International Journal of Molecular Sciences

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Open AccessReview

From End to End: tRNA Editing at 5'- and 3'-Terminal Positions

by Heike Betat, Yicheng Long, Jane E. Jackman and Mario Mörl

Int. J. Mol. Sci. 2014, 15(12), 23975-23998; https://doi.org/10.3390/ijms151223975 - 22 Dec 2014

Cited by 25 | Viewed by 12599

During maturation, tRNA molecules undergo a series of individual processing steps, ranging from exo- and endonucleolytic trimming reactions at their 5'- and 3'-ends, specific base modifications and intron removal to the addition of the conserved 3'-terminal CCA sequence. Especially in mitochondria, this plethora [...] Read more.

During maturation, tRNA molecules undergo a series of individual processing steps, ranging from exo- and endonucleolytic trimming reactions at their 5'- and 3'-ends, specific base modifications and intron removal to the addition of the conserved 3'-terminal CCA sequence. Especially in mitochondria, this plethora of processing steps is completed by various editing events, where base identities at internal positions are changed and/or nucleotides at 5'- and 3'-ends are replaced or incorporated. In this review, we will focus predominantly on the latter reactions, where a growing number of cases indicate that these editing events represent a rather frequent and widespread phenomenon. While the mechanistic basis for 5'- and 3'-end editing differs dramatically, both reactions represent an absolute requirement for generating a functional tRNA. Current in vivo and in vitro model systems support a scenario in which these highly specific maturation reactions might have evolved out of ancient promiscuous RNA polymerization or quality control systems. Full article

(This article belongs to the Special Issue Functions of Transfer RNAs)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Editing events in the tRNA acceptor stem. (A) Cloverleaf secondary structure and (B) tertiary structure of a tRNA; the anticodon is indicated in green, positions of acceptor stem editing are shown in blue (5'-editing) and red (3'-editing). While the 3'-editing events occur in the conventional 5'-3'-polymerization direction of nucleic acids, the 5'-editing shows a highly unusual 3'-5'-direction, indicating that highly specialized polymerases are involved in this reaction. In contrast, the 3'-editing reaction can in principle be catalyzed by an RNA polymerase with conventional directionality, like the poly(A) polymerase, the CCA-adding enzyme, or the TRAMP complex (TRAMP: Trf4 or Trf5/Air2/Mtr4 polyadenylation; Trf: topoisomerase I-related function, poly(A) polymerase activity; Air: arginine methyltransferase-interacting RING finger, RNA binding protein; Mtr4: mRNA transport). Full article ">Figure 2
Known and predicted 5'-end editing pathways. 5'-end editing of mt-tRNA is associated with the presence of mismatched nucleotides at any of the first three positions of the tRNA acceptor stem (indicated by the red X). The top pathway shows the two-step editing process that repairs these mismatches, first by removal of the 5'-mismatched residues (by unknown enzymes) and next by repair of the resulting 5'-truncated tRNA by 3'–5'-polymerases known as TLPs (Thg1-like proteins; Thg1: tRNAHis guanylyltransferase), thus generating a fully base paired acceptor stem (Watson–Crick base pairs indicated by the green bars). The bottom pathway shows an alternative process that could also utilize similar 3'–5'-polymerases to edit tRNA in the absence of bona fide mismatches, such as in organisms where these types of mismatched nucleotides have not been identified in tRNA genes. In these cases, several possible pathways, as indicated, could conceivably generate 5'-truncated substrates to be repaired by Thg1/TLP orthologs found in these species. Full article ">Figure 3
A specialized 5'-editing pathway that acts on tRNAHis. In Bacteria and Archaea, tRNAHis species are almost always encoded with an additional 5'-G nucleotide that is retained during 5'-end maturation by RNase P (indicated by the blue lightning bolt), ensuring the presence of the 5'-G-1 residue that is required to recognize the tRNAHis by its canonical Histidyl-tRNA synthetase (HisRS). However, in most eukaryotes, the pre-tRNAHis does not encode the additional G-1 and eukaryotic Thg1 enzymes catalyze a specialized 5'-end editing reaction to post-transcriptionally add G-1 to the tRNA and thus ensure proper recognition. Recently, some eukaryotes have been identified that lack a bona fide Thg1 ortholog, and in these cases, the lack of the 5'-edited tRNAHis is accommodated by the presence of a non-canonical HisRS that is capable of efficiently aminoacylating the tRNA even in the absence of G-1. The G-1 nucleotide is indicated in red, and the N+1 nucleotide that represents the normal 5'-end for all other tRNA species is highlighted in blue. Full article ">Figure 4
The TRAMP4 complex as an editing enzyme. Both TRAMP4 and TRAMP5 are important poly(A) polymerases involved in RNA quality control. Left: By the distributive addition of short poly(A) tails to a misfolded transcript (indicated by the red asterisks), these RNAs are tagged for subsequent degradation; Right: TRAMP4 also adds a restricted number of A residues to 3'-terminally truncated tRNA transcripts. If more A residues are added, leading to poly(A) tails, the tRNA is also labeled for subsequent degradation. However, as TRAMP4 is also a distributive polymerase that leaves the RNA substrate after the addition of a single A residue, the CCA-adding enzyme has a chance to bind to the restored tRNA, adding the CCA-terminus. In this case, TRAMP4 does not add a poly(A) tail, and the tRNA that now carries a CCA-end is functional and can participate in translation. Hence, the single A addition catalyzed by TRAMP4 corresponds to a true tRNA 3'-editing event. It is very likely that during evolution, similar RNA polymerases involved in RNA surveillance were recruited as editing enzymes. Full article ">

