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Biomolecules, Volume 9, Issue 10 (October 2019)

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Cover Story (view full-size image) Lysyl oxidases (LOX and LOX-likes [LOXLs] isoenzymes) are copper-dependent enzymes involved in the [...] Read more.
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Open AccessArticle
Mechanical Tillage Diversely Affects Glomalin Content, Water Stable Aggregates and AM Fungal Community in the Soil Profiles of Two Differently Managed Olive Orchards
Biomolecules 2019, 9(10), 639; https://doi.org/10.3390/biom9100639 - 22 Oct 2019
Abstract
This work was designed to investigate the effect of mechanical tillage on glomalin content, arbuscular mycorrhizal fungi (AMF) abundance and diversity, and the concentration of water stable aggregates (WSA), in two adjacent olive groves located in Basilicata (Italy) that were managed over the [...] Read more.
This work was designed to investigate the effect of mechanical tillage on glomalin content, arbuscular mycorrhizal fungi (AMF) abundance and diversity, and the concentration of water stable aggregates (WSA), in two adjacent olive groves located in Basilicata (Italy) that were managed over the course of 11 years in accordance with different horticultural models (conventional and sustainable). Soil sampling was performed at four depths between the trees within a row and between rows. In the end, WSA was found to be a highly sensitive indicator (especially in the “macro” fraction) of the effect of management on soil structure, showing the highest statistically significant values within the sustainable system. In the same regard, the diversity of the AM fungal community was negatively affected by conventional practices; on the other hand, a higher concentration of glomalin in the first 20 cm layer of the conventional system is here reported for the first time, as a likely result of disruption of the mycelium provoked by the mechanical tillage. Full article
(This article belongs to the Section Chemical Biology)
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Open AccessArticle
The Novel Direct Modulatory Effects of Perampanel, an Antagonist of AMPA Receptors, on Voltage-Gated Sodium and M-type Potassium Currents
Biomolecules 2019, 9(10), 638; https://doi.org/10.3390/biom9100638 - 22 Oct 2019
Abstract
Perampanel (PER) is a selective blocker of AMPA receptors showing efficacy in treating various epileptic disorders including brain tumor-related epilepsy and also potential in treating motor neuron disease. However, besides its inhibition of AMPA-induced currents, whether PER has any other direct ionic effects [...] Read more.
Perampanel (PER) is a selective blocker of AMPA receptors showing efficacy in treating various epileptic disorders including brain tumor-related epilepsy and also potential in treating motor neuron disease. However, besides its inhibition of AMPA-induced currents, whether PER has any other direct ionic effects in different types of neurons remains largely unknown. We investigated the effects of PER and related compounds on ionic currents in different types of cells, including hippocampal mHippoE-14 neurons, motor neuron-like NSC-34 cells and U87 glioma cells. We found that PER differentially and effectively suppressed the amplitude of voltage-gated Na+ currents (INa) in mHippoE-14 cells. The IC50 values required to inhibit peak and late INa were 4.12 and 0.78 μM, respectively. PER attenuated tefluthrin-induced increases in both amplitude and deactivating time constant of INa. Importantly, PER also inhibited the amplitude of M-type K+ currents (IK(M)) with an IC50 value of 0.92 μM. The suppression of IK(M) was attenuated by the addition of flupirtine or ZnCl2 but not by L-quisqualic acid or sorafenib. Meanwhile, in cell-attached configuration, PER (3 μM) decreased the activity of M-type K+ channels with no change in single-channel conductance but shifting the activation curve along the voltage axis in a rightward direction. Supportively, PER suppressed IK(M) in NSC-34 cells and INa in U87 glioma cells. The inhibitory effects of PER on both INa and IK(M), independent of its antagonistic effect on AMPA receptors, may be responsible for its wide-spectrum of effects observed in neurological clinical practice. Full article
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Open AccessArticle
Antioxidant Barrier, Redox Status, and Oxidative Damage to Biomolecules in Patients with Colorectal Cancer. Can Malondialdehyde and Catalase Be Markers of Colorectal Cancer Advancement?
Biomolecules 2019, 9(10), 637; https://doi.org/10.3390/biom9100637 - 22 Oct 2019
Abstract
This study is the first to assess the diagnostic utility of redox biomarkers in patients with colorectal cancer (CRC). Antioxidant barrier (Cu,Zn-superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), uric acid (UA), reduced glutathione (GSH)), redox status (total antioxidant (TAC)/oxidant [...] Read more.
This study is the first to assess the diagnostic utility of redox biomarkers in patients with colorectal cancer (CRC). Antioxidant barrier (Cu,Zn-superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), uric acid (UA), reduced glutathione (GSH)), redox status (total antioxidant (TAC)/oxidant status (TOS), ferric reducing ability (FRAP)), and oxidative damage products (advanced glycation end products (AGE), advanced oxidation protein products (AOPP), malondialdehyde (MDA)) were measured in serum/plasma samples of 50 CRC patients. The activity of SOD was significantly higher whereas the activity of CAT, GPx and GR was considerably lower in CRC patients compared to the control group (p < 0.0001). Levels of UA, TOS, and OSI and concentrations of AGE, AOPP, and MDA were significantly higher, and the levels of GSH, TAC, and FRAP were considerably lower in CRC patients compared to the healthy controls (p < 0.0001). AUC for CAT with respect to presence of lymph node metastasis was 0.7450 (p = 0.0036), whereas AUC for MDA according to the depth of tumour invasion was 0.7457 (p = 0.0118). CRC is associated with enzymatic/non-enzymatic redox imbalance as well as increased oxidative damage to proteins and lipids. Redox biomarkers can be potential diagnostic indicators of CRC advancement. Full article
(This article belongs to the Special Issue Oxidative Damage on Biomolecules and Antioxidants)
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Open AccessArticle
Increased Expression of Adherens Junction Components in Mouse Liver following Bile Duct Ligation
Biomolecules 2019, 9(10), 636; https://doi.org/10.3390/biom9100636 - 22 Oct 2019
Abstract
Adherens junctions, consisting of cadherins and catenins, are a group of cell-to-cell junctions that mediate mechanistic linkage between neighboring cells. By doing so, adherens junctions ensure direct intercellular contact and play an indispensable role in maintaining tissue architecture. Considering these critical functions, it [...] Read more.
Adherens junctions, consisting of cadherins and catenins, are a group of cell-to-cell junctions that mediate mechanistic linkage between neighboring cells. By doing so, adherens junctions ensure direct intercellular contact and play an indispensable role in maintaining tissue architecture. Considering these critical functions, it is not surprising that adherens junctions are frequently involved in disease. In the present study, the effects of bile duct ligation—a surgical procedure to experimentally induce cholestatic and fibrotic liver pathology—on hepatic adherens junctions were investigated in mice. In essence, it was found that liver mRNA and protein levels of E-cadherin, β-catenin and γ-catenin drastically increase following bile duct ligation. These results could suggest a cytoprotective role for hepatic adherens junctions following bile duct ligation. Full article
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Open AccessArticle
GLYI4 Plays A Role in Methylglyoxal Detoxification and Jasmonate-Mediated Stress Responses in Arabidopsis thaliana
Biomolecules 2019, 9(10), 635; https://doi.org/10.3390/biom9100635 - 22 Oct 2019
Abstract
Plant hormones play a central role in various physiological functions and in mediating defense responses against (a)biotic stresses. In response to primary metabolism alteration, plants can produce also small molecules such as methylglyoxal (MG), a cytotoxic aldehyde. MG is mostly detoxified by the [...] Read more.
