). (C). Schematic representation of MT dynamics in the growth cone. An exploring MT enters the P domain following the F-actin path (Ci), but at the same time it, couples to F-actin and thus, to F-actin retrograde flow (Cii). (D). Schematic representation of molecular clutch formation. Recruitment of many integrin receptors (orange) result in adhesive molecules binding (green), and reduced F-actin retrograde flow and growth cone advance (Di), while few integrin receptors result in adhesion collapse, and no advance is observed (Dii). (E). Schematic representation of F-actin and MT binding proteins involved in F-actin/MT coupling in growth cone filopodia. Full article ">Figure 2
Guidance cues are associated with the glial scar and glial bridge. (A). Schematic representation of non-regenerative (NR) and regenerative (R) spinal cord response after injury. Injured axons from NR animal models (upper panel) fail to form a growth cone but instead form a retracting bulb guiding axonal degeneration, while a glial scar is formed in the injury site, which expresses mainly repulsive guidance cues. Injured axons from R animal models (lower panel) generate a growth cone, which travels through the ablation gap following a glial bridge formed by bipolar glial cells, that also expresses guidance cues. (B). Schematic representation of a peripheral nervous system axon after injury. Damaged axons can regenerate by extending through the ablation gap, following a glial bridge formed by Schwann cells and macrophages, which express different guidance cues that restrict a corridor for axons to grow. Full article ">

22 pages, 1119 KiB

Open AccessReview

Purinergic Signalling in Allogeneic Haematopoietic Stem Cell Transplantation and Graft-versus-Host Disease

by Peter Cuthbertson, Nicholas J. Geraghty, Sam R. Adhikary, Katrina M. Bird, Stephen J. Fuller, Debbie Watson and Ronald Sluyter

Int. J. Mol. Sci. 2021, 22(15), 8343; https://doi.org/10.3390/ijms22158343 - 3 Aug 2021

Cited by 8 | Viewed by 3741

Abstract

Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for blood cancers and other haematological disorders. However, allo-HSCT leads to graft-versus-host disease (GVHD), a severe and often lethal immunological response, in the majority of transplant recipients. Current therapies for GVHD are limited [...] Read more.

Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for blood cancers and other haematological disorders. However, allo-HSCT leads to graft-versus-host disease (GVHD), a severe and often lethal immunological response, in the majority of transplant recipients. Current therapies for GVHD are limited and often reduce the effectiveness of allo-HSCT. Therefore, pro- and anti-inflammatory factors contributing to disease need to be explored in order to identify new treatment targets. Purinergic signalling plays important roles in haematopoiesis, inflammation and immunity, and recent evidence suggests that it can also affect haematopoietic stem cell transplantation and GVHD development. This review provides a detailed assessment of the emerging roles of purinergic receptors, most notably P2X7, P2Y₂ and A_2A receptors, and ectoenzymes, CD39 and CD73, in GVHD. Full article

(This article belongs to the Special Issue Transplant Immunology in Allogeneic Hematopoietic Stem Cell Transplantation)

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Purinergic signalling molecules associated with graft-versus-host disease. The purinergic receptors P2X7, P2Y2 and A2A are activated by adenosine 5′-triphosphate (ATP), uridine triphosphate (UTP)/ATP or adenosine, respectively. P2X7 plays a pro-inflammatory role and A2A plays an anti-inflammatory role while P2Y2 may be involved in both roles. CD39 converts ATP to adenosine 5′-diphosphate (ADP) and ADP to adenosine 5′-monophosphate (AMP). CD73 converts AMP to adenosine. Antagonists for P2X7 (A-438079, Brilliant blue G (BBG), KN-62, and pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid (PPADS) and stavudine), CD39 (α,β-methylene ADP (APCP) and polyoxotungstate-1 (POM-1)), CD73 (APCP) and A2A (Caffeine, SCH58261 and ZM241385) have been used to study the roles of these molecules in GVHD. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>. Full article ">Figure 2
The role of the purinergic signalling molecules during graft-versus-host disease. Tissue damage from pre-transplant conditioning or GVHD results in adenosine 5′-triphosphate (ATP) release. This ATP activates P2X7 on host antigen presenting cells (APCs) and donor T cells leading to donor T cell enhanced activation, proliferation and survival. ATP can also activate P2X7 on regulatory T cells, reducing their function and survival. P2X7 activation on myeloid derived suppressor cells can reduce their suppressive capacity (not shown). Combined these pathways result in worsened GVHD. ATP can also activate P2Y2 on host dendritic cells and monocytes initiating the migration of these cells to inflamed tissues. Alternatively, ATP can be converted to ADP then AMP by CD39 present on mesenchymal stem cells or regulatory T cells. CD73 receptors on these same cells, or other cells (not shown) convert AMP to adenosine which activates A2A receptors on donor T cells, reducing T cell function. This conversion of ATP to adenosine results in reduced GVHD. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>. Full article ">

13 pages, 33620 KiB

Open AccessArticle

alfaNET: A Database of Alfalfa-Bacterial Stem Blight Protein–Protein Interactions Revealing the Molecular Features of the Disease-causing Bacteria

by Raghav Kataria and Rakesh Kaundal

Int. J. Mol. Sci. 2021, 22(15), 8342; https://doi.org/10.3390/ijms22158342 - 3 Aug 2021

Cited by 7 | Viewed by 3010

Abstract

Alfalfa has emerged as one of the most important forage crops, owing to its wide adaptation and high biomass production worldwide. In the last decade, the emergence of bacterial stem blight (caused by Pseudomonas syringae pv. syringae ALF3) in alfalfa has caused around [...] Read more.

Alfalfa has emerged as one of the most important forage crops, owing to its wide adaptation and high biomass production worldwide. In the last decade, the emergence of bacterial stem blight (caused by Pseudomonas syringae pv. syringae ALF3) in alfalfa has caused around 50% yield losses in the United States. Studies are being conducted to decipher the roles of the key genes and pathways regulating the disease, but due to the sparse knowledge about the infection mechanisms of Pseudomonas, the development of resistant cultivars is hampered. The database alfaNET is an attempt to assist researchers by providing comprehensive Pseudomonas proteome annotations, as well as a host–pathogen interactome tool, which predicts the interactions between host and pathogen based on orthology. alfaNET is a user-friendly and efficient tool and includes other features such as subcellular localization annotations of pathogen proteins, gene ontology (GO) annotations, network visualization, and effector protein prediction. Users can also browse and search the database using particular keywords or proteins with a specific length. Additionally, the BLAST search tool enables the user to perform a homology sequence search against the alfalfa and Pseudomonas proteomes. With the successful implementation of these attributes, alfaNET will be a beneficial resource to the research community engaged in implementing molecular strategies to mitigate the disease. alfaNET is freely available for public use at http://bioinfo.usu.edu/alfanet/. Full article

(This article belongs to the Special Issue Genomics: Infectious Disease and Host-Pathogen Interaction)

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Sitemap architecture of the alfaNET database. Full article ">Figure 2
Interface of the host-pathogen interactome tool. Full article ">Figure 3
Advanced search module of alfaNET displaying the default parameters. Full article ">Figure 4
Examples of annotation modules of alfaNET: (a) protein annotations; (b) subcellular localizations; (c) functional domain mappings. Full article ">Figure 4 Cont.
Examples of annotation modules of alfaNET: (a) protein annotations; (b) subcellular localizations; (c) functional domain mappings. Full article ">Figure 5
BLAST search module: (a) BLAST search interface; (b) visualization of BLAST alignments using BlasterJS. Full article ">Figure 6
Visualization of the interactions for up-regulated genes of alfalfa interacting with P. syringae. Green nodes are host proteins and red nodes are pathogen proteins. Edges in grey represent the interactions from the interolog-based method. Full article ">Figure 7
Visualization of the interactions for down-regulated genes of alfalfa interacting with P. syringae. Green nodes are host proteins and red nodes are pathogen proteins. Edges in grey represent the interactions from the interolog-based method. Full article ">

15 pages, 1773 KiB

Open AccessReview

Application of Carbon Nanoparticles in Oncology and Regenerative Medicine

by Katarzyna Lisik and Anita Krokosz

Int. J. Mol. Sci. 2021, 22(15), 8341; https://doi.org/10.3390/ijms22158341 - 3 Aug 2021

Cited by 24 | Viewed by 3765

Abstract

Currently, carbon nanoparticles play a large role as carriers of various types of drugs, and also have applications in other fields of medicine, e.g., in tissue engineering, where they are used to reconstruct bone tissue. They also contribute to the early detection of [...] Read more.