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Open AccessReview

Inhalation of Silver Nanomaterials—Seeing the Risks

by Ioannis G. Theodorou, Mary P. Ryan, Teresa D. Tetley and Alexandra E. Porter

Int. J. Mol. Sci. 2014, 15(12), 23936-23974; https://doi.org/10.3390/ijms151223936 - 22 Dec 2014

Cited by 44 | Viewed by 12044

Abstract

Demand for silver engineered nanomaterials (ENMs) is increasing rapidly in optoelectronic and in health and medical applications due to their antibacterial, thermal, electrical conductive, and other properties. The continued commercial up-scaling of ENM production and application needs to be accompanied by an understanding [...] Read more.

Demand for silver engineered nanomaterials (ENMs) is increasing rapidly in optoelectronic and in health and medical applications due to their antibacterial, thermal, electrical conductive, and other properties. The continued commercial up-scaling of ENM production and application needs to be accompanied by an understanding of the occupational health, public safety and environmental implications of these materials. There have been numerous in vitro studies and some in vivo studies of ENM toxicity but their results are frequently inconclusive. Some of the variability between studies has arisen due to a lack of consistency between experimental models, since small differences between test materials can markedly alter their behaviour. In addition, the propensity for the physicochemistry of silver ENMs to alter, sometimes quite radically, depending on the environment they encounter, can profoundly alter their bioreactivity. Consequently, it is important to accurately characterise the materials before use, at the point of exposure and at the nanomaterial-tissue, or “nanobio”, interface, to be able to appreciate their environmental impact. This paper reviews current literature on the pulmonary effects of silver nanomaterials. We focus our review on describing whether, and by which mechanisms, the chemistry and structure of these materials can be linked to their bioreactivity in the respiratory system. In particular, the mechanisms by which the physicochemical properties (e.g., aggregation state, morphology and chemistry) of silver nanomaterials change in various biological milieu (i.e., relevant proteins, lipids and other molecules, and biofluids, such as lung surfactant) and affect subsequent interactions with and within cells will be discussed, in the context not only of what is measured but also of what can be visualized. Full article

(This article belongs to the Special Issue Nanotoxicology and Lung Diseases)

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1999 KiB

Open AccessShort Note

Candida parapsilosis Biofilm Identification by Raman Spectroscopy

by Ota Samek, Katarina Mlynariková, Silvie Bernatová, Jan Ježek, Vladislav Krzyžánek, Martin Šiler, Pavel Zemánek, Filip Růžička, Veronika Holá and Martina Mahelová

Int. J. Mol. Sci. 2014, 15(12), 23924-23935; https://doi.org/10.3390/ijms151223924 - 22 Dec 2014

Cited by 42 | Viewed by 10936

Abstract

Colonies of Candida parapsilosis on culture plates were probed directly in situ using Raman spectroscopy for rapid identification of specific strains separated by a given time intervals (up to months apart). To classify the Raman spectra, data analysis was performed using the approach [...] Read more.

Colonies of Candida parapsilosis on culture plates were probed directly in situ using Raman spectroscopy for rapid identification of specific strains separated by a given time intervals (up to months apart). To classify the Raman spectra, data analysis was performed using the approach of principal component analysis (PCA). The analysis of the data sets generated during the scans of individual colonies reveals that despite the inhomogeneity of the biological samples unambiguous associations to individual strains (two biofilm-positive and two biofilm-negative) could be made. Full article

(This article belongs to the Special Issue Laser Application in Life Sciences)