Plant hormones play a central role in various physiological functions and in mediating defense responses against (a)biotic stresses. In response to primary metabolism alteration, plants can produce also small molecules such as methylglyoxal (MG), a cytotoxic aldehyde. MG is mostly detoxified by the combined actions of the enzymes glyoxalase I (GLYI) and glyoxalase II (GLYII) that make up the glyoxalase system. Recently, by a genome-wide association study performed in Arabidopsis, we identified GLYI4 as a novel player in the crosstalk between jasmonate (JA) and salicylic acid (SA) hormone pathways. Here, we investigated the impact of GLYI4 knock-down on MG scavenging and on JA pathway. In glyI4 mutant plants, we observed a general stress phenotype, characterized by compromised MG scavenging, accumulation of reactive oxygen species (ROS), stomatal closure, and reduced fitness. Accumulation of MG in glyI4 plants led to lower efficiency of the JA pathway, as highlighted by the increased susceptibility of the plants to the pathogenic fungus Plectospherella cucumerina. Moreover, MG accumulation brought about a localization of GLYI4 to the plasma membrane, while MeJA stimulus induced a translocation of the protein into the cytoplasmic compartment. Collectively, the results are consistent with the hypothesis that GLYI4 is a hub in the MG and JA pathways. Full article
(This article belongs to the Special Issue Phytohormones)
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Open AccessArticle
CDK12 Activity-Dependent Phosphorylation Events in Human Cells
Biomolecules 2019, 9(10), 634; https://doi.org/10.3390/biom9100634 - 22 Oct 2019
Abstract
We asked whether the C-terminal repeat domain (CTD) kinase, CDK12/CyclinK, phosphorylates substrates in addition to the CTD of RPB1, using our CDK12analog-sensitive HeLa cell line to investigate CDK12 activity-dependent phosphorylation events in human cells. Characterizing the phospho-proteome before and after selective inhibition [...] Read more.
We asked whether the C-terminal repeat domain (CTD) kinase, CDK12/CyclinK, phosphorylates substrates in addition to the CTD of RPB1, using our CDK12analog-sensitive HeLa cell line to investigate CDK12 activity-dependent phosphorylation events in human cells. Characterizing the phospho-proteome before and after selective inhibition of CDK12 activity by the analog 1-NM-PP1, we identified 5,644 distinct phospho-peptides, among which were 50 whose average relative amount decreased more than 2-fold after 30 min of inhibition (none of these derived from RPB1). Half of the phospho-peptides actually showed >3-fold decreases, and a dozen showed decreases of 5-fold or more. As might be expected, the 40 proteins that gave rise to the 50 affected phospho-peptides mostly function in processes that have been linked to CDK12, such as transcription and RNA processing. However, the results also suggest roles for CDK12 in other events, notably mRNA nuclear export, cell differentiation and mitosis. While a number of the more-affected sites resemble the CTD in amino acid sequence and are likely direct CDK12 substrates, other highly-affected sites are not CTD-like, and their decreased phosphorylation may be a secondary (downstream) effect of CDK12 inhibition. Full article
(This article belongs to the Section Molecular Biology)
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Open AccessArticle
Disclosing the Impact of Carcinogenic SF3b Mutations on Pre-mRNA Recognition Via All-Atom Simulations
Biomolecules 2019, 9(10), 633; https://doi.org/10.3390/biom9100633 - 21 Oct 2019
Viewed by 102
Abstract
The spliceosome accurately promotes precursor messenger-RNA splicing by recognizing specific noncoding intronic tracts including the branch point sequence (BPS) and the 3’-splice-site (3’SS). Mutations of Hsh155 (yeast)/SF3B1 (human), which is a protein of the SF3b factor involved in BPS recognition and induces altered [...] Read more.
The spliceosome accurately promotes precursor messenger-RNA splicing by recognizing specific noncoding intronic tracts including the branch point sequence (BPS) and the 3’-splice-site (3’SS). Mutations of Hsh155 (yeast)/SF3B1 (human), which is a protein of the SF3b factor involved in BPS recognition and induces altered BPS binding and 3’SS selection, lead to mis-spliced mRNA transcripts. Although these mutations recur in hematologic malignancies, the mechanism by which they change gene expression remains unclear. In this study, multi-microsecond-long molecular-dynamics simulations of eighth distinct ∼700,000 atom models of the spliceosome Bact complex, and gene sequencing of SF3B1, disclose that these carcinogenic isoforms destabilize intron binding and/or affect the functional dynamics of Hsh155/SF3B1 only when binding non-consensus BPSs, as opposed to the non-pathogenic variants newly annotated here. This pinpoints a cross-talk between the distal Hsh155 mutation and BPS recognition sites. Our outcomes unprecedentedly contribute to elucidating the principles of pre-mRNA recognition, which provides critical insights on the mechanism underlying constitutive/alternative/aberrant splicing. Full article
(This article belongs to the Section Biomacromolecules)
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Open AccessArticle
The Antifungal Effect of Garlic Essential Oil on Phytophthora nicotianae and the Inhibitory Component Involved
Biomolecules 2019, 9(10), 632; https://doi.org/10.3390/biom9100632 - 21 Oct 2019
Viewed by 104
Abstract
This study explored the chemical compositions of garlic essential oil, the inhibitory activity of garlic essential oil and diallyl disulfide (DADS) against Phytophthora nicotianae, and the effects on mycelial plasma membrane permeability and P. nicotianae inhibition. In total, 29 compounds were detected [...] Read more.