Currently, carbon nanoparticles play a large role as carriers of various types of drugs, and also have applications in other fields of medicine, e.g., in tissue engineering, where they are used to reconstruct bone tissue. They also contribute to the early detection of cancer cells, and can act as markers in imaging diagnostics. Their antibacterial and anti-inflammatory properties are also known. This feature is particularly important in dental implantology, where various types of bacterial infections and implant rejection often occur. The search for newer and more effective treatments may lead to future use of nanoparticles on a large scale. In this work, the current state of knowledge on the possible use of nanotubes, nanodiamonds, and fullerenes in therapy is reviewed. Both advantages and disadvantages of the use of carbon nanoparticles in therapy and diagnostics have been indicated. Full article

(This article belongs to the Special Issue Biologically Synthesized Nanomaterials and Their Nanocomposites for Biomedical Applications)

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24 pages, 40798 KiB

Open AccessReview

Dendritic Cells and CCR7 Expression: An Important Factor for Autoimmune Diseases, Chronic Inflammation, and Cancer

by Emma Probst Brandum, Astrid Sissel Jørgensen, Mette Marie Rosenkilde and Gertrud Malene Hjortø

Int. J. Mol. Sci. 2021, 22(15), 8340; https://doi.org/10.3390/ijms22158340 - 3 Aug 2021

Cited by 59 | Viewed by 10529

Abstract

Chemotactic cytokines—chemokines—control immune cell migration in the process of initiation and resolution of inflammatory conditions as part of the body’s defense system. Many chemokines also participate in pathological processes leading up to and exacerbating the inflammatory state characterizing chronic inflammatory diseases. In this [...] Read more.

Chemotactic cytokines—chemokines—control immune cell migration in the process of initiation and resolution of inflammatory conditions as part of the body’s defense system. Many chemokines also participate in pathological processes leading up to and exacerbating the inflammatory state characterizing chronic inflammatory diseases. In this review, we discuss the role of dendritic cells (DCs) and the central chemokine receptor CCR7 in the initiation and sustainment of selected chronic inflammatory diseases: multiple sclerosis (MS), rheumatoid arthritis (RA), and psoriasis. We revisit the binary role that CCR7 plays in combatting and progressing cancer, and we discuss how CCR7 and DCs can be harnessed for the treatment of cancer. To provide the necessary background, we review the differential roles of the natural ligands of CCR7, CCL19, and CCL21 and how they direct the mobilization of activated DCs to lymphoid organs and control the formation of associated lymphoid tissues (ALTs). We provide an overview of DC subsets and, briefly, elaborate on the different T-cell effector types generated upon DC–T cell priming. In the conclusion, we promote CCR7 as a possible target of future drugs with an antagonistic effect to reduce inflammation in chronic inflammatory diseases and an agonistic effect for boosting the reactivation of the immune system against cancer in cell-based and/or immune checkpoint inhibitor (ICI)-based anti-cancer therapy. Full article

(This article belongs to the Special Issue Role of Dendritic Cells in Inflammation 2.0)

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CCL19, CCL21, and Tailless-CCL21 (Tailless) induce differential signaling through their common receptor CCR7. Overall, CCL19 is a strong agonist of both G-protein signaling, β-arrestin recruitment, and chemotaxis, whereas CCL21 is a weak agonist. Upon cleavage by DC-released proteases, CCL21 is turned into Tailless-CCL21, which resembles CCL19 and, thus, is a strong agonist. Full article ">Figure 2
Based on their differential tissue expression and signaling properties, the chemokines CCL19 and CCL21 exert different functions in vivo. CCL21 is believed to be the major lymph node (LN)-homing chemokine, directing the LN localization of activated CCR7+ dendritic cells (DCs) through afferent lymphatics. As both CCL19 and CCL21 are present in high endothelial venules (HEVs) and T cells enter the LN through the HEVs, both CCL19 and CCL21 appear to be important for directing the localization of CCR7+ T cell subsets (naïve, memory, and Treg) to the LN. CCL19 secreted by active DCs in the LN partakes in the subsequent scanning and DC–T cell priming process, directing T cells to interact with the DCs. The rapid internalization of CCR7 occurring upon receptor engagement with CCL19 allows for a swift DC–T cell interaction and allowing for the scanning process to occur. Full article ">Figure 3
DC subsets and T-cell differentiation. Various DC types exist that partake in regulating immune reactions. Depending on the context, DCs may induce the activation of different T cell subsets. cDC1s can present Ags on MHCII to naïve CD4+ T cells to induce Th1 response or cross-present Ags on MHCI to cytotoxic CD8+ T cells, both resulting in cell-based immunity. In mice, cDC1s and cDC2s can induce Th1 cell and Th17 cell skewing, respectively, whereas human cDC2s can induce both Th1 cell and Th17 cell skewing [<a href="#B69-ijms-22-08340" class="html-bibr">69</a>]. cDC2s induces the T-cell differentiation of naïve CD4+ T cells through MHCII Ag presentation, giving rise to the T cell subsets: Th1, Th17, Th2, Tfh, and Treg, which induce cell-based immunity, autoimmune immunity, antibody based immunity, T cell help to B cells, and tolerogenic immunity, respectively. pDCs express both MHCI and MHCII and patrol the bloodstream and peripheral lymphoid organs. When exposed to viral and bacterial infections, pDCs produce type I interferon (IFN). MoDCs are divided into classical, intermediate and non-classical subsets, of which the classical seem to be pro-inflammatory, while the non-classical subset exerts patrolling functions. The pro-inflammatory moDCs induce cell based- and antibody-based immunity via CD8+ T cells and B cells, respectively. Full article ">Figure 4
CCR7 and dendritic cells (DCs) in multiple sclerosis (MS): In healthy individuals, CCR7 and CCL19 are involved in the normal trafficking of memory T cells into the brain. In MS, elevated levels of CCL19 can be found in the CSF (cerebrospinal fluid). MS is associated with an increase in T cell infiltrates and an increase in pro-inflammatory CCR7+ DCs in the CSF. Blocking CCR7 reduces the binding of T cells to the endothelium and recruitment into the CSF. CCR7 signaling in DCs leads to the release of IL-12 and IL-23, which, in turn, induces T-cell activation. Deficiency of CCR7 ligands reduce T-cell activation and protects against EAE in the EAE mouse model. Full article ">Figure 5
Dendritic cells (DCs) and the CCR7 axis in rheumatoid arthritis: DCs can be recruited into the joint but can also differentiate and mature from precursors or monocytes in response to locally produced cytokines. Blocking of the DC LN migration (by blocking MMP-9) prevents T- and B-cell activation, and displays protection against early RA in a mouse model. DCs from RA lesions can display increased CCL19 production and the presence of CCL21 and CCL19 chemokines are associated with the formation of lymphocytic aggregates both with and without B cells. These aggregates can support T- and B-cell activation in the later stages of RA. CCR7 KO protects against the formation of the lymphocytic aggregates in a mouse model and can also reduce the production of autoantibodies. CCL21 and CCL19 are also associated with microenvironmental changes, which can support the synovial inflammation. Full article ">Figure 6
Dendritic cells (DCs) in psoriasis. Psoriasis can be triggered by various factors, including trauma, infections, injury, and medication. The increased proliferation of keratinocytes leads to thickening of the epidermis, while the corneocytes fail to stack normally. During epidermal injury, keratinocytes secrete CCL20, leading to the recruitment of CCR6+ monocytes that, together with dermal DCs, are critical for psoriasis development. Inflammatory myeloid DCs produce the cytokines IL-12 and IL-23, resulting in the activation and expansion of Th17 cells and leading to an autoimmune response. The formation of inducible skin-associated lymphoid tissue (iSALT) structures occurs in psoriatic lesions. CCR7 and CCL19 partake in the establishment of these structures. iSALT primary contains CD3+ T cells, CD11c+ LAMP3/DC-LAMP+ DC, and, to a minor extent, CXCR5+ B cells. Keratinocytes produce the antimicrobial peptide LL-37, which can form complexes with DNA and RNA from dying cells. These complexes bind to toll-like receptors (TLRs) in myeloid DCs and pDCs leading to the activation of these cells. Full article ">

12 pages, 3618 KiB

Open AccessArticle

Hierarchical Structure of Protein Sequence

by Alexei N. Nekrasov, Yuri P. Kozmin, Sergey V. Kozyrev, Rustam H. Ziganshin, Alexandre G. de Brevern and Anastasia A. Anashkina

Int. J. Mol. Sci. 2021, 22(15), 8339; https://doi.org/10.3390/ijms22158339 - 3 Aug 2021

Cited by 7 | Viewed by 2873

Abstract

Most non-communicable diseases are associated with dysfunction of proteins or protein complexes. The relationship between sequence and structure has been analyzed for a long time, and the analysis of the sequences organization in domains and motifs remains an actual research area. Here, we [...] Read more.