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Figure 1
(A) Image of Candida parapsilosis colonies cultivated on agar for 48 h (the arrow shows selected colony for Raman analysis). The colony size is about 2 mm; (B) SEM (Scanning electron microscopy) image of Candida parapsilosis (cultivation for 48 h on a glass substrate), detailed image was obtained using combined preparation of the two techniques—chemical fixation and freeze drying (using ACE600 Leica microsystems). Full article ">Figure 2
Raman spectra of Candida parapsilosis. Full article ">Figure 3
(a) Raman spectra of Candida orthopsilosis; and (b) Candida metapsilosis. Full article ">Figure 4
Scores plot of the first two principal components relation for four Candida parapsilosis strains (BC 11, BC 16, BC 45 and BC 90) cultured for 48 h. Using the two principle components, one can clearly separate the clusters of spectra associated with the biofilm-positive (BC 11, BC 16) and biofilm-negative (BC 45, BC 90) strains. Data sets were recorded on 30 July 2013. Full article ">Figure 5
Scores plot of the first two principal components relation for four Candida parapsilosis strains (BC 11, BC 16, BC 45 and BC 90) cultured for 48 h. Using the two principle components, one can clearly separate the clusters of spectra associated with the biofilm-positive (BC 11, BC 16) and biofilm-negative (BC 45, BC 90) strains. Data sets were recorded on 19 December 2013. Full article ">Figure 6
3-D-scores plot of principle component relation (PC1, PC2, and PC4) for four Candida parapsilosis strains (BC 11, BC 16, BC 45 and BC 90) cultured for 48 h. Using the three principle components, one can clearly separate the clusters of spectra associated with the biofilm-positive (BC 11, BC 16) and biofilm-negative (BC 45, BC 90) strains. Green data sets (on the left) and red (on the right side) include all the data measured at three different days (30 July, 19 December 2013 and 13 February 2014). Inspecting this data show that the clustering of the data sets—for two-biofilm positive and two biofilm-negative strains can be separated by the two clear clusters, although the first and the last data sets were recorded about six months apart (July 2013 and February 2014). This demonstrates the solid reproducibility in the Raman fingerprints of these biofilm-positive and negative strains. Explained variances of PC1 (97%), PC2 (2%), and PC4 (0.5%). Full article ">Figure 7
Plot of loadings of (a) PC1; and (b) PC2 corresponding to <a href="#ijms-15-23924-f005" class="html-fig">Figure 5</a>. Different features corresponding to the lipids, proteins and DNA can be identified having the largest variability within the data (see <a href="#ijms-15-23924-t001" class="html-table">Table 1</a>). This illustrates the contribution of the wavenumbers to PC1 and PC2. Thus, the loadings clarify what spectral bands can distinguish biofilm positive and biofilm negative strains. Full article ">Figure 8
Excitation laser focused by 50× objective onto the central part of the C. parapsilosis colony. Note the steep decrease in colony height outside the flat center of growing colony visualized by unfocused colony surface. The vertical size of the laser spot is about 20 µm. Full article ">

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Open AccessArticle

PLGA Biodegradable Nanoparticles Containing Perphenazine or Chlorpromazine Hydrochloride: Effect of Formulation and Release

by Mohammed Halayqa and Urszula Domańska

Int. J. Mol. Sci. 2014, 15(12), 23909-23923; https://doi.org/10.3390/ijms151223909 - 22 Dec 2014

Cited by 66 | Viewed by 10073

Abstract

In our study, poly(dl-lactide-co-glycolide) (PLGA) nanoparticles loaded with perphenazine (PPH) and chlorpromazine hydrochloride (CPZ-HCl) were formulated by emulsion solvent evaporation technique. The effect of various processing variables, including PLGA concentration, theoretical drug loading, poly(vinyl alcohol) (PVA) concentration and the power of sonication were [...] Read more.

In our study, poly(dl-lactide-co-glycolide) (PLGA) nanoparticles loaded with perphenazine (PPH) and chlorpromazine hydrochloride (CPZ-HCl) were formulated by emulsion solvent evaporation technique. The effect of various processing variables, including PLGA concentration, theoretical drug loading, poly(vinyl alcohol) (PVA) concentration and the power of sonication were assessed systematically to obtain higher encapsulation efficiency and to minimize the nanoparticles size. By the optimization formulation process, the nanoparticles were obtained in submicron size from 325.5 ± 32.4 to 374.3 ± 10.1 nm for nanoparticles loaded with PPH and CPZ-HCl, respectively. Nanoparticles observed by scanning electron microscopy (SEM) presented smooth surface and spherical shape. The encapsulation efficiency of nanoparticles loaded with PPH and CPZ-HCl were 83.9% and 71.0%, respectively. The drug loading were 51.1% and 39.4% for PPH and CPZ-HCl, respectively. Lyophilized nanoparticles with different PLGA concentration 0.8%, 1.3% and 1.6% (w/v) in formulation process were evaluated for in vitro release in phosphate buffered saline (pH = 7.4) by using dialysis bags. The release profile for both drugs have shown that the rate of PPH and CPZ-HCl release were dependent on a size and amount of drugs in the nanoparticles. Full article

(This article belongs to the Special Issue Bioactive Nanoparticles 2014)

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SEM images of PPH-loaded PLGA prepared with PLGA concentration (a) 0.8% (w/v); (b) 1.3% (w/v); and (c) 1.6% (w/v) at TDL 20% (w/w). Full article ">Figure 1 Cont.
SEM images of PPH-loaded PLGA prepared with PLGA concentration (a) 0.8% (w/v); (b) 1.3% (w/v); and (c) 1.6% (w/v) at TDL 20% (w/w). Full article ">Figure 2
SEM images of CPZ-HCL-loaded PLGA prepared with PLGA concentration (a) 0.8% (w/v); (b) 1.3% (w/v); (c) 1.6% (w/v) at TDL 20% (w/w). Full article ">Figure 3
The in vitro PPH release profile from NPs at T = 310.15 K in phosphate buffered saline, pH 7.4 for the same amount of drug: Results for different concentration of PLGA (w/v): (▲) 0.8%, (♦) 1.3%, (■) 1.6%. Full article ">Figure 4
The in vitro CPZ-HCl release profile from NPs at T = 310.15 K in phosphate buffered saline, pH 7.4 for the same amount of drug: Results for different concentration of PLGA (w/v): (▲) 0.8%, (♦) 1.3%, (■) 1.6%. Full article ">

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Open AccessReview

The Promise of Novel Molecular Markers in Bladder Cancer

by Jahan Miremami and Natasha Kyprianou

Int. J. Mol. Sci. 2014, 15(12), 23897-23908; https://doi.org/10.3390/ijms151223897 - 22 Dec 2014

Cited by 33 | Viewed by 7208

Abstract

Bladder cancer is the fourth most common malignancy in the US and is associated with the highest cost per patient. A high likelihood of recurrence, mandating stringent surveillance protocols, has made the development of urinary markers a focus of intense pursuit with the [...] Read more.