This study explored the chemical compositions of garlic essential oil, the inhibitory activity of garlic essential oil and diallyl disulfide (DADS) against Phytophthora nicotianae, and the effects on mycelial plasma membrane permeability and P. nicotianae inhibition. In total, 29 compounds were detected in garlic essential oil, of which 26 were detected by gas chromatography‒mass spectrometry (GC-MS) and 21 by headspace solid-phase microextraction (HS-SPME) GC-MS. DADS (60.12% and 19.09%) and trisulfide di-2-propenyl (14.18% and 17.98%) were the major components identified by HS-SPME GC-MS and GC-MS analysis, respectively. Half-inhibitory concentration (Ec50, antagonism) and minimum inhibitory concentration (MIC, fumigation) of DADS against P. nicotianae were 150.83 μL/L and 20 μL/L, respectively, while Ec50 of garlic essential oil was 1108.25 μL/L. Mycelial membrane permeability gradually increased in a concentration-dependent manner, and cell death increased at 450 μL/L DADS. Furthermore, DADS treatment significantly reduced the incidence of tobacco black shank and the number of P. nicotianae pathogens in rhizosphere soil. DADS also promoted root development of tobacco seedlings at low concentrations, which was inhibited at high concentrations. Therefore, DADS may play an important role in the antifungal effect against P. nicotianae by destroying mycelial cell membrane integrity, causing an increase in cell membrane permeability, and leading to cell death. Full article
(This article belongs to the Special Issue Perspectives of Essential Oils)
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Open AccessArticle
Synthesis of Novel Analogs of Thieno[2,3-d] Pyrimidin-4(3H)-ones as Selective Inhibitors of Cancer Cell Growth
Biomolecules 2019, 9(10), 631; https://doi.org/10.3390/biom9100631 - 21 Oct 2019
Viewed by 110
Abstract
New 2,3-disubstituted thieno[2,3-d]pyrimidin-4(3H)-ones were synthesized via a one-pot reaction from 2H-thieno[2,3-d] [1,3]oxazine-2,4(1H)-diones, aromatic aldehydes, and benzylamine or 4-hydroxylbezylamine. The obtained compounds were tested in vitro for cancer cell growth inhibition. Compound 19 can [...] Read more.
New 2,3-disubstituted thieno[2,3-d]pyrimidin-4(3H)-ones were synthesized via a one-pot reaction from 2H-thieno[2,3-d] [1,3]oxazine-2,4(1H)-diones, aromatic aldehydes, and benzylamine or 4-hydroxylbezylamine. The obtained compounds were tested in vitro for cancer cell growth inhibition. Compound 19 can inhibit all four types of tested cancer cells, i.e., MCF-7, A549, PC-9, and PC-3 cells. Most of the compounds inhibited the proliferation of A549 and MCF-7 cells. Compound 15 exhibited the strongest anti-proliferative effect against A549 cell lines with IC50 values of 0.94 μM, and with no toxicity to normal human liver cells. Its potency was further proved by cell clone formation assay, Hoechst 33258 staining, and evaluation on the effects of apoptosis-related proteins. Full article
(This article belongs to the Special Issue Bioactives from Marine Products)
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Open AccessArticle
Sex-Specific Associations of Brain-Derived Neurotrophic Factor and Cardiorespiratory Fitness in the General Population
Biomolecules 2019, 9(10), 630; https://doi.org/10.3390/biom9100630 - 20 Oct 2019
Viewed by 197
Abstract
The brain-derived neurotrophic factor (BDNF) was initially considered to be neuron-specific. Meanwhile, this neurotrophin is peripherally also secreted by skeletal muscle cells and increases due to exercise. Whether BDNF is related to cardiorespiratory fitness (CRF) is currently unclear. We analyzed the association of [...] Read more.
The brain-derived neurotrophic factor (BDNF) was initially considered to be neuron-specific. Meanwhile, this neurotrophin is peripherally also secreted by skeletal muscle cells and increases due to exercise. Whether BDNF is related to cardiorespiratory fitness (CRF) is currently unclear. We analyzed the association of serum BDNF levels with CRF in the general population (Study of Health in Pomerania (SHIP-TREND) from Northeast Germany; n = 1607, 51% female; median age 48 years). Sex-stratified linear regression models adjusted for age, height, smoking, body fat, lean mass, physical activity, and depression analyzed the association between BDNF and maximal oxygen consumption (VO2peak), maximal oxygen consumption normalized for body weight (VO2peak/kg), and oxygen consumption at the anaerobic threshold (VO2@AT). In women, 1 mL/min higher VO2peak, VO2peak/kg, and VO2@AT were associated with a 2.43 pg/mL (95% confidence interval [CI]: 1.16 to 3.69 pg/mL; p = 0.0002), 150.66 pg/mL (95% CI: 63.42 to 237.90 pg/mL; p = 0.0007), and 2.68 pg/mL (95% CI: 0.5 to 4.8 pg/mL; p = 0.01) higher BDNF serum concentration, respectively. No significant associations were found in men. Further research is needed to understand the sex-specific association between CRF and BDNF. Full article
(This article belongs to the Section Biological Factors)
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Open AccessArticle
The HER2 S310F Mutant Can Form an Active Heterodimer with the EGFR, Which Can Be Inhibited by Cetuximab but Not by Trastuzumab as well as Pertuzumab
Biomolecules 2019, 9(10), 629; https://doi.org/10.3390/biom9100629 - 19 Oct 2019
Viewed by 125
Abstract
G309 or S310 mutations on the HER2 extracellular domain II induce receptor activation. Clinically, S310F is most frequent among HER2 extracellular domain mutations and patients with the S310F mutation without HER2 amplification responded to trastuzumab with or without the pertuzumab combination. However, the [...] Read more.
G309 or S310 mutations on the HER2 extracellular domain II induce receptor activation. Clinically, S310F is most frequent among HER2 extracellular domain mutations and patients with the S310F mutation without HER2 amplification responded to trastuzumab with or without the pertuzumab combination. However, the ability of S310F mutant to form homodimers or heterodimers with wild-type HER2 and other HER receptors, or their reactivity to trastuzumab and pertuzumab treatments, has not been reported. We overexpressed S310F as well as G309A, G309E and S310Y HER2 mutants and tested their reactivity to trastuzumab and pertuzumab. All mutants reacted to trastuzumab, but S310F mutant did not react to pertuzumab along with S310Y or G309E mutants. Thereafter, we tested the effects of trastuzumab and pertuzumab on 5637 cell line expressing both wild-type HER2 and S310F mutant. The ligand-independent HER2 homodimerization blocking antibody, trastuzumab, did not inhibit the activation of the HER2 receptor, suggesting that the S310F HER2 mutant did not form homodimers or heterodimers with wild-type HER2. Because 5637 cells overexpressed the EGFR, the effects of cetuximab and gefitinib were determined, and both inhibited the activation of HER2 and significantly reduced cell growth. Because pertuzumab did not inhibit the phosphorylation of HER2 while it bound to wild-type HER2, EGFR-mediated phosphorylation is expected to occur on the S310F mutant. To confirm whether the S310F mutant HER2 retained its affinity to the EGFR, single molecule interaction analyses using TIRF microscopy were performed, which showed that S310F mutant successfully formed complexes with EGFR. In conclusion, HER2 S310F mutant can form an active heterodimer with the EGFR and it can be inhibited by cetuximab, but not by trastuzumab in combination with pertuzumab. Full article
(This article belongs to the Special Issue Advances in Antibody Therapy of Cancer)
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Open AccessArticle
A Novel Dermaseptin Isolated from the Skin Secretion of Phyllomedusa tarsius and Its Cationicity-Enhanced Analogue Exhibiting Effective Antimicrobial and Anti-Proliferative Activities
Biomolecules 2019, 9(10), 628; https://doi.org/10.3390/biom9100628 - 18 Oct 2019
Viewed by 236
Abstract
A novel dermaseptin peptide, dermaseptin-PT9 (DPT9), was isolated and identified from Phyllomedusa tarsius by the combination of molecular cloning and LC-MS analysis. Chemically synthesised DPT9 was broadly effective against the tested microorganisms through the disruption of cell membranes and showed weak haemolytic activity [...] Read more.