Most non-communicable diseases are associated with dysfunction of proteins or protein complexes. The relationship between sequence and structure has been analyzed for a long time, and the analysis of the sequences organization in domains and motifs remains an actual research area. Here, we propose a mathematical method for revealing the hierarchical organization of protein sequences. The method is based on the pentapeptide as a unit of protein sequences. Employing the frequency of occurrence of pentapeptides in sequences of natural proteins and a special mathematical approach, this method revealed a hierarchical structure in the protein sequence. The method was applied to 24,647 non-homologous protein sequences with sizes ranging from 50 to 400 residues from the NRDB90 database. Statistical analysis of the branching points of the graphs revealed 11 characteristic values of y (the width of the inscribed function), showing the relationship of these multiple fragments of the sequences. Several examples illustrate how fragments of the protein spatial structure correspond to the elements of the hierarchical structure of the protein sequence. This methodology provides a promising basis for a mathematically-based classification of the elements of the spatial organization of proteins. Elements of the hierarchical structure of different levels of the hierarchy can be used to solve biotechnological and medical problems. Full article

(This article belongs to the Special Issue Medical Genetics, Genomics and Bioinformatics – 2021)

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23 pages, 1235 KiB

Open AccessReview

The Prion-Like Spreading of Alpha-Synuclein in Parkinson’s Disease: Update on Models and Hypotheses

by Asad Jan, Nádia Pereira Gonçalves, Christian Bjerggaard Vaegter, Poul Henning Jensen and Nelson Ferreira

Int. J. Mol. Sci. 2021, 22(15), 8338; https://doi.org/10.3390/ijms22158338 - 3 Aug 2021

Cited by 53 | Viewed by 10098

Abstract

The pathological aggregation of the presynaptic protein α-synuclein (α-syn) and propagation through synaptically coupled neuroanatomical tracts is increasingly thought to underlie the pathophysiological progression of Parkinson’s disease (PD) and related synucleinopathies. Although the precise molecular mechanisms responsible for the spreading of pathological α-syn [...] Read more.

The pathological aggregation of the presynaptic protein α-synuclein (α-syn) and propagation through synaptically coupled neuroanatomical tracts is increasingly thought to underlie the pathophysiological progression of Parkinson’s disease (PD) and related synucleinopathies. Although the precise molecular mechanisms responsible for the spreading of pathological α-syn accumulation in the CNS are not fully understood, growing evidence suggests that de novo α-syn misfolding and/or neuronal internalization of aggregated α-syn facilitates conformational templating of endogenous α-syn monomers in a mechanism reminiscent of prions. A refined understanding of the biochemical and cellular factors mediating the pathological neuron-to-neuron propagation of misfolded α-syn will potentially elucidate the etiology of PD and unravel novel targets for therapeutic intervention. Here, we discuss recent developments on the hypothesis regarding trans-synaptic propagation of α-syn pathology in the context of neuronal vulnerability and highlight the potential utility of novel experimental models of synucleinopathies. Full article

(This article belongs to the Special Issue Alpha-Synuclein in Neurodegeneration)

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A schematic representation of hypothesized α-syn aggregation and spreading from the ENS towards the CNS via vagus nerve. Environmental factors, including changes in the gut microbiota (dysbiosis), are hypothesized to initiate pathological processes within the enteric nerve cell plexus, provoking mucosal inflammation and oxidative stress and thereby inducing abnormal aggregation of α-syn. Increased permeability of the intestinal barrier (‘leaky gut’) will ultimately provide a route of transmission for the ENS-formed α-syn seeds into the brain. Structures are not drawn to scale. The illustration was created in <a href="http://biorender.com" target="_blank">biorender.com</a> (accessed on 3 August 2021). Full article ">Figure 2
Schematic depiction of hypothesized α-syn neuron-to-neuron transmission and intracellular redox imbalance resulting in neurodegeneration. Under normal homeostatic conditions, neuronal α-syn exists in soluble non-aggregated conformations and the anti-oxidant (AOX) scavenging mechanisms are at equilibrium with intracellular reactive oxygen species (ROS) generation. Misfolded α-syn perturbs cellular redox balance in favor of excessive ROS, which is further aggravated by additional susceptibility/risk factors (e.g., genetic risk factors and ageing) that promote pathological α-syn aggregation and proteopathic stress [<a href="#B1-ijms-22-08338" class="html-bibr">1</a>,<a href="#B2-ijms-22-08338" class="html-bibr">2</a>]. Subsequently, cell-to-cell transmission of α-syn seeds from the affected neurons (depicted as donor neuron) via the neuroanatomical projections onto additional neuronal populations results in transmission of α-syn pathology into the recipient neurons. In the receiving neuron, the newly internalized seeds recruit endogenous soluble α-syn and further template a vicious cycle of α-syn aggregation and neurotoxicity. In established (i.e., long-term) α-syn neuronal pathology, there is profound dysregulation of AOX/ROS balance which is associated with loss of synaptic terminals and neuronal demise. The neuroglial cells modulate these processes by providing trophic support (e.g., glia derived neurotrophic factor- GDNF) which serves to maintain pro-survival local microenvironment [<a href="#B185-ijms-22-08338" class="html-bibr">185</a>,<a href="#B186-ijms-22-08338" class="html-bibr">186</a>,<a href="#B187-ijms-22-08338" class="html-bibr">187</a>,<a href="#B188-ijms-22-08338" class="html-bibr">188</a>,<a href="#B189-ijms-22-08338" class="html-bibr">189</a>]. However, relentless disease progression and ensuing neurodegeneration are strong triggers for neuroinflammatory response. Structures are not drawn to scale. The illustration was created in <a href="http://biorender.com" target="_blank">biorender.com</a> (accessed on 3 August 2021). Full article ">

13 pages, 2683 KiB

Open AccessArticle

Regulation Network of Colorectal-Cancer-Specific Enhancers in the Progression of Colorectal Cancer

by Bohan Chen, Yiping Ma, Jinfang Bi, Wenbin Wang, Anshun He, Guangsong Su, Zhongfang Zhao, Jiandang Shi and Lei Zhang

Int. J. Mol. Sci. 2021, 22(15), 8337; https://doi.org/10.3390/ijms22158337 - 3 Aug 2021

Cited by 5 | Viewed by 3515

Abstract

Enhancers regulate multiple genes via higher-order chromatin structures, and they further affect cancer progression. Epigenetic changes in cancer cells activate several cancer-specific enhancers that are silenced in normal cells. These cancer-specific enhancers are potential therapeutic targets of cancer. However, the functions and regulation [...] Read more.