Bladder cancer is the fourth most common malignancy in the US and is associated with the highest cost per patient. A high likelihood of recurrence, mandating stringent surveillance protocols, has made the development of urinary markers a focus of intense pursuit with the hope of decreasing the burden this disease places on patients and the healthcare system. To date, routine use of markers is not recommended for screening or diagnosis. Interests include the development of a single urinary marker that can be used in place of or as an adjunct to current screening and surveillance techniques, as well identifying a molecular signature for an individual’s disease that can help predict progression, prognosis, and potential therapeutic response. Markers have shown potential value in improving diagnostic accuracy when used as an adjunct to current modalities, risk-stratification of patients that could aid the clinician in determining aggressiveness of surveillance, and allowing for a decrease in invasive surveillance procedures. This review discusses the current understanding of emerging biomarkers, including miRNAs, gene signatures and detection of circulating tumor cells in the blood, and their potential clinical value in bladder cancer diagnosis, as prognostic indicators, and surveillance tools, as well as limitations to their incorporation into medical practice. Full article

(This article belongs to the Special Issue Emerging Classes of Biomarkers for Molecular Diagnostics)

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Open AccessReview

Emerging Biomarkers in Heart Failure and Cardiac Cachexia

by Goran Loncar, Daniel Omersa, Natasa Cvetinovic, Aleksandra Arandjelovic and Mitja Lainscak

Int. J. Mol. Sci. 2014, 15(12), 23878-23896; https://doi.org/10.3390/ijms151223878 - 22 Dec 2014

Cited by 34 | Viewed by 9041

Abstract

Biomarkers are objective tools with an important role for diagnosis, prognosis and therapy optimization in patients with heart failure (HF). To date, natriuretic peptides are closest to optimal biomarker standards for clinical implications in HF. Therefore, the efforts to identify and test new [...] Read more.

Biomarkers are objective tools with an important role for diagnosis, prognosis and therapy optimization in patients with heart failure (HF). To date, natriuretic peptides are closest to optimal biomarker standards for clinical implications in HF. Therefore, the efforts to identify and test new biomarkers in HF are reasonable and justified. Along the natural history of HF, cardiac cachexia may develop, and once at this stage, patient performance and prognosis is particularly poor. For these reasons, numerous biomarkers reflecting hormonal, inflammatory and oxidative stress pathways have been investigated, but only a few convey relevant information. The complex pathophysiology of HF appears far too complex to be embraced by a single biomarker; thus, a combined approach appears reasonable. With these considerations, we have reviewed the recent developments in the field to highlight key candidates with diagnostic, prognostic and therapy optimization properties, either alone or in combination. Full article

(This article belongs to the Special Issue Emerging Classes of Biomarkers for Molecular Diagnostics)

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Open AccessReview

Characterization and Study of Transgenic Cultivars by Capillary and Microchip Electrophoresis

by Elena Domínguez Vega and Maria Luisa Marina

Int. J. Mol. Sci. 2014, 15(12), 23851-23877; https://doi.org/10.3390/ijms151223851 - 22 Dec 2014

Cited by 16 | Viewed by 8332

Abstract

Advances in biotechnology have increased the demand for suitable analytical techniques for the analysis of genetically modified organisms. Study of the substantial equivalence, discrimination between transgenic and non-transgenic cultivars, study of the unintended effects caused by a genetic modification or their response to [...] Read more.

Advances in biotechnology have increased the demand for suitable analytical techniques for the analysis of genetically modified organisms. Study of the substantial equivalence, discrimination between transgenic and non-transgenic cultivars, study of the unintended effects caused by a genetic modification or their response to diverse situations or stress conditions (e.g., environmental, climatic, infections) are some of the concerns that need to be addressed. Capillary electrophoresis (CE) is emerging as an alternative to conventional techniques for the study and characterization of genetically modified organisms. This article reviews the most recent applications of CE for the analysis and characterization of transgenic cultivars in the last five years. Different strategies have been described depending on the level analyzed (DNA, proteins or metabolites). Capillary gel electrophoresis (CGE) has shown to be particularly useful for the analysis of DNA fragments amplified by PCR. Metabolites and proteins have been mainly separated using capillary zone electrophoresis (CZE) using UV and MS detection. Electrophoretic chips have also proven their ability in the analysis of transgenic cultivars and a section describing the new applications is also included. Full article

(This article belongs to the Special Issue Detection and Safety Assessment of Genetically Modified Organisms)