A novel dermaseptin peptide, dermaseptin-PT9 (DPT9), was isolated and identified from Phyllomedusa tarsius by the combination of molecular cloning and LC-MS analysis. Chemically synthesised DPT9 was broadly effective against the tested microorganisms through the disruption of cell membranes and showed weak haemolytic activity towards horse erythrocytes. It also exhibited anti-proliferative effect against various human cancer cells. Moreover, an analogue with enhanced cationicity, K8, 23-DPT9, in which Asp8 and Glu23 were substituted by lysine residues, had a markedly increased antimicrobial effect against all tested microorganisms and disrupted microbial cell membranes. This analogue also showed no haemolysis at its effective antimicrobial concentrations. In addition, K8, 23-DPT9 displayed an enhanced anti-proliferative effect against cancer cells, while displayed weak activity against the normal human cell line, HMEC-1. Full article
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Open AccessArticle
Effects of Sterols on the Interaction of SDS, Benzalkonium Chloride, and A Novel Compound, Kor105, with Membranes
Biomolecules 2019, 9(10), 627; https://doi.org/10.3390/biom9100627 - 18 Oct 2019
Viewed by 136
Abstract
Sterols change the biophysical properties of lipid membranes. Here, we analyzed how sterols affect the activity of widely used antimicrobial membrane-active compounds, sodium dodecyl sulfate (SDS) and benzalkonium chloride (BAC). We also tested a novel benzalkonium-like substance, Kor105. Our data suggest that benzalkonium [...] Read more.
Sterols change the biophysical properties of lipid membranes. Here, we analyzed how sterols affect the activity of widely used antimicrobial membrane-active compounds, sodium dodecyl sulfate (SDS) and benzalkonium chloride (BAC). We also tested a novel benzalkonium-like substance, Kor105. Our data suggest that benzalkonium and Kor105 disturb the ordering of the membrane lipid packaging, and this disturbance is dampened by cholesterol. The disturbance induced by Kor105 is stronger than that induced by BAC because of the higher rigidity of the Kor105 molecule due to a shorter linker between the phenyl group and quaternary nitrogen. On the contrary, individual SDS molecules do not cause the disturbance. Thus, in the tested range of concentrations, SDS–membrane interaction is not influenced by cholesterol. To study how sterols influence the biological effects of these chemicals, we used yeast strains lacking Lam1–4 proteins. These proteins transport sterols from the plasma membrane into the endoplasmic reticulum. We found that the mutants are resistant to BAC and Kor105 but hypersensitive to SDS. Together, our findings show that sterols influence the interaction of SDS versus benzalkonium chloride and Kor105 with the membranes in a completely different manner. Full article
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Open AccessArticle
Anti-Cancer Effects of Synergistic Drug–Bacterium Combinations on Induced Breast Cancer in BALB/c Mice
Biomolecules 2019, 9(10), 626; https://doi.org/10.3390/biom9100626 - 18 Oct 2019
Viewed by 113
Abstract
Cancer development and progression are extremely complex due to the alteration of various genes and pathways. In most cases, multiple agents are required to control cancer progression. The purpose of this study is to investigate, using a mouse model, the synergistic interactions of [...] Read more.
Cancer development and progression are extremely complex due to the alteration of various genes and pathways. In most cases, multiple agents are required to control cancer progression. The purpose of this study is to investigate, using a mouse model, the synergistic interactions of anti-cancer agents, 1′-S-1′-acetoxychavicol acetate (ACA), Mycobacterium indicus pranii (MIP), and cisplatin (CDDP) in double and triple combinations to treat chemo-sensitize and immune-sensitize breast cancer. Changes in tumor volume and body weight were monitored. Organs were harvested and stained using hematoxylin–eosin for histopathological assessment. Milliplex enzyme-linked immunosorbent assay (ELISA) was performed to determine cytokine levels, while immunohistochemistry (IHC) was conducted on tumor biopsies to verify systemic drug effects. In vivo mouse models showed tumor regression with maintenance of regular body weight for all the different treatment regimens. IHC results provided conclusive evidence indicating that combination regimens were able to down-regulate nuclear factor kappa-B activation and reduce the expression of its regulated pro-inflammatory proteins. Reduction of pro-inflammatory cytokines (e.g., IL-6, TNF-α, and IFN-ɣ) levels were observed when using the triple combination, which indicated that the synergistic drug combination was able to significantly control cancer progression. In conclusion, ACA, MIP, and CDDP together serve as promising candidates for further development and for subsequent clinical trials against estrogen-sensitive breast cancer. Full article
(This article belongs to the Special Issue Antitumor Agents from Natural Sources)
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Open AccessArticle
Interaction of a Polyarginine Peptide with Membranes of Different Mechanical Properties
Biomolecules 2019, 9(10), 625; https://doi.org/10.3390/biom9100625 - 18 Oct 2019
Viewed by 114
Abstract
The membrane translocation efficiency of cell penetrating peptides (CPPs) has been largely studied, and poly-arginines have been highlighted as particularly active CPPs, especially upon negatively charged membranes. Here we inquire about the influence of membrane mechanical properties in poly-arginine adsorption, penetration and translocation, [...] Read more.
The membrane translocation efficiency of cell penetrating peptides (CPPs) has been largely studied, and poly-arginines have been highlighted as particularly active CPPs, especially upon negatively charged membranes. Here we inquire about the influence of membrane mechanical properties in poly-arginine adsorption, penetration and translocation, as well as the subsequent effect on the host membrane. For this, we selected anionic membranes exhibiting different rigidity and fluidity, and exposed them to the nona-arginine KR9C. Three different membrane compositions were investigated, all of them having 50% of the anionic lipid 1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DOPG), thus, ensuring a high affinity of the peptide for membrane surfaces. The remaining 50% was a saturated PC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), an unsaturated PC (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) or a mixture of DOPC with cholesterol. Peptide-membrane interactions were studied using four complementary models for membranes: Langmuir monolayers, Large Unilamellar Vesicles, Black Lipid Membranes and Giant Unilamellar Vesicles. The patterns of interaction of KR9C varied within the different membrane compositions. The peptide strongly adsorbed on membranes with cholesterol, but did not incorporate or translocate them. KR9C stabilized phase segregation in DPPC/DOPG films and promoted vesicle rupture. DOPC/DOPG appeared like the better host for peptide translocation: KR9C adsorbed, inserted and translocated these membranes without breaking them, despite softening was observed. Full article
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Open AccessArticle
Electro-Acupuncture Alleviates Cisplatin-Induced Anorexia in Rats by Modulating Ghrelin and Monoamine Neurotransmitters
Biomolecules 2019, 9(10), 624; https://doi.org/10.3390/biom9100624 - 18 Oct 2019
Viewed by 107
Abstract
Anorexia is common in patients with cancer, mostly as a side effect of chemotherapy. The effect of electro-acupuncture (EA) on ameliorating cancer-related symptoms have been studied in animal models and in clinical trials. The aim of this study was to determine optimal conditions [...] Read more.