Enhancers regulate multiple genes via higher-order chromatin structures, and they further affect cancer progression. Epigenetic changes in cancer cells activate several cancer-specific enhancers that are silenced in normal cells. These cancer-specific enhancers are potential therapeutic targets of cancer. However, the functions and regulation networks of colorectal-cancer-specific enhancers are still unknown. In this study, we profile colorectal-cancer-specific enhancers and reveal their regulation network through the analysis of HiChIP data that were derived from a colorectal cancer cell line and Hi-C and RNA-seq data that were derived from tissue samples by in silico analysis and in vitro experiments. Enhancer–promoter loops in colorectal cancer cells containing colorectal-cancer-specific enhancers are involved in more than 50% of the topological associated domains (TADs) changed in colorectal cancer cells compared to normal colon cells. In addition, colorectal-cancer-specific enhancers interact with 152 genes that are significantly and highly expressed in colorectal cancer cells. These colorectal-cancer-specific enhancer target genes include ITGB4, RECQL4, MSLN, and GDF15. We propose that the regulation network of colorectal-cancer-specific enhancers plays an important role in the progression of colorectal cancer. Full article

(This article belongs to the Section Molecular Oncology)

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16 pages, 2039 KiB

Open AccessArticle

Inflammasome Signaling Regulates the Microbial–Neuroimmune Axis and Visceral Pain in Mice

by Mònica Aguilera, Valerio Rossini, Ana Hickey, Donjete Simnica, Fiona Grady, Valeria D. Felice, Amy Moloney, Lauren Pawley, Aine Fanning, Lorraine McCarthy, Siobhan M. O’Mahony, John F. Cryan, Ken Nally, Fergus Shanahan and Silvia Melgar

Int. J. Mol. Sci. 2021, 22(15), 8336; https://doi.org/10.3390/ijms22158336 - 3 Aug 2021

Cited by 11 | Viewed by 3623

Abstract

Interactions between the intestinal microbiota, immune system and nervous system are essential for homeostasis in the gut. Inflammasomes contribute to innate immunity and brain–gut interactions, but their role in microbiota–neuro–immune interactions is not clear. Therefore, we investigated the effect of the inflammasome on [...] Read more.

Interactions between the intestinal microbiota, immune system and nervous system are essential for homeostasis in the gut. Inflammasomes contribute to innate immunity and brain–gut interactions, but their role in microbiota–neuro–immune interactions is not clear. Therefore, we investigated the effect of the inflammasome on visceral pain and local and systemic neuroimmune responses after antibiotic-induced changes to the microbiota. Wild-type (WT) and caspase-1/11 deficient (Casp1 KO) mice were orally treated for 2 weeks with an antibiotic cocktail (Abx, Bacitracin A and Neomycin), followed by quantification of representative fecal commensals (by qPCR), cecal short chain fatty acids (by HPLC), pathways implicated in the gut–neuro-immune axis (by RT-qPCR, immunofluorescence staining, and flow cytometry) in addition to capsaicin-induced visceral pain responses. Abx-treatment in WT-mice resulted in an increase in colonic macrophages, central neuro-immune interactions, colonic inflammasome and nociceptive receptor gene expression and a reduction in capsaicin-induced visceral pain. In contrast, these responses were attenuated in Abx-treated Casp1 KO mice. Collectively, the data indicate an important role for the inflammasome pathway in functional and inflammatory gastrointestinal conditions where pain and alterations in microbiota composition are prominent. Full article

(This article belongs to the Special Issue Diet-Microbiota and Host Interactions in Intestinal Health and Inflammation)

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Characterization and quantification of total bacteria, the phylum Firmicutes, Actinobacteria, Bacteroidetes and Proteobacteria, and the genera Clostridium XIVa and Lactobacillus by qPCR in control and antibiotic-treated WT and Casp1 KO mice. (A) Representative cycle threshold (CT) values for total bacterial qPCR detection. (B) Percentage (%) of the composition including Firmicutes, Actinobacteria (Bifidobacterium spp), Bacteroidetes (Bacteroides spp) and Proteobacteria (E. coli), representing the commensal microbiota from the total bacterial abundance and (C) abundance of Clostridium cluster XIVa and (D) Lactobacillus Order of the Firmicutes phylum. (A) Data are presented as mean (SD), n = 7–10/group. (B) Data are presented as percentage of each phylum from the fold change of the total bacterial abundance. (C,D) Data are median (interquartile range) (SD). Full article ">Figure 2
Representative RT-qPCR expression of genes associated with (A) inflammasome (Pycard, Aim2, Nlrp3, Nlrc4, Nlrp6, Il-1β and Il-18); (B) toll-like receptors (TLR2, -4, -5 and -7); antimicrobial peptides (AMPs-Rentlβ and Reg3γ), the mucus layer component Muc2 and (C) macrophage M1/M2 signature (F4/80, Fitz1, Arg1, Il-6 and Il-12β), the colon of WT and Casp1 KO antibiotic-treated groups. Data are mean (SEM). n= 7–9/group. *: p < 0.05, **: p <0.001. Dashed line indicates a background value of 1 of WT and Casp1 KO-control groups. Abx: Antibiotic. Full article ">Figure 3
Colonic lamina propria (LP) cells isolated from control and antibiotic-treated WT and Casp1 KO mice and stained with (A) CD45 (Leukocytes); (B) Ly6G (neutrophils); (C) CD3 (CD3+ T cells); (D) CD4 (CD4+ T cells); (E) CD11c (dendritic cells); and (F) F4/80 (macrophages). Bars represent the percentage of the indicated cell population. Representative figure of two individual experiments. Data are mean (SEM). n = 4–5/group. *: p < 0.05. Full article ">Figure 4
Representative RT-qPCR expression of colon nociceptive markers; (A) the endocannabinoid system (CB1, CB2 and Faah), the protease-activated receptor 2 (PAR2, Fr2l1) and the serotonin transporter (Scl6a4); (B) the opioid peptide pro-enkephalin (Penk), the neurothrophin (NGFβ), the vanilloid system (transient receptor potential, Trpv3 and Trpv4) and calcitonin-related polypeptide alpha (Calca). Data are mean (SEM). n = 7–9/group. *: p <0.05, **: p < 0.001. Abx: antibiotic. Dashed line indicates background value of 1 of WT and Casp1 KO-control groups. (C) Colonic lamina propria (LP) cells isolated from control and antibiotic-treated WT and Casp1 KO mice and stained with CD45 and GFAP. Bars represent the percentage of the indicated cell population. Representative figure of two individual experiments. Data are mean (SD). n = 4/group. *: p < 0.05. Full article ">Figure 5
Representative percentage (%) of (A) microglia and (B) astrocytes cell average in the anterior cingulate cortex (ACC) of WT and Casp1 KO control and antibiotic-treated groups. Data are mean (SD). n = 3/group. *: p < 0.05. Full article ">Figure 6
Representative number of visceral pain behaviors during 30 min per each experimental group upon intracolonic administration of capsaicin. Data mean (SD), * p < 0.05 compared to the WT control group. n = 7–11/group, including in all groups, males (range 2 to 8) and females (range 3 to 5). Full article ">

15 pages, 6595 KiB

Open AccessReview

Hypoxia in Cancer and Fibrosis: Part of the Problem and Part of the Solution

by Yair Romero and Arnoldo Aquino-Gálvez

Int. J. Mol. Sci. 2021, 22(15), 8335; https://doi.org/10.3390/ijms22158335 - 3 Aug 2021

Cited by 14 | Viewed by 4300

Abstract

Adaptive responses to hypoxia are involved in the progression of lung cancer and pulmonary fibrosis. However, it has not been pointed out that hypoxia may be the link between these diseases. As tumors or scars expand, a lack of oxygen results in the [...] Read more.

Adaptive responses to hypoxia are involved in the progression of lung cancer and pulmonary fibrosis. However, it has not been pointed out that hypoxia may be the link between these diseases. As tumors or scars expand, a lack of oxygen results in the activation of the hypoxia response, promoting cell survival even during chronic conditions. The role of hypoxia-inducible factors (HIFs) as master regulators of this adaptation is crucial in both lung cancer and idiopathic pulmonary fibrosis, which have shown the active transcriptional signature of this pathway. Emerging evidence suggests that interconnected feedback loops such as metabolic changes, fibroblast differentiation or extracellular matrix remodeling contribute to HIF overactivation, making it an irreversible phenomenon. This review will focus on the role of HIF signaling and its possible overlapping in order to identify new opportunities in therapy and regeneration. Full article

(This article belongs to the Special Issue Hypoxia Signaling in Human Diseases)

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21 pages, 841 KiB

Open AccessReview

The Role of the IL-6 Cytokine Family in Epithelial–Mesenchymal Plasticity in Cancer Progression

by Andrea Abaurrea, Angela M. Araujo and Maria M. Caffarel

Int. J. Mol. Sci. 2021, 22(15), 8334; https://doi.org/10.3390/ijms22158334 - 3 Aug 2021

Cited by 51 | Viewed by 6172

Abstract

Epithelial–mesenchymal plasticity (EMP) plays critical roles during embryonic development, wound repair, fibrosis, inflammation and cancer. During cancer progression, EMP results in heterogeneous and dynamic populations of cells with mixed epithelial and mesenchymal characteristics, which are required for local invasion and metastatic dissemination. Cancer [...] Read more.