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Figure 1
Electrophoregrams obtained by CE-electrochemoluminiscence (ECL) after multiplex PCR for roundup ready soybeans (RRS) certified reference material standards containing 0%, 0.1% and 1.0% of RRS. Reprinted with permission from [<a href="#B33-ijms-15-23851" class="html-bibr">33</a>] Full article ">Figure 2
(A) Schematic diagram of the multiplex Microdroplet PCR Implemented Capillary gel electrophoresis (CGE) (MPIC) assay. (1) Target-specific DNA products with universal tails on each side were generated in multiplex PCR preamplification, using bipartite primers; (2) The products of the multiplex preamplification were purified and used as templates in the second step, universal microdroplet PCR amplification, using universal primer pairs; (3) The microdroplet PCR was performed, employing the universal primer pair Uni-F/R in the emulsion droplets; (4) The w/o emulsion was centrifuged to gather all microdroplets, and DNA fragments were purified for subsequent CGE-based detection; (5) Detectable amplicons were analyzed by CGE; (B) Capillary gel electrophoresis analysis of the 24-plex MPIC assay for detection of 14 different GM events using 24 bipartite primer pairs. Amplicons were produced from a GM DNA solution mixture containing 1.6% of each of the 14 different GM events. The peaks of the amplicon are indicated by blue arrows. The profile shown under the electrophoregram is the simulated gel image corresponding to the results of the capillary gel electrophoresis analysis. Reprinted with permission from [<a href="#B36-ijms-15-23851" class="html-bibr">36</a>]. Full article ">Figure 3
CE-TOF base peak electropherogram of the digested protein extract from conventional and transgenic soybean. Reprinted with permission from [<a href="#B42-ijms-15-23851" class="html-bibr">42</a>]. Full article ">Figure 4
Changes in the level of representative primary metabolites after aphid introduction. Data for the metabolite per mg of soybean leaves (wet weight) at 0–96 h after aphid introduction are plotted. Solid and dashed lines indicate the metabolite concentrations in Tohoku 149 (n = 5) and Suzuyutaka (n = 5), respectively. The data points are mean values and the error bars indicate the standard error. Asterisk (**) denotes statistical significance with p < 0.01. Reprinted with permission from [<a href="#B44-ijms-15-23851" class="html-bibr">44</a>]. Full article ">Figure 5
Representative electropherograms obtained by multi-channel microchip electrophoresis of rice knockout DNA sample sunder (A) constant field strength (CFS) and (B) programmed field strength gradient (PFSG). Reprinted with permission from [<a href="#B57-ijms-15-23851" class="html-bibr">57</a>]. Full article ">

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Open AccessArticle

Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

by Simon Lindhoud, Adrie H. Westphal, Carlo P. M. Van Mierlo, Antonie J. W. G. Visser and Jan Willem Borst

Int. J. Mol. Sci. 2014, 15(12), 23836-23850; https://doi.org/10.3390/ijms151223836 - 19 Dec 2014

Cited by 25 | Viewed by 7371

Abstract

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive [...] Read more.

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway. Full article

(This article belongs to the Special Issue Förster Resonance Energy Transfer (FRET) 2015)

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Fluorescence lifetimes with corresponding negative and positive amplitudes characterize the time-resolved fluorescence of the acceptor upon donor excitation. (Top) The time dependence of experimental (grey) and fitted (black) fluorescence intensity of apoflavodoxin labeled with A488 (donor) and A568 (acceptor) (see the Experimental Section for details on the theoretical background, protein labeling and data acquisition and analysis). The decay of reference compound erythrosine B has a fluorescence lifetime of 89 ps (red line); (Bottom) The weighted residuals between experimental and fitted curves (grey lines). (a) Acceptor fluorescence of folded, double-labeled apoflavodoxin in 0 M GuHCl. The rise time is 0.51 ns (confidence limits at the 0.67 confidence level are 0.50 and 0.53 ns); decay time is 3.85 ns (confidence limits at the 0.67 confidence level are 3.84 and 3.86 ns). The absolute value of the amplitude ratio |A−/A+| is 0.44. The fit quality criterion χ2 is 1.226; and (b) Acceptor fluorescence of unfolded, double-labeled apoflavodoxin in 4.12 M GuHCl. The rise time is 1.57 ns (confidence limits at the 0.67 confidence level are 1.41 and 1.78 ns); decay time is 3.71 ns (confidence limits at the 0.67 confidence level are 3.67 and 3.73 ns). The absolute value of the amplitude ratio |A−/A+| is 0.134. The fit quality criterion χ2 is 1.085. Full article ">Figure 2
Denaturant-dependencies of rise and decay times of acceptor fluorescence upon donor excitation track folding of apoflavodoxin labeled with A488 (donor) and A568 (acceptor). In all panels, black dots represent fitted values and grey triangles represent confidence limits. (a) The rise time of acceptor fluorescence and (b) decay time of acceptor fluorescence reveal the biphasic dependencies on GuHCl; (c) The amplitude of the acceptor fluorescence rise time (A−) changes in a monophasic manner as a function of GuHCl; (d) The amplitude of acceptor fluorescence decay (A+) is virtually constant as a function of denaturant concentration; and (e) The absolute ratio of the amplitudes of fluorescence rise and decay time (|A−/A+|) shows a monophasic dependence on denaturant concentration. Full article ">Figure 3
The rate constants of energy transfer change as a function of denaturant concentration. Rate constants are obtained through the relation ki = 1/τi. kda is the decay rate of the donor fluorescence in presence of an acceptor, as obtained from the acceptor rise time (open circles), kd is the decay rate of the donor in the absence of an acceptor (open squares) and is obtained from [<a href="#B8-ijms-15-23836" class="html-bibr">8</a>], as described in <a href="#app1-ijms-15-23836" class="html-app">Supplementary Information SI2</a>, and kt is the rate of energy transfer, as obtained from the relation kt = kda − kd (Equation (5); black dots). Full article ">Figure 4
The distance between donor and acceptor fluorophores changes as a function of denaturant concentration and reveals conformational changes during apoflavodoxin (un)folding. The Förster transfer distance (R0; open squares) changes as a function of denaturant concentration in a folding state-dependent manner. Calculated distances (rda; black dots) between donor Alexa Fluor 488 and acceptor Alexa Fluor 568 track the features of apoflavodoxin (un)folding. Values of R0 are obtained by using parameters as described in <a href="#app1-ijms-15-23836" class="html-app">Supplementary Information SI3</a>. Values of rda are obtained by using Equation (7). Full article ">