Anorexia is common in patients with cancer, mostly as a side effect of chemotherapy. The effect of electro-acupuncture (EA) on ameliorating cancer-related symptoms have been studied in animal models and in clinical trials. The aim of this study was to determine optimal conditions for the application of EA to alleviate anorexia, followed by the study of molecular mechanisms affecting its therapeutics. Anorexia was induced in male Wistar rats by injecting cisplatin, which was then followed by EA treatment at CV12, the acupuncture point located in the center of the abdominal midline. Body weight and food intake were measured daily throughout the duration of the study. The levels of monoamine neurotransmitters in the plasma were quantitatively analyzed by HPLC-ECD. Gastrointestinal hormone concentrations were elucidated with ELISA kits. RT-qPCR was performed to evaluate the mRNA expression of ghrelin (GHRL), neuropeptide Y (NPY), and pro-opiomelanocortin. The expression of c-Fos in the nucleus tractus solitarii was detected using western blotting analysis. The optimal conditions of EA to alleviate anorexia in rats was determined to be 1 unit for intensity and 10 Hz for frequency. EA treatment at CV12 reduced the levels of plasma monoamine neurotransmitters 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, dopamine, and norepinephrine; as well as stimulated the expression of GHRL and NPY to alleviate cisplatin-induced anorexia in rats. EA stimulation at CV12 could be used to treat cisplatin-induced anorexia in rats. Full article
(This article belongs to the Special Issue Peptides: Molecular and Biotechnological Aspects)
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Open AccessFeature PaperReview
Epigenetic Regulation of Auxin Homeostasis
Biomolecules 2019, 9(10), 623; https://doi.org/10.3390/biom9100623 - 18 Oct 2019
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Abstract
Epigenetic regulation involves a myriad of mechanisms that regulate the expression of loci without altering the DNA sequence. These different mechanisms primarily result in modifications of the chromatin topology or DNA chemical structure that can be heritable or transient as a dynamic response [...] Read more.
Epigenetic regulation involves a myriad of mechanisms that regulate the expression of loci without altering the DNA sequence. These different mechanisms primarily result in modifications of the chromatin topology or DNA chemical structure that can be heritable or transient as a dynamic response to environmental cues. The phytohormone auxin plays an important role in almost every aspect of plant life via gradient formation. Auxin maxima/minima result from a complex balance of metabolism, transport, and signaling. Although epigenetic regulation of gene expression during development has been known for decades, the specific mechanisms behind the spatiotemporal dynamics of auxin levels in plants are only just being elucidated. In this review, we gather current knowledge on the epigenetic mechanisms regulating the expression of genes for indole-3-acetic acid (IAA) metabolism and transport in Arabidopsis and discuss future perspectives of this emerging field. Full article
(This article belongs to the Special Issue Advances in Molecular Biology of Action of Auxins)
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Open AccessArticle
Caveolin-1 Regulates P2Y2 Receptor Signaling during Mechanical Injury in Human 1321N1 Astrocytoma
Biomolecules 2019, 9(10), 622; https://doi.org/10.3390/biom9100622 - 18 Oct 2019
Viewed by 136
Abstract
Caveolae-associated protein caveolin-1 (Cav-1) plays key roles in cellular processes such as mechanosensing, receptor coupling to signaling pathways, cell growth, apoptosis, and cancer. In 1321N1 astrocytoma cells Cav-1 interacts with the P2Y2 receptor (P2Y2R) to modulate its downstream signaling. P2Y [...] Read more.
Caveolae-associated protein caveolin-1 (Cav-1) plays key roles in cellular processes such as mechanosensing, receptor coupling to signaling pathways, cell growth, apoptosis, and cancer. In 1321N1 astrocytoma cells Cav-1 interacts with the P2Y2 receptor (P2Y2R) to modulate its downstream signaling. P2Y2R and its signaling machinery also mediate pro-survival actions after mechanical injury. This study determines if Cav-1 knockdown (KD) affects P2Y2R signaling and its pro-survival actions in the 1321N1 astrocytoma cells mechanical injury model system. KD of Cav-1 decreased its expression in 1321N1 cells devoid of or expressing hHAP2Y2R by ~88% and ~85%, respectively. Cav-1 KD had no significant impact on P2Y2R expression. Post-injury densitometric analysis of pERK1/2 and Akt activities in Cav-1-positive 1321N1 cells (devoid of or expressing a hHAP2Y2R) revealed a P2Y2R-dependent temporal increase in both kinases. These temporal increases in pERK1/2 and pAkt were significantly decreased in Cav-1 KD 1321N1 (devoid of or expressing a hHAP2Y2R). Cav-1 KD led to an ~2.0-fold and ~2.4-fold decrease in the magnitude of the hHAP2Y2R-mediated pERK1/2 and pAkt kinases’ activity, respectively. These early-onset hHAP2Y2R-mediated signaling responses in Cav-1-expressing and Cav-1 KD 1321N1 correlated with changes in cell viability (via a resazurin-based method) and apoptosis (via caspase-9 expression). In Cav-1-positive 1321N1 cells, expression of hHAP2Y2R led to a significant increase in cell viability and decreased apoptotic (caspase-9) activity after mechanical injury. In contrast, hHAP2Y2R-elicited changes in viability and apoptotic (caspase-9) activity were decreased after mechanical injury in Cav-1 KD 1321N1 cells expressing hHAP2Y2R. These findings support the importance of Cav-1 in modulating P2Y2R signaling during mechanical injury and its protective actions in a human astrocytoma cell line, whilst shedding light on potential new venues for brain injury or trauma interventions. Full article
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Open AccessArticle
Beyond Chelation: EDTA Tightly Binds Taq DNA Polymerase, MutT and dUTPase and Directly Inhibits dNTPase Activity
Biomolecules 2019, 9(10), 621; https://doi.org/10.3390/biom9100621 - 17 Oct 2019
Viewed by 237
Abstract
EDTA is commonly used as an efficient chelator of metal ion enzyme cofactors. It is highly soluble, optically inactive and does not interfere with most chemicals used in standard buffers making EDTA a common choice to generate metal-free conditions for biochemical and biophysical [...] Read more.