Epithelial–mesenchymal plasticity (EMP) plays critical roles during embryonic development, wound repair, fibrosis, inflammation and cancer. During cancer progression, EMP results in heterogeneous and dynamic populations of cells with mixed epithelial and mesenchymal characteristics, which are required for local invasion and metastatic dissemination. Cancer development is associated with an inflammatory microenvironment characterized by the accumulation of multiple immune cells and pro-inflammatory mediators, such as cytokines and chemokines. Cytokines from the interleukin 6 (IL-6) family play fundamental roles in mediating tumour-promoting inflammation within the tumour microenvironment, and have been associated with chronic inflammation, autoimmunity, infectious diseases and cancer, where some members often act as diagnostic or prognostic biomarkers. All IL-6 family members signal through the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway and are able to activate a wide array of signalling pathways and transcription factors. In general, IL-6 cytokines activate EMP processes, fostering the acquisition of mesenchymal features in cancer cells. However, this effect may be highly context dependent. This review will summarise all the relevant literature related to all members of the IL-6 family and EMP, although it is mainly focused on IL-6 and oncostatin M (OSM), the family members that have been more extensively studied. Full article

(This article belongs to the Special Issue The Epithelial-to-Mesenchymal Transition (EMT) in Cancers)

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19 pages, 18705 KiB

Open AccessArticle

Effects of Urolithin A on Mitochondrial Parameters in a Cellular Model of Early Alzheimer Disease

by Carsten Esselun, Ellen Theyssen and Gunter P. Eckert

Int. J. Mol. Sci. 2021, 22(15), 8333; https://doi.org/10.3390/ijms22158333 - 3 Aug 2021

Cited by 30 | Viewed by 5538

Abstract

(1) Background: Ellagitannins are natural products occurring in pomegranate and walnuts. They are hydrolyzed in the gut to release ellagic acid, which is further metabolized by the microflora into urolithins, such as urolithin A (UA). Accumulation of damaged mitochondria is a hallmark of [...] Read more.

(1) Background: Ellagitannins are natural products occurring in pomegranate and walnuts. They are hydrolyzed in the gut to release ellagic acid, which is further metabolized by the microflora into urolithins, such as urolithin A (UA). Accumulation of damaged mitochondria is a hallmark of aging and age-related neurodegenerative diseases. In this study, we investigated the neuroprotective activity of the metabolite UA against mitochondrial dysfunction in a cellular model of early Alzheimer disease (AD). (2) Methods: In the present study we used SH-SY5Y-APP695 cells and its corresponding controls (SH-SY5Ymock) to assess UA’s effect on mitochondrial function. Using these cells we investigated mitochondrial respiration (OXPHOS), mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) production, autophagy and levels of reactive oxygen species (ROS) in cells treated with UA. Furthermore, we assessed UA’s effect on the expression of genes related to mitochondrial bioenergetics, mitochondrial biogenesis, and autophagy via quantitative real-time PCR (qRT-PCR). (3) Results: Treatment of SH-SY5Y-APP695 cells suggests changes to autophagy corresponding with qRT-PCR results. However, LC3B-I, LC3B-II, and p62 levels were unchanged. UA (10 µM) reduced MMP, and ATP-levels. Treatment of cells with UA (1 µM) for 24 h did not affect ROS production or levels of Aβ, but significantly increased expression of genes for mitochondrial biogenesis and OXPHOS. Mitochondrial Transcription Factor A (TFAM) expression was specifically increased in SH-SY5Y-APP695. Both cell lines showed unaltered levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which is commonly associated with mitochondrial biogenesis. Results imply that biogenesis might be facilitated by estrogen-related receptor (ESRR) genes. (4) Conclusion: Urolithin A shows no effect on autophagy in SH-SY5Y-APP695 cells and its effect on mitochondrial function is limited. Instead, data suggests that UA treatment induces hormetic effects as it induces transcription of several genes related to mitochondrial biogenesis. Full article

(This article belongs to the Special Issue Natural Products and Neuroprotection 3.0)

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Chemical structure of compound Urolithin A. Full article ">Figure 2
(A): Oxygen consumption of 206 SY5Ymock cells and SY5Y-APP695 cells treated with ctrl or 1 µM UA. N = 9–15. Activity of OXPHOS complexes were assessed via addition of several substrates, inhibitors or uncouplers. Which substance was added in which stage of the experiment is marked with “X”. (B,C) Mitochondrial membrane potential measured of SY5Ymock cells and SY5Y-APP695 cells treated with 1 µM UA (B) or 10 µM UA (C). N = 7–11; (D,E) ATP levels of SY5Ymock and SY5Y-APP695 cells treated with 1 µM UA (D) or 10 µM (E). N = 11–13 Displayed are means ± SEM. Statistical significance was tested via two-way ANOVA followed by a false discovery rate correction according to Benjamini, Krieger, and Yekutieli. Significance is displayed as: ns p > 0.05, * p < 0.05, ** p < 0.01; *** p < 0.001, **** p < 0.0001. List of all statistical parameters and comparisons can be found in <a href="#app1-ijms-22-08333" class="html-app">Supplementary Table S1</a>. Full article ">Figure 3
(A) Citrate synthase activity of SY5Ymock and SY5Y-APP695 samples. Cells were incubated with either 1 µM UA or its DMSO control (ctrl). Enzyme activity waws adjusted to protein content of the samples. N = 7–13. (B) ROS level measured in form of DCFDA/H2DCFDA fluorescence in SY5Ymock and SY5Y-APP695 cells. N = 8; (C,D) Mitochondrial membrane potential of SY5Ymock and SY5Y-APP695 cells treated with 1 µM UA (C) or 10 µM UA (D) whose complex I activity was inhibited via addition of 25 µM rotenone. Dashed line corresponds to MMP of uninhibited SY5Ymock/APP695 control cells. N = 7–11; (E,F) ATP levels of SY5Ymock and SY5Y-APP695 cells treated with 1 µM UA (E) or 10 µM (F) whose complex I activity was inhibited via addition of 25 µM rotenone. The dashed line shows the basal ATP level of SY5Ymock/-APP695 cells not treated with 20 µM rotenone. Dashed line corresponds to MMP of uninhibited SY5Ymock/APP695 control cells. N = 7–14; Displayed are means ± SEM. Statistical significance was tested via one-way ANOVA followed by a false discovery rate correction according to Benjamini, Krieger and Yekutieli. Significance is displayed as: ns p > 0.05, * p < 0.05, *** p < 0.001 and **** p < 0.0001. List of all statistical parameters and comparisons can be found in <a href="#app1-ijms-22-08333" class="html-app">Supplementary Table S1</a>. Full article ">Figure 4
Relative normalized mRNA expression of relevant genes relevant to OXPHOS and mitochondrial biogenesis. Genes related to OXPHOS: (A) citrate synthases (CS), (B) complex I (NDUFV1), (C) complex IV (COX5D), and (D) complex V (ATP5D). Genes related to mitochondrial biogenesis: (E) Sirtuin 1 (SIRT1), (F) cAMP response element-binding protein ranscription factor (CREB), (G) Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), (H) nuclear respiratory factor 1 (NRF1), (I) mitochondrial transcription factor A (TFAM), (J) GA Binding Protein Transcription Factor Subunit Alpha (GABPα/NRF2), (K) estrogen-related receptor alpha (ESRRα), (L) estrogen-related receptor gamma (ESRRγ). Displayed are data of SY5Ymock and SY5Y-APP695 cells which were measured seperately from each other. Data of both cell lines is adjusted to aged SY5Ymock ctrl group = 100%; N = 8–10; Displayed are means ± SEM; Statistical significance was tested via two-way ANOVA followed by a false discovery rate correction (FDRC) according to Benjamini, Krieger, and Yekutieli. Results were normalized to the mRNA expression levels of three housekeeping genes (beta-actine (ACTβ), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Phosphoglycerate Kinase 1 (PGK1)) according to the MIQE guidelines. Significance is displayed as: ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. List of all statistical parameters and comparisons can be found in <a href="#app1-ijms-22-08333" class="html-app">Supplementary Table S1</a>. Full article ">Figure 5
(A) Expression of microtubule-associated proteins 1A/1B light chain 3 (MAP1LC3) gene related to autophagy; (B) Expression of Sequestosome 1 (SQSTM1) gene related to autophagy. Displayed are data of SY5Ymock (left side) and SY5Y-APP695 cells (right side) which were measured separately from each other. Data of both cell lines is adjusted to SY5Ymock ctrl group = 100%; N = 8–10; Displayed are means ± SEM. Results were normalized to the mRNA expression levels of three housekeeping genes (beta-actine (ACTβ), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Phosphoglycerate Kinase 1 (PGK1)) according to the MIQE guidelines. (C) Fluorescence of marker dye binding to autophagosomes of SY5Ymock and SY5Y-APP695 cells. Displayed are means ± SEM. N = 10. (D) Western blots of LC3B-I, LC3B-II, and p62 in SY5Ymock and SY5Y-APP695 treated with 1 µM UA or ctrl. β-Actine was used for housekeeping and results have been adjusted to it (<a href="#ijms-22-08333-f002" class="html-fig">Figure 2</a>E–G). Gels were loaded with 15 µg protein. (E,F) Western blot results from LC3B-I and LC3B-II adjusted to β-actine. Displayed are means ± SEM. N = 9. (G) Western blot results from p62 adjusted to β-actine. Displayed are means ± SEM. N = 8. Cells were incubated with either 1 µM UA or its DMSO control (ctrl). Statistical significance was tested via two-way ANOVA followed by a false discovery rate correction according to Benjamini, Krieger, and Yekutieli. Significance is displayed as: ns p > 0.05, * p < 0.05, ** p < 0.01 and **** p < 0.0001. List of all statistical parameters and comparisons can be found in <a href="#app1-ijms-22-08333" class="html-app">Supplementary Table S1</a>. Full article ">