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Open AccessReview

The Role of Reactive Oxygen Species in Microvascular Remodeling

by Marius C. Staiculescu, Christopher Foote, Gerald A. Meininger and Luis A. Martinez-Lemus

Int. J. Mol. Sci. 2014, 15(12), 23792-23835; https://doi.org/10.3390/ijms151223792 - 19 Dec 2014

Cited by 49 | Viewed by 8730

Abstract

The microcirculation is a portion of the vascular circulatory system that consists of resistance arteries, arterioles, capillaries and venules. It is the place where gases and nutrients are exchanged between blood and tissues. In addition the microcirculation is the major contributor to blood [...] Read more.

The microcirculation is a portion of the vascular circulatory system that consists of resistance arteries, arterioles, capillaries and venules. It is the place where gases and nutrients are exchanged between blood and tissues. In addition the microcirculation is the major contributor to blood flow resistance and consequently to regulation of blood pressure. Therefore, structural remodeling of this section of the vascular tree has profound implications on cardiovascular pathophysiology. This review is focused on the role that reactive oxygen species (ROS) play on changing the structural characteristics of vessels within the microcirculation. Particular attention is given to the resistance arteries and the functional pathways that are affected by ROS in these vessels and subsequently induce vascular remodeling. The primary sources of ROS in the microcirculation are identified and the effects of ROS on other microcirculatory remodeling phenomena such as rarefaction and collateralization are briefly reviewed. Full article

(This article belongs to the Special Issue Oxidative Stress in Cardiovascular Disease 2015)

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Open AccessArticle

When Isolated at Full Receptivity, in Vitro Fertilized Wheat (Triticum aestivum, L.) Egg Cells Reveal [Ca²⁺]_cyt Oscillation of Intracellular Origin

by Zsolt Pónya, Ilaria Corsi, Richárd Hoffmann, Melinda Kovács, Anikó Dobosy, Attila Zoltán Kovács, Mauro Cresti and Beáta Barnabás

Int. J. Mol. Sci. 2014, 15(12), 23766-23791; https://doi.org/10.3390/ijms151223766 - 19 Dec 2014

Cited by 4 | Viewed by 6623

Abstract

During in vitro fertilization of wheat (Triticum aestivum, L.) in egg cells isolated at various developmental stages, changes in cytosolic free calcium ([Ca²⁺]_cyt) were observed. The dynamics of [Ca²⁺]_cyt elevation varied, reflecting the difference [...] Read more.

During in vitro fertilization of wheat (Triticum aestivum, L.) in egg cells isolated at various developmental stages, changes in cytosolic free calcium ([Ca²⁺]_cyt) were observed. The dynamics of [Ca²⁺]_cyt elevation varied, reflecting the difference in the developmental stage of the eggs used. [Ca²⁺]_cyt oscillation was exclusively observed in fertile, mature egg cells fused with the sperm cell. To determine how [Ca²⁺]_cyt oscillation in mature egg cells is generated, egg cells were incubated in thapsigargin, which proved to be a specific inhibitor of the endoplasmic reticulum (ER) Ca²⁺-ATPase in wheat egg cells. In unfertilized egg cells, the addition of thapsigargin caused an abrupt transient increase in [Ca²⁺]_cyt in the absence of extracellular Ca²⁺, suggesting that an influx pathway for Ca²⁺ is activated by thapsigargin. The [Ca²⁺]_cyt oscillation seemed to require the filling of an intracellular calcium store for the onset of which, calcium influx through the plasma membrane appeared essential. This was demonstrated by omitting extracellular calcium from (or adding GdCl₃ to) the fusion medium, which prevented [Ca²⁺]_cyt oscillation in mature egg cells fused with the sperm. Combined, these data permit the hypothesis that the first sperm-induced transient increase in [Ca²⁺]_cyt depletes an intracellular Ca²⁺ store, triggering an increase in plasma membrane Ca²⁺ permeability, and this enhanced Ca²⁺ influx results in [Ca²⁺]_cyt oscillation. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms of Sperm-Egg Interaction)