EDTA is commonly used as an efficient chelator of metal ion enzyme cofactors. It is highly soluble, optically inactive and does not interfere with most chemicals used in standard buffers making EDTA a common choice to generate metal-free conditions for biochemical and biophysical investigations. However, the controversy in the literature on metal-free enzyme activities achieved using EDTA or by other means called our attention to a putative effect of EDTA beyond chelation. Here, we show that EDTA competes for the nucleotide binding site of the nucleotide hydrolase dUTPase by developing an interaction network within the active site similar to that of the substrate. To achieve these findings, we applied kinetics and molecular docking techniques using two different dUTPases. Furthermore, we directly measured the binding of EDTA to dUTPases and to two other dNTPases, the Taq polymerase and MutT using isothermal titration calorimetry. EDTA binding proved to be exothermic and mainly enthalpy driven with a submicromolar dissociation constant considerably lower than that of the enzyme:substrate or the Mg:EDTA complexes. Control proteins, including an ATPase, did not interact with EDTA. Our findings indicate that EDTA may act as a selective inhibitor against dNTP hydrolyzing enzymes and urge the rethinking of the utilization of EDTA in enzymatic experiments. Full article
(This article belongs to the Section Biochemistry)
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Open AccessArticle
YAP Inhibition by Nuciferine via AMPK-Mediated Downregulation of HMGCR Sensitizes Pancreatic Cancer Cells to Gemcitabine
Biomolecules 2019, 9(10), 620; https://doi.org/10.3390/biom9100620 - 17 Oct 2019
Viewed by 172
Abstract
Nuciferine, a major aporphine alkaloid constituent of lotus leaves, is a raw material for obesity treatment. Extensive studies have revealed that obesity is associated with pancreatic cancer (PC). However, it has not been clarified whether nuciferine could be used in PC treatment or [...] Read more.
Nuciferine, a major aporphine alkaloid constituent of lotus leaves, is a raw material for obesity treatment. Extensive studies have revealed that obesity is associated with pancreatic cancer (PC). However, it has not been clarified whether nuciferine could be used in PC treatment or prevention. Here, we show that nuciferine could enhance the sensitivity of PC cells to gemcitabine in both cultured cells and the xenograft mouse model. The mechanism study demonstrated that nuciferine induced YAP Ser127 phosphorylation [pYAP(Ser127)] through AMPK-mediated 3-hydroxy-3-methyl-glutaryl-coA reductase (HMGCR) downregulation. Remarkably, wild-type YAP overexpression or YAP Ser127 mutant could resist to nuciferine and no longer sensitize PC cells to gemcitabine. Knockdown of AMPK attenuated pYAP(Ser127) induced by nuciferine. Moreover, knockdown of AMPK reversed nuciferine-mediated HMGCR downregulation. Notably, HMGCR inhibiting could restrain YAP by phosphorylation Ser 127, and therefore enhance the efficiency of gemcitabine in PC cells. In line with this consistent, overexpression of HMGCR reduced growth inhibition caused by nuciferine and/or gemcitabine treatment in PC cells. In summary, these results provide an effective supplementary agent and suggest a therapeutic strategy to reduce gemcitabine resistance in PC. Full article
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Open AccessReview
Status of Plant Protein-Based Green Scaffolds for Regenerative Medicine Applications
Biomolecules 2019, 9(10), 619; https://doi.org/10.3390/biom9100619 - 17 Oct 2019
Viewed by 172
Abstract
In recent decades, regenerative medicine has merited substantial attention from scientific and research communities. One of the essential requirements for this new strategy in medicine is the production of biocompatible and biodegradable scaffolds with desirable geometric structures and mechanical properties. Despite such promise, [...] Read more.
In recent decades, regenerative medicine has merited substantial attention from scientific and research communities. One of the essential requirements for this new strategy in medicine is the production of biocompatible and biodegradable scaffolds with desirable geometric structures and mechanical properties. Despite such promise, it appears that regenerative medicine is the last field to embrace green, or environmentally-friendly, processes, as many traditional tissue engineering materials employ toxic solvents and polymers that are clearly not environmentally friendly. Scaffolds fabricated from plant proteins (for example, zein, soy protein, and wheat gluten), possess proper mechanical properties, remarkable biocompatibility and aqueous stability which make them appropriate green biomaterials for regenerative medicine applications. The use of plant-derived proteins in regenerative medicine has been especially inspired by green medicine, which is the use of environmentally friendly materials in medicine. In the current review paper, the literature is reviewed and summarized for the applicability of plant proteins as biopolymer materials for several green regenerative medicine and tissue engineering applications. Full article
(This article belongs to the Special Issue New Targets and Strategies in Regenerative Medicine)
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Open AccessArticle
Identification and Isolation of Active Compounds from Astragalus membranaceus that Improve Insulin Secretion by Regulating Pancreatic Β-Cell Metabolism
Biomolecules 2019, 9(10), 618; https://doi.org/10.3390/biom9100618 - 17 Oct 2019
Viewed by 145
Abstract
In type 2 diabetes (T2D), insufficient secretion of insulin from the pancreatic β-cells contributes to high blood glucose levels, associated with metabolic dysregulation. Interest in natural products to complement or replace existing antidiabetic medications has increased. In this study, we examined the effect [...] Read more.
In type 2 diabetes (T2D), insufficient secretion of insulin from the pancreatic β-cells contributes to high blood glucose levels, associated with metabolic dysregulation. Interest in natural products to complement or replace existing antidiabetic medications has increased. In this study, we examined the effect of Astragalus membranaceus extract (ASME) and its compounds 19 on glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells. ASME and compounds 19 isolated from A. membranaceus stimulated insulin secretion in INS-1 cells without inducing cytotoxicity. A further experiment showed that compounds 2, 3, and 5 enhanced the phosphorylation of total insulin receptor substrate-2 (IRS-2), phosphatidylinositol 3-kinase (PI3K), and Akt, and activated pancreatic and duodenal homeobox-1 (PDX-1) and peroxisome proliferator-activated receptor-γ (PPAR-γ), which are associated with β-cell function and insulin secretion. The data suggest that two isoflavonoids (2 and 3) and a nucleoside (compound 5), isolated from the roots of A. membranaceus, have the potential to improve insulin secretion in β-cells, representing the first step towards the development of potent antidiabetic drugs. Full article
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Open AccessArticle
An Efficient Agrobacterium Mediated Transformation of Pineapple with GFP-Tagged Protein Allows Easy, Non-Destructive Screening of Transgenic Pineapple Plants
Biomolecules 2019, 9(10), 617; https://doi.org/10.3390/biom9100617 - 17 Oct 2019
Viewed by 139
Abstract
Quite a few studies have been conducted to improve the Agrobacterium-mediated transformation of pineapple, which is the second most important commercial tropical fruit crop worldwide. However, pineapple transformation remains challenging, due to technical difficulties, the lengthy regeneration process, and a high labor [...] Read more.