15 pages, 779 KiB

Open AccessReview

Dual Nature of Relationship between Mycobacteria and Cancer

by Marek Fol, Piotr Koziński, Jakub Kulesza, Piotr Białecki and Magdalena Druszczyńska

Int. J. Mol. Sci. 2021, 22(15), 8332; https://doi.org/10.3390/ijms22158332 - 3 Aug 2021

Cited by 12 | Viewed by 3867

Abstract

Although the therapeutic effect of mycobacteria as antitumor agents has been known for decades, recent epidemiological and experimental studies have revealed that mycobacterium-related chronic inflammation may be a possible mechanism of cancer pathogenesis. Mycobacterium tuberculosis and non-tuberculous Mycobacterium avium complex infections have been [...] Read more.

Although the therapeutic effect of mycobacteria as antitumor agents has been known for decades, recent epidemiological and experimental studies have revealed that mycobacterium-related chronic inflammation may be a possible mechanism of cancer pathogenesis. Mycobacterium tuberculosis and non-tuberculous Mycobacterium avium complex infections have been implicated as potentially contributing to the etiology of lung cancer, whereas Mycobacterium ulcerans has been correlated with skin carcinogenesis. The risk of tumor development with chronic mycobacterial infections is thought to be a result of many host effector mechanisms acting at different stages of oncogenesis. In this paper, we focus on the nature of the relationship between mycobacteria and cancer, describing the clinical significance of mycobacteria-based cancer therapy as well as epidemiological evidence on the contribution of chronic mycobacterial infections to the increased lung cancer risk. Full article

(This article belongs to the Special Issue The Consequences of Infections on the Host Immune Microenvironment)

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17 pages, 2983 KiB

Open AccessCase Report

A Novel Germline Mutation of ADA2 Gene in Two “Discordant” Homozygous Female Twins Affected by Adenosine Deaminase 2 Deficiency: Description of the Bone-Related Phenotype

by Silvia Vai, Erika Marin, Roberta Cosso, Francesco Saettini, Sonia Bonanomi, Alessandro Cattoni, Iacopo Chiodini, Luca Persani and Alberto Falchetti

Int. J. Mol. Sci. 2021, 22(15), 8331; https://doi.org/10.3390/ijms22158331 - 3 Aug 2021

Cited by 1 | Viewed by 2517

Abstract

Adenosine Deaminase 2 Deficiency (DADA2) syndrome is a rare monogenic disorder prevalently linked to recessive inherited loss of function mutations in the ADA2/CECR1 gene. It consists of an immune systemic disease including autoinflammatory vasculopathies, with a frequent onset at infancy/early childhood age. DADA2 [...] Read more.

Adenosine Deaminase 2 Deficiency (DADA2) syndrome is a rare monogenic disorder prevalently linked to recessive inherited loss of function mutations in the ADA2/CECR1 gene. It consists of an immune systemic disease including autoinflammatory vasculopathies, with a frequent onset at infancy/early childhood age. DADA2 syndrome encompasses pleiotropic manifestations such as stroke, systemic vasculitis, hematologic alterations, and immunodeficiency. Although skeletal abnormalities have been reported in patients with this disease, clear information about skeletal health, with appropriate biochemical-clinical characterization/management, its evolution over time and any appropriate clinical management is still insufficient. In this paper, after a general introduction shortly reviewing the pathophysiology of Ada2 enzymatic protein, its potential role in bone health, we describe a case study of two 27 year-old DADA2 monozygotic female twins exhibiting bone mineral density and bone turnover rate abnormalities over the years of their clinical follow-up. Full article

(This article belongs to the Special Issue Osteoporosis)

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(A) Normal integral metabolic purine salvage pathway enables the regulation and availability of purines; (B) Disruption of ADA activity prevents the physiological continuation of the purine pathways with increased levels of Ad and dAd. In grey letters, the deoxy-metabolites branches are reported. ADA2 inactivation determines the stop of the dAD to d-Inosine and Ad to Inosine progression, and consequently the downstream final product represented by uric acid, with upstream accumulation of both dAd and Ad metabolites. Full article ">Figure 2
Pedigree of family ADA2-AUXMI1. Full article ">Figure 3
(A–D). II-1 Tw1 LS- (A), TH/FN- (B), and T. B.-DXA (C) scans. A e B report also the previous relative findings, from 2011 to 2019, showing the trend of BMD over the years. Asterisk refer to values that are significantly different from the previous ones (result calculated directly by the software of the device). (D) II-1 Tw1. It reports also LS, TH and FN Z-score. In bold are shown the Z-scores (LS) which, in accordance with ISCD, diagnosed the reduced bone mass compared. Full article ">Figure 4
(A–D). II-2 Tw2 LS- (A), TH/FN- (B), and T. B.-DXA (C) scans. A e B report also the previous relative findings, from 2011 to 2019, showing the trend of BMD over the years. Asterisk refer to values that are significantly different from the previous ones (result calculated directly by the software of the device). (D) II-2 Tw2. It reports also LS, TH and FN Z-score. In bold are shown the Z-scores (LS) which, in accordance with ISCD, diagnosed the reduced bone mass compared. Full article ">Figure 5
(A) II-1 Tw1 TB% fat composition results, 2019 vs. 2020. (A1) reports anthropometric results from 2011 up to 2020, and (A2) summarizes DXA TB parameters (BMD, BMC, T- and Z-scores, and total fat percentage) from 2011 up to 2020. (B) II-2 Tw2 TB% fat composition results, 2018, 2019 and 2020. (B1) reports anthropometric results from 2011 up to 2020, and (B2) summarizes DXA TB parameters (BMD, BMC, T- and Z-scores, and total fat percentage) from 2011 up to 2019. Full article ">Figure 6
BTMs trend over time (U-NTX is not described here, which is instead described in <a href="#ijms-22-08331-t002" class="html-table">Table 2</a>). The dashed line in the graphs represents the upper limit of the range for each parameter. On the right side of each figure are the respective units of measurement, indicated in the parenthesis next to each BTM. The line below each graph shows the year in which the relevant biochemical assessments were carried out. The * symbol indicates the start of amino-bisphosphonate therapy (stopped at the end of 2020). Full article ">Figure 7
Within the solid box, a partial DNA sequence of ADA2 gene is reported, while in the dashed box the corresponding amino acid sequence of ADA2 protein is depicted. The larger and underlined letters indicate the gene mutation (T) and amino acid substitution in the corresponding protein (L), respectively. Full article ">

21 pages, 3423 KiB

Open AccessArticle

Synthesis and Characterization of New Biodegradable Injectable Thermosensitive Smart Hydrogels for 5-Fluorouracil Delivery

by Adam Kasiński, Monika Zielińska-Pisklak, Sebastian Kowalczyk, Andrzej Plichta, Anna Zgadzaj, Ewa Oledzka and Marcin Sobczak

Int. J. Mol. Sci. 2021, 22(15), 8330; https://doi.org/10.3390/ijms22158330 - 3 Aug 2021

Cited by 13 | Viewed by 3413

Abstract

In this paper, injectable, thermosensitive smart hydrogel local drug delivery systems (LDDSs) releasing the model antitumour drug 5-fluorouracil (5-FU) were developed. The systems were based on biodegradable triblock copolymers synthesized via ring opening polymerization (ROP) of ε-caprolactone (CL) in the presence of poly(ethylene [...] Read more.