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1214 KiB

Open AccessArticle

Inhibitory Effects of Benzaldehyde Derivatives from the Marine Fungus Eurotium sp. SF-5989 on Inflammatory Mediators via the Induction of Heme Oxygenase-1 in Lipopolysaccharide-Stimulated RAW264.7 Macrophages

by Kyoung-Su Kim, Xiang Cui, Dong-Sung Lee, Wonmin Ko, Jae Hak Sohn, Joung Han Yim, Ren-Bo An, Youn-Chul Kim and Hyuncheol Oh

Int. J. Mol. Sci. 2014, 15(12), 23749-23765; https://doi.org/10.3390/ijms151223749 - 19 Dec 2014

Cited by 31 | Viewed by 7420

Abstract

Two benzaldehyde derivatives, flavoglaucin (1) and isotetrahydro-auroglaucin (2), were isolated from the marine fungus Eurotium sp. SF-5989 through bioassay- and ¹H NMR-guided investigation. In this study, we evaluated the anti-inflammatory effects of these compounds in lipopolysaccharide (LPS)-stimulated RAW264.7 [...] Read more.

Two benzaldehyde derivatives, flavoglaucin (1) and isotetrahydro-auroglaucin (2), were isolated from the marine fungus Eurotium sp. SF-5989 through bioassay- and ¹H NMR-guided investigation. In this study, we evaluated the anti-inflammatory effects of these compounds in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. We demonstrated that compounds 1 and 2 markedly inhibited LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE₂) production by suppressing inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression without affecting cell viability. We also demonstrated that the compounds reduced the secretion of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Furthermore, compounds 1 and 2 inhibited LPS-induced nuclear factor-κB (NF-κB) activation by suppressing phosphorylation of IkappaB (IκB). These results indicated that the anti-inflammatory effects of these benzaldehyde derivatives in LPS-stimulated RAW264.7 macrophages were due to the inactivation of the NF-κB pathway. In addition, compounds 1 and 2 induced heme oxygenase-1 (HO-1) expression through the nuclear transcription factor-E2–related factor 2 (Nrf2) translocation. The inhibitory effects of compounds 1 and 2 on the production of pro-inflammatory mediators and on NF-κB binding activity were reversed by HO-1 inhibitor tin protoporphyrin (SnPP). Thus, the anti-inflammatory effects of compounds 1 and 2 also correlated with their ability of inducing HO-1 expression. Full article

(This article belongs to the Section Biochemistry)

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Open AccessReview

Regulation of Angiogenesis by Aminoacyl-tRNA Synthetases

by Adam C. Mirando, Christopher S. Francklyn and Karen M. Lounsbury

Int. J. Mol. Sci. 2014, 15(12), 23725-23748; https://doi.org/10.3390/ijms151223725 - 19 Dec 2014

Cited by 29 | Viewed by 9160

Abstract

In addition to their canonical roles in translation the aminoacyl-tRNA synthetases (ARSs) have developed secondary functions over the course of evolution. Many of these activities are associated with cellular survival and nutritional stress responses essential for homeostatic processes in higher eukaryotes. In particular, [...] Read more.

In addition to their canonical roles in translation the aminoacyl-tRNA synthetases (ARSs) have developed secondary functions over the course of evolution. Many of these activities are associated with cellular survival and nutritional stress responses essential for homeostatic processes in higher eukaryotes. In particular, six ARSs and one associated factor have documented functions in angiogenesis. However, despite their connection to this process, the ARSs are mechanistically distinct and exhibit a range of positive or negative effects on aspects of endothelial cell migration, proliferation, and survival. This variability is achieved through the appearance of appended domains and interplay with inflammatory pathways not found in prokaryotic systems. Complete knowledge of the non-canonical functions of ARSs is necessary to understand the mechanisms underlying the physiological regulation of angiogenesis. Full article

(This article belongs to the Special Issue Functions of Transfer RNAs)

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Figure 1
Mechanisms of angiogenesis by extracellularly acting ARS. Left-Full-length tyrosyl-tRNA synthetase (YARS) is secreted from the cell by an unknown mechanism. Outside the cell it is cleaved by polymorphonuclear leukocyte (PMN)-elastase or other protease molecules into N- and C-terminal fragments which stimulate angiogenesis or immune responses respectively; Center-Full-length tryptophanyl-tRNA synthetase (WARS) or mini-WARS are generated from alternative splicing of WARS mRNA and are subsequently secreted into the extracellular space by unknown mechanisms. Full-length WARS is further processed by PMN-elastase or other proteases to form fragments like mini-WARS. The WARS fragments disrupt endothelial cell–cell contacts and angiogenic signaling molecules, eliciting an angiostatic effect; Right-Full-length threonyl-tRNA synthetase (TARS) is secreted from the cell by unknown processes and stimulates vessel migration. The mechanisms of these effects have yet to be determined. Full article ">Figure 2
Mechanisms of angiogenesis by intracellularly acting ARS. (1) seryl-tRNA synthetase (SARS) is transported into the nucleus via a canonical nuclear localization sequence (NLS) sequence and binds to the vascular endothelial growth factor A gene (vegfaa) promoter. The binding of SARS inhibits c-Myc-mediated expression of vascular endothelial growth factor A (VEGFA) through modification of vegfaa epigenetics. This effect is angiostatic; (2) glutamyl-prolyl-tRNA synthetase (EPRS) is phosphorylated upon treatment of the cell with IFNγ, releasing it from the multisynthetase complex (MSC). Phospho-EPRS binds with L13a, GAPDH, and NSAP1 to form the interferon-γ-activated inhibitor of translation (GAIT) complex. This complex then binds to mRNA containing stem-loop, 3' UTR GAIT elements via EPRS WHEP domains, thereby blocking their translation. As VEGFA contains a GAIT element, this effect is angiostatic. Full article ">Figure 3
The biphasic responses of aminoacyl-tRNA synthetase interacting multifunctional protein 1 (AIMP1) and endothelial monocyte activating polypeptide II (EMAP II) on angiogenesis. (1) Caspase 7 cleaves the C-terminal EMAP II peptide from AIMP1, releasing EMAP II from the multi-synthetase complex; (2) Alternatively, AIMP1 is released by unknown processes; (3) Both EMAP II and AIMP1 are secreted from the cell by mechanisms that remain to be determined; (4) At low concentrations (perhaps corresponding to early portions of the AIMP1/EMAP II signaling responses in vivo) EMAPII and AIMP1 bind to surface receptors, activating various kinase cascades; (5) These kinase cascades induce the expression of tumor necrosis factor α (TNFα); (6) TNFα is secreted from the cell and mediates angiogenesis through canonical TNFα pathways; (7) At high concentrations (perhaps corresponding to prolonged exposure in vivo), AIMP1 and EMAP II induce the expression of the TNFα-receptor (TNFR); and (8) Over-stimulation of TNFR by TNFα induces apoptosis through signaling by the TNFR death-domain, resulting in an anti-angiogenic response. Full article ">