Quite a few studies have been conducted to improve the Agrobacterium-mediated transformation of pineapple, which is the second most important commercial tropical fruit crop worldwide. However, pineapple transformation remains challenging, due to technical difficulties, the lengthy regeneration process, and a high labor requirement. There have not been any studies specifically addressing the introduction of GFP-tagged genes into pineapples through Agrobacterium-mediated transformation, which would enable easy, non-destructive expression detection. It would also allow expression localization at the organelle level, which is not possible with GUS a reporter gene that encodes β-glucuronidase or a herbicide resistance reporter gene. Here, we report a method for the introduction of GFP-tagged genes into pineapples through Agrobacterium-mediated transformation. We used embryonic calli for transformation, and plants were regenerated through somatic embryogenesis. In this study, we optimized the incubation time for Agrobacterium infection, the co-cultivation time, the hygromycin concentration for selection, and the callus growth conditions after selection. Our strategy reduced the time required to obtain transgenic plants from 7.6 months to 6.1 months. The expression of GFP-tagged AcWRKY28 was observed in the nuclei of transgenic pineapple root cells. This method allows easy, non-destructive expression detection of transgenic constructs at the organelle level. These findings on pineapple transformation will help accelerate pineapple molecular breeding efforts to introduce new desirable traits. Full article
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Open AccessArticle
Parvifloron D from Plectranthus strigosus: Cytotoxicity Screening of Plectranthus spp. Extracts
Biomolecules 2019, 9(10), 616; https://doi.org/10.3390/biom9100616 - 17 Oct 2019
Viewed by 147
Abstract
The Plectranthus genus is commonly used in traditional medicine due to its potential to treat several illnesses, including bacterial infections and cancer. As such, aiming to screen the antibacterial and cytotoxic activities of extracts, sixteen selected Plectranthus species with medicinal potential were studied. [...] Read more.
The Plectranthus genus is commonly used in traditional medicine due to its potential to treat several illnesses, including bacterial infections and cancer. As such, aiming to screen the antibacterial and cytotoxic activities of extracts, sixteen selected Plectranthus species with medicinal potential were studied. In total, 31 extracts obtained from 16 Plectranthus spp. were tested for their antibacterial and anticancer properties. Well diffusion method was used for preliminary antibacterial screening. The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of the five most active acetonic extracts (P. aliciae, P. japonicus, P. madagascariensis var. “Lynne”, P. stylesii, and P. strigosus) were determined. After preliminary toxicity evaluation on Artemia salina L., their cytotoxic properties were assessed on three human cancer cell lines (HCT116, MCF-7, and H460). These were also selected for mechanism of resistance studies (on NCI-H460/R and DLD1-TxR cells). An identified compound—parvifloron D—was tested in a pair of sensitive and MDR-Multidrug resistance cancer cells (NCI-H460 and NCI-H460/R) and in normal bronchial fibroblasts MRC-5. The chemical composition of the most active extract was studied through high performance liquid chromatography with a diode array detector (HPLC-DAD/UV) and liquid chromatography–mass spectrometry (LC–MS). Overall, P. strigosus acetonic extract showed the strongest antimicrobial and cytotoxic potential that could be explained by the presence of parvifloron D, a highly cytotoxic diterpene. This study provides valuable information on the use of the Plectranthus genus as a source of bioactive compounds, namely P. strigosus with the potential active ingredient the parvifloron D. Full article
(This article belongs to the Special Issue Antitumor Agents from Natural Sources)
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Open AccessReview
Fructans as Immunomodulatory and Antiviral Agents: The Case of Echinacea
Biomolecules 2019, 9(10), 615; https://doi.org/10.3390/biom9100615 - 16 Oct 2019
Viewed by 140
Abstract
Throughout history, medicinal purposes of plants have been studied, documented, and acknowledged as an integral part of human healthcare systems. The development of modern medicine still relies largely on this historical knowledge of the use and preparation of plants and their extracts. Further [...] Read more.
Throughout history, medicinal purposes of plants have been studied, documented, and acknowledged as an integral part of human healthcare systems. The development of modern medicine still relies largely on this historical knowledge of the use and preparation of plants and their extracts. Further research into the human microbiome highlights the interaction between immunomodulatory responses and plant-derived, prebiotic compounds. One such group of compounds includes the inulin-type fructans (ITFs), which may also act as signaling molecules and antioxidants. These multifunctional compounds occur in a small proportion of plants, many of which have recognized medicinal properties. Echinacea is a well-known medicinal plant and products derived from it are sold globally for its cold- and flu-preventative and general health-promoting properties. Despite the well-documented phytochemical profile of Echinacea plants and products, little research has looked into the possible role of ITFs in these products. This review aims to highlight the occurrence of ITFs in Echinacea derived formulations and the potential role they play in immunomodulation. Full article
Open AccessArticle
Effect of Marination Time on the Antioxidant Properties of Peptides Extracted from Organic Dry-Fermented Beef
Biomolecules 2019, 9(10), 614; https://doi.org/10.3390/biom9100614 - 16 Oct 2019
Viewed by 146
Abstract
In this study, we evaluated the effect of marination time on changes in the antioxidant properties of peptides extracted from bovine semimembranosus muscle. We measured antiradical scavenging capacity and reducing power of the peptides using a spectrophotometric decolorization method; inhibition of lipid oxidation [...] Read more.
In this study, we evaluated the effect of marination time on changes in the antioxidant properties of peptides extracted from bovine semimembranosus muscle. We measured antiradical scavenging capacity and reducing power of the peptides using a spectrophotometric decolorization method; inhibition of lipid oxidation was also assessed by estimating the level of malondialdehyde formed. According to our results, there was no benefit from the doubling of marinating time (from 24 to 48 h) as part of the preprocessing of beef. Samples from S1 batch (24 h marination) showed better antioxidant properties than those from S2 batch. We also tested various color parameters as a reflection of the inhibition of oxidative processes, in which case, the most favorable parameters from the consumer point of view were found to be lightness and redness. The effect of marination time on the degree of proteolytic changes was estimated using peptidomic approach. The degradation of myoglobin, hemoglobin, creatine kinase-type M, and beta-enolase—as the most sensitive proteins to proteolytic degradation—was observed during the 62 days of processing. It seems that the prolongation of marination time as a preprocessing step intensifies the hydrolytic degradation of proteins and peptides during the processing step. This results in the loss (or it has no effect) of antioxidative properties in organic dry-fermented beef. Full article
(This article belongs to the Special Issue Peptides: Molecular and Biotechnological Aspects)
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Open AccessArticle
Suppression of Sclerotinia sclerotiorum by the Induction of Systemic Resistance and Regulation of Antioxidant Pathways in Tomato Using Fengycin Produced by Bacillus amyloliquefaciens FZB42
Biomolecules 2019, 9(10), 613; https://doi.org/10.3390/biom9100613 - 16 Oct 2019
Viewed by 237
Abstract
Lipopeptides from Bacillus species exhibit promising biological control activity against plant pathogens. This study aimed to explore the potential of purified fengycin to induce systemic resistance in tomato against Sclerotinia sclerotiorum. Bacillus amyloliquefaciens FZB42, its mutant AK1S, and their corresponding metabolites showed [...] Read more.