In this paper, injectable, thermosensitive smart hydrogel local drug delivery systems (LDDSs) releasing the model antitumour drug 5-fluorouracil (5-FU) were developed. The systems were based on biodegradable triblock copolymers synthesized via ring opening polymerization (ROP) of ε-caprolactone (CL) in the presence of poly(ethylene glycol) (PEG) and zirconium(IV) acetylacetonate (Zr(acac)₄), as co-initiator and catalyst, respectively. The structure, molecular weight (M_n) and molecular weight distribution (Đ) of the synthesized materials was studied in detail using nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC) techniques; the optimal synthesis conditions were determined. The structure corresponded well to the theoretical assumptions. The produced hydrogels demonstrated a sharp sol–gel transition at temperature close to physiological value, forming a stable gel with good mechanical properties at 37 °C. The kinetics and mechanism of in vitro 5-FU release were characterized by zero order, first order, Higuchi and Korsmeyer–Peppas mathematical models. The obtained results indicate good release control; the kinetics were generally defined as first order according to the predominant diffusion mechanism; and the total drug release time was approximately 12 h. The copolymers were considered to be biodegradable and non-toxic; the resulting hydrogels appear to be promising as short-term LDDSs, potentially useful in antitumor therapy. Full article

(This article belongs to the Special Issue Challenges, Opportunities, and Innovation in Local Drug Delivery)

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27 pages, 2917 KiB

Open AccessReview

Cannabinoids Drugs and Oral Health—From Recreational Side-Effects to Medicinal Purposes: A Systematic Review

by Luigi Bellocchio, Alessio Danilo Inchingolo, Angelo Michele Inchingolo, Felice Lorusso, Giuseppina Malcangi, Luigi Santacroce, Antonio Scarano, Ioana Roxana Bordea, Denisa Hazballa, Maria Teresa D’Oria, Ciro Gargiulo Isacco, Ludovica Nucci, Rosario Serpico, Gianluca Martino Tartaglia, Delia Giovanniello, Maria Contaldo, Marco Farronato, Gianna Dipalma and Francesco Inchingolo

Int. J. Mol. Sci. 2021, 22(15), 8329; https://doi.org/10.3390/ijms22158329 - 3 Aug 2021

Cited by 12 | Viewed by 8177

Abstract

Background: marijuana, the common name for cannabis sativa preparations, is one of the most consumed drug all over the world, both at therapeutical and recreational levels. With the legalization of medical uses of cannabis in many countries, and even its recreational use in [...] Read more.

Background: marijuana, the common name for cannabis sativa preparations, is one of the most consumed drug all over the world, both at therapeutical and recreational levels. With the legalization of medical uses of cannabis in many countries, and even its recreational use in most of these, the prevalence of marijuana use has markedly risen over the last decade. At the same time, there is also a higher prevalence in the health concerns related to cannabis use and abuse. Thus, it is mandatory for oral healthcare operators to know and deal with the consequences and effects of cannabis use on oral cavity health. This review will briefly summarize the components of cannabis and the endocannabinoid system, as well as the cellular and molecular mechanisms of biological cannabis action in human cells and biologic activities on tissues. We will also look into oropharyngeal tissue expression of cannabinoid receptors, together with a putative association of cannabis to several oral diseases. Therefore, this review will elaborate the basic biology and physiology of cannabinoids in human oral tissues with the aim of providing a better comprehension of the effects of its use and abuse on oral health, in order to include cannabinoid usage into dental patient health records as well as good medicinal practice. Methods: the paper selection was performed by PubMed/Medline and EMBASE electronic databases, and reported according to the PRISMA guidelines. The scientific products were included for qualitative analysis. Results: the paper search screened a total of 276 papers. After the initial screening and the eligibility assessment, a total of 32 articles were considered for the qualitative analysis. Conclusions: today, cannabis consumption has been correlated to a higher risk of gingival and periodontal disease, oral infection and cancer of the oral cavity, while the physico-chemical activity has not been completely clarified. Further investigations are necessary to evaluate a therapeutic efficacy of this class of drugs for the promising treatment of several different diseases of the salivary glands and oral diseases. Full article

(This article belongs to the Special Issue Endocannabinoid System in Health and Disease: Current Situation and Future Perspectives 4.0)

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16 pages, 32655 KiB

Open AccessArticle

Evidence for Protein–Protein Interaction between Dopamine Receptors and the G Protein-Coupled Receptor 143

by Beatriz Bueschbell, Prashiela Manga, Erika Penner and Anke C. Schiedel

Int. J. Mol. Sci. 2021, 22(15), 8328; https://doi.org/10.3390/ijms22158328 - 3 Aug 2021

Cited by 7 | Viewed by 4237

Abstract

Protein-protein interactions between G protein-coupled receptors (GPCRs) can augment their functionality and increase the repertoire of signaling pathways they regulate. New therapeutics designed to modulate such interactions may allow for targeting of a specific GPCR activity, thus reducing potential for side effects. Dopamine [...] Read more.

Protein-protein interactions between G protein-coupled receptors (GPCRs) can augment their functionality and increase the repertoire of signaling pathways they regulate. New therapeutics designed to modulate such interactions may allow for targeting of a specific GPCR activity, thus reducing potential for side effects. Dopamine receptor (DR) heteromers are promising candidates for targeted therapy of neurological conditions such as Parkinson’s disease since current treatments can have severe side effects. To facilitate development of such therapies, it is necessary to identify the various DR binding partners. We report here a new interaction partner for DRD₂ and DRD₃, the orphan receptor G protein-coupled receptor 143 (GPR143), an atypical GPCR that plays multiple roles in pigment cells and is expressed in several regions of the brain. We previously demonstrated that the DRD₂/ DRD₃ antagonist pimozide also modulates GPR143 activity. Using confocal microscopy and two FRET methods, we observed that the DRs and GPR143 colocalize and interact at intracellular membranes. Furthermore, co-expression of wildtype GPR143 resulted in a 57% and 67% decrease in DRD₂ and DRD₃ activity, respectively, as determined by β-Arrestin recruitment assay. GPR143-DR dimerization may negatively modulate DR activity by changing affinity for dopamine or delaying delivery of the DRs to the plasma membrane. Full article

(This article belongs to the Collection Feature Papers in Molecular Pharmacology)

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FRET of GPR143-YFP and DRD2-CFP in COS7 cells. The sensitized emission method was used to detect interaction between GPR143 (YFP channel) and DRD2 (CFP channel). FRET signal, corrected by CoA and CoB parameters, and FRET efficiency (color scale on the far right) are shown. White arrows indicate regions where FRET signal is localized. Controls are shown in <a href="#app1-ijms-22-08328" class="html-app">Figure S2</a>. Scale bar = 20 μm. Full article ">Figure 2
FRET of GPR143-YFP and DRD3-CFP in COS7 cells. A sensitized emission method was used to detect interaction between GPR143 (YFP channel) and DRD3 (CFP channel). FRET signal, corrected by CoA and CoB parameters, and FRET efficiency (color scale on the far right) are shown. White arrows indicate regions where the FRET signal is localized. Controls are shown in <a href="#app1-ijms-22-08328" class="html-app">Figure S2</a>. Scale bar = 20 μm. Full article ">Figure 3
Quantification of acceptor photobleaching FRET. Wt or pmGPR143-YFP, A2AAR-YFP or GPR18-YFP and DRs-CFP (DRD2 or DRD3) were co-transfected in COS7s. (A) Ratio of emission intensity after:before bleaching was determined. Controls = single transfected COS7s, DRs-CFP + GPR18-YFP, and CFP-YFP fusion protein; (B) FRET efficiency was quantified for co-transfected COS7s. Controls = Single transfected cells, DRs-CFP + GPR18-YFP, and CFP-YFP fusion protein. Data represent means ± SEM of three independent experiments; on average, 92 ± 9 ROIs were analyzed per sample. Significant differences between controls (single transfected and +GPR18 samples) and treatment samples including the positive control CFP-YFP fusion protein were observed. Wt and pmGPR143 and A2AAR-transfected samples did not differ from the CFP-YFP fusion-protein, except for pmGPR143+DRD2. Values refer to limited regions (see <a href="#app1-ijms-22-08328" class="html-app">Figures S3 and S4</a>). * = p < 0.0001, ns = not significant. Full article ">Figure 4
Dopamine response in CHO β-Arrestin cells expressing DRD2 and DRD3 co-transfected with a second GPCR. β-Arrestin assays were performed on dopamine treated CHO cells expressing DRD2 (A) or DRD3 (B), co-transfected with a second GPCR. The data were baseline corrected and correspond to 2–3 independent experiments performed in duplicates or triplicates. (A) Significant differences were observed between the DRD2 alone and the other dimer pairs (vs. wtGPR143+DRD2 p = 0.0113; vs. pmGPR143+DRD2 p = 0.0052 and vs. A2AAR+DRD2 p = 0.0052), except for GPR18+DRD2 where no significant difference was observed (p = 0.8082). (B) Significant differences were also observed between DRD3 alone and the other dimer pairs (* = p < 0.0001 for all), except for GPR18+DRD3 where no significant difference (ns = not significant) was observed (p = 0.1192). Full article ">Figure 5
Dopamine response of CHO β-Arrestin DRD2 and DRD3 cells co-transfected with different concentrations of wtGPR143-, pmGPR143-, A2AAR- and GPR18-YFP in the β-Arrestin recruitment assay. CHO β-Arrestin DRD2 and DRD3 cells were transfected with different amounts of plasmids containing receptor cDNA (8, 4, 2, 0.2, and 0.02 µg) and treated with 10 µM dopamine (or buffer). Data were normalized to buffer (0%), and DRs only (0 µg, 100%) and correspond to mean ± SEM of two to three independent experiments performed in triplicates or quadruplicates. (A) Coexpression of wtGPR143 had a significant effect on DRD2 at all concentrations (p < 0.0001). (B) Coexpression of pmGPR143 had a significant effect overall on DRD2 activity (p < 0.0127). The 0 µg vs. 0.02 µg sample differed less (p = 0.003), while the other concentrations differed more significantly (p < 0.0001 for 0 µg vs. 0.2–8 µg). (C) Coexpression of A2AAR did not have a significant effect on DRD2 for 0 µg vs. 0.02 µg (p = 0.1037), while significant differences were observed for the other concentrations (0.2 µg p = 0.0445; vs. 2 µg p = 0.0222; vs. 4 µg p = 0.0066; vs. 8 µg p = 0.0066). (D) Coexpression of GPR18 had no significant effect on DRD2 (p > 0.05 for all). (E) Coexpression of wtGPR143 had a significant effect on DRD3 at concentrations of 4 µg and 8 µg (p = 0.0301, p = 0.0024). (F) Coexpression of pmGPR143 had a significant effect on DRD3 overall (p < 0.0001). Post-hoc comparisons indicated significant differences in activity for concentrations greater than 2 µg (vs. 2 µg p = 0.012; vs. 4 µg p = 0.0006, vs. 8 µg p = 0.0004). (G) Coexpression of A2AAR had a significant effect on DRD3. Post-hoc comparisons indicated that the transfection of 0.02 µg and 0.2 µg significantly differed from the 0 µg sample (p = 0.071; p = 0.0066, respectively), while higher concentrations had a greater effect (vs. 2–8 µg p < 0.0001 for all). (H) Coexpression of GPR18 had no significant effect on DRD3 (p > 0.05 for all, except for buffer). * = p≤ 0.05; ** = p ≤ 0.01. ns = not significant. Full article ">

20 pages, 775 KiB

Open AccessReview

The Roles of CCCH Zinc-Finger Proteins in Plant Abiotic Stress Tolerance

by Guoliang Han, Ziqi Qiao, Yuxia Li, Chengfeng Wang and Baoshan Wang

Int. J. Mol. Sci. 2021, 22(15), 8327; https://doi.org/10.3390/ijms22158327 - 3 Aug 2021

Cited by 70 | Viewed by 6373

Abstract

Zinc-finger proteins, a superfamily of proteins with a typical structural domain that coordinates a zinc ion and binds nucleic acids, participate in the regulation of growth, development, and stress adaptation in plants. Most zinc fingers are C2H2-type or CCCC-type, named after the configuration [...] Read more.

Zinc-finger proteins, a superfamily of proteins with a typical structural domain that coordinates a zinc ion and binds nucleic acids, participate in the regulation of growth, development, and stress adaptation in plants. Most zinc fingers are C2H2-type or CCCC-type, named after the configuration of cysteine (C) and histidine (H); the less-common CCCH zinc-finger proteins are important in the regulation of plant stress responses. In this review, we introduce the domain structures, classification, and subcellular localization of CCCH zinc-finger proteins in plants and discuss their functions in transcriptional and post-transcriptional regulation via interactions with DNA, RNA, and other proteins. We describe the functions of CCCH zinc-finger proteins in plant development and tolerance to abiotic stresses such as salt, drought, flooding, cold temperatures and oxidative stress. Finally, we summarize the signal transduction pathways and regulatory networks of CCCH zinc-finger proteins in their responses to abiotic stress. CCCH zinc-finger proteins regulate the adaptation of plants to abiotic stress in various ways, but the specific molecular mechanisms need to be further explored, along with other mechanisms such as cytoplasm-to-nucleus shuttling and post-transcriptional regulation. Unraveling the molecular mechanisms by which CCCH zinc-finger proteins improve stress tolerance will facilitate the breeding and genetic engineering of crops with improved traits. Full article

(This article belongs to the Collection Feature Papers in Molecular Plant Sciences)

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13 pages, 9399 KiB

Open AccessReview

Renal and Red Marrow Dosimetry in Peptide Receptor Radionuclide Therapy: 20 Years of History and Ahead

by Stephan Walrand and François Jamar

Int. J. Mol. Sci. 2021, 22(15), 8326; https://doi.org/10.3390/ijms22158326 - 3 Aug 2021

Cited by 12 | Viewed by 2894

Abstract

The development of dosimetry and studies in peptide receptor radionuclide therapy (PRRT) over the past two decades are reviewed. Differences in kidney and bone marrow toxicity reported between ⁹⁰Y, ¹⁷⁷Lu and external beam radiotherapy (EBRT) are discussed with regard to the [...] Read more.

The development of dosimetry and studies in peptide receptor radionuclide therapy (PRRT) over the past two decades are reviewed. Differences in kidney and bone marrow toxicity reported between ⁹⁰Y, ¹⁷⁷Lu and external beam radiotherapy (EBRT) are discussed with regard to the physical properties of these beta emitter radionuclides. The impact of these properties on the response to small and large tumors is also considered. Capacities of the imaging modalities to assess the dosimetry to target tissues are evaluated. Studies published in the past two years that confirm a red marrow uptake in ¹⁷⁷Lu-DOTATATE therapy, as already observed 20 years ago in ⁸⁶Y-DOTATOC PET studies, are analyzed in light of the recent developments in the transferrin transport mechanism. The review enlightens the importance (i) of using state-of-the-art imaging modalities, (ii) of individualizing the activity to be injected with regard to the huge tissue uptake variability observed between patients, (iii) of challenging the currently used but inappropriate blood-based red marrow dosimetry and (iv) of considering individual tandem therapy. Last, a smart individually optimized tandem therapy taking benefit of the bi-orthogonal toxicity-response pattern of ¹⁷⁷Lu-DOTATATE and of ⁹⁰Y-DOTATOC is proposed. Full article

(This article belongs to the Special Issue Somatostatin 2.0)

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