Lipopeptides from Bacillus species exhibit promising biological control activity against plant pathogens. This study aimed to explore the potential of purified fengycin to induce systemic resistance in tomato against Sclerotinia sclerotiorum. Bacillus amyloliquefaciens FZB42, its mutant AK1S, and their corresponding metabolites showed in vitro inhibition of S. sclerotiorum mycelium. Fengycin derived from an AK1S mutant was purified and identified through HPLC and MALDI-TOF-MS, respectively. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed structural deformities in the fungal mycelium. Moreover, fengycin induced the accumulation of reactive oxygen species (ROS) in S. sclerotiorum mycelium and downregulated the expression of ROS-scavenging genes viz., superoxide dismutase (SsSOD1), peroxidase (SsPO), and catalase (SsCAT1) compared to the untreated control. Furthermore, the lesion size was dramatically reduced in fengycin-treated tomato plants compared to plants infected with S. sclerotiorum only in a greenhouse experiment. Additionally, the transcriptional regulation of defense-related genes GST, SOD, PAL, HMGR, and MPK3 showed the highest upsurge in expression at 48 h post-inoculation (hpi). However, their expression was subsequently decreased at 96 hpi in fengycin + S. sclerotiorum treatment compared to the plants treated with fengycin only. Conversely, the expression of PPO increased in a linear manner up to 96 hpi. Full article
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Open AccessArticle
Reliable Generation of Native-Like Decoys Limits Predictive Ability in Fragment-Based Protein Structure Prediction
Biomolecules 2019, 9(10), 612; https://doi.org/10.3390/biom9100612 - 15 Oct 2019
Viewed by 176
Abstract
Our previous work with fragment-assembly methods has demonstrated specific deficiencies in conformational sampling behaviour that, when addressed through improved sampling algorithms, can lead to more reliable prediction of tertiary protein structure when good fragments are available, and when score values can be relied [...] Read more.
Our previous work with fragment-assembly methods has demonstrated specific deficiencies in conformational sampling behaviour that, when addressed through improved sampling algorithms, can lead to more reliable prediction of tertiary protein structure when good fragments are available, and when score values can be relied upon to guide the search to the native basin. In this paper, we present preliminary investigations into two important questions arising from more difficult prediction problems. First, we investigated the extent to which native-like conformational states are generated during multiple runs of our search protocols. We determined that, in cases of difficult prediction, native-like decoys are rarely or never generated. Second, we developed a scheme for decoy retention that balances the objectives of retaining low-scoring structures and retaining conformationally diverse structures sampled during the course of the search. Our method succeeds at retaining more diverse sets of structures, and, for a few targets, more native-like solutions are retained as compared to our original, energy-based retention scheme. However, in general, we found that the rate at which native-like structural states are generated has a much stronger effect on eventual distributions of predictive accuracy in the decoy sets, as compared to the specific decoy retention strategy used. We found that our protocols show differences in their ability to access native-like states for some targets, and this may explain some of the differences in predictive performance seen between these methods. There appears to be an interaction between fragment sets and move operators, which influences the accessibility of native-like structures for given targets. Our results point to clear directions for further improvements in fragment-based methods, which are likely to enable higher accuracy predictions. Full article
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Open AccessArticle
Tuning Structure and Dynamics of Blue Copper Azurin Junctions via Single Amino-Acid Mutations
Biomolecules 2019, 9(10), 611; https://doi.org/10.3390/biom9100611 - 15 Oct 2019
Viewed by 199
Abstract
In the growing field of biomolecular electronics, blue-copper Azurin stands out as one of the most widely studied protein in single-molecule contacts. Interestingly, despite the paramount importance of the structure/dynamics of molecular contacts in their transport properties, these factors remain largely unexplored from [...] Read more.
In the growing field of biomolecular electronics, blue-copper Azurin stands out as one of the most widely studied protein in single-molecule contacts. Interestingly, despite the paramount importance of the structure/dynamics of molecular contacts in their transport properties, these factors remain largely unexplored from the theoretical point of view in the context of single Azurin junctions. Here we address this issue using all-atom Molecular Dynamics (MD) of Pseudomonas Aeruginosa Azurin adsorbed to a Au(111) substrate. In particular, we focus on the structure and dynamics of the free/adsorbed protein and how these properties are altered upon single-point mutations. The results revealed that wild-type Azurin adsorbs on Au(111) along two well defined configurations: one tethered via cysteine groups and the other via the hydrophobic pocket surrounding the Cu 2 + . Surprisingly, our simulations revealed that single amino-acid mutations gave rise to a quenching of protein vibrations ultimately resulting in its overall stiffening. Given the role of amino-acid vibrations and reorientation in the dehydration process at the protein-water-substrate interface, we suggest that this might have an effect on the adsorption process of the mutant, giving rise to new adsorption configurations. Full article
(This article belongs to the Special Issue Biomolecular Electronics)
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Open AccessReview
Emerging Roles of Lysyl Oxidases in the Cardiovascular System: New Concepts and Therapeutic Challenges
Biomolecules 2019, 9(10), 610; https://doi.org/10.3390/biom9100610 - 14 Oct 2019
Viewed by 213
Abstract
Lysyl oxidases (LOX and LOX-likes (LOXLs) isoenzymes) belong to a family of copper-dependent enzymes classically involved in the covalent cross-linking of collagen and elastin, a pivotal process that ensures extracellular matrix (ECM) stability and provides the tensile and elastic characteristics of connective tissues. [...] Read more.
Lysyl oxidases (LOX and LOX-likes (LOXLs) isoenzymes) belong to a family of copper-dependent enzymes classically involved in the covalent cross-linking of collagen and elastin, a pivotal process that ensures extracellular matrix (ECM) stability and provides the tensile and elastic characteristics of connective tissues. Besides this structural role, in the last years, novel biological properties have been attributed to these enzymes, which can critically influence cardiovascular function. LOX and LOXLs control cell proliferation, migration, adhesion, differentiation, oxidative stress, and transcriptional regulation and, thereby, their dysregulation has been linked to a myriad of cardiovascular pathologies. Lysyl oxidase could modulate virtually all stages of the atherosclerotic process, from endothelial dysfunction and plaque progression to calcification and rupture of advanced and complicated plaques, and contributes to vascular stiffness in hypertension. The alteration of LOX/LOXLs expression underlies the development of other vascular pathologies characterized by a destructive remodeling of the ECM, such as aneurysm and artery dissections, and contributes to the adverse myocardial remodeling and dysfunction in hypertension, myocardial infarction, and obesity. This review examines the most recent advances in the study of LOX and LOXLs biology and their pathophysiological role in cardiovascular diseases with special emphasis on their potential as therapeutic targets. Full article
(This article belongs to the Special Issue Lysyl Oxidases: Novel Roles in Disease and Therapeutic Opportunities)
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