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Open AccessArticle

Lunasin Sensitivity in Non-Small Cell Lung Cancer Cells Is Linked to Suppression of Integrin Signaling and Changes in Histone Acetylation

by Junichi Inaba, Elizabeth J. McConnell and Keith R. Davis

Int. J. Mol. Sci. 2014, 15(12), 23705-23724; https://doi.org/10.3390/ijms151223705 - 18 Dec 2014

Cited by 20 | Viewed by 12107

Abstract

Lunasin is a plant derived bioactive peptide with both cancer chemopreventive and therapeutic activity. We recently showed lunasin inhibits non-small cell lung cancer (NSCLC) cell proliferation in a cell-line-specific manner. We now compared the effects of lunasin treatment of lunasin-sensitive (H661) and lunasin-insensitive [...] Read more.

Lunasin is a plant derived bioactive peptide with both cancer chemopreventive and therapeutic activity. We recently showed lunasin inhibits non-small cell lung cancer (NSCLC) cell proliferation in a cell-line-specific manner. We now compared the effects of lunasin treatment of lunasin-sensitive (H661) and lunasin-insensitive (H1299) NSCLC cells with respect to lunasin uptake, histone acetylation and integrin signaling. Both cell lines exhibited changes in histone acetylation, with H661 cells showing a unique increase in H4K16 acetylation. Proximity ligation assays demonstrated lunasin interacted with integrins containing αv, α5, β1 and β3 subunits to a larger extent in the H661 compared to H1299 cells. Moreover, lunasin specifically disrupted the interaction of β1 and β3 subunits with the downstream signaling components phosphorylated Focal Adhesion Kinase (pFAK), Kindlin and Intergrin Linked Kinase in H661 cells. Immunoblot analyses demonstrated lunasin treatment of H661 resulted in reduced levels of pFAK, phosphorylated Akt and phosphorylated ERK1/2 whereas no changes were observed in H1299 cells. Silencing of αv expression in H661 cells confirmed signaling through integrins containing αv is essential for proliferation. Moreover, lunasin was unable to further inhibit proliferation in αv-silenced H661 cells. This indicates antagonism of integrin signaling via αv-containing integrins is an important component of lunasin’s mechanism of action. Full article

(This article belongs to the Special Issue Bioactive Proteins and Peptides Derived from Food)

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Internalization of lunasin into non-small cell lung cancer (NSCLC) cells. Cells were treated for 24 h with either vehicle (Control) or 100 µM lunasin prior to processing for immunocytochemistry. Full article ">Figure 2
(A) Interaction of lunasin with core histones H3 and H4 in NSCLC cells. Cells were treated with 100 µM lunasin for 24 h before performing proximity ligation assays (PLA) assays using antibodies specific for lunasin, H3 and H4; (B) Quantitation of PLA fluorescence in NSCLC cells. Fluorescence is expressed as relative fluorescence per cell. Data shown are the mean ± SD obtained in three independent experiments where 40 cells per treatment were imaged in each experiment; asterisks indicate a statistically significant difference (p < 0.05) between treatments. Full article ">Figure 3
(A) Immunoblot analysis of acetylated histones in lunasin-treated and untreated NSCLC cells; (B) Relative histone acetylation in lunasin-treated and untreated NSCLC cells. Cells were treated with 100 μM lunasin or vehicle for 48 h. Total histones were isolated and subjected to immunoblot analyses using antibodies specific for the indicated histone acetylation marks. Relative histone acetylation was determined by image analyses of immunoblots using Image J software. Data shown are the mean ± SD of immunoblots obtained in three independent experiments; asterisks indicate a statistically significant difference (p < 0.05) between treatments. Full article ">Figure 4
(A) Analysis of integrin subunit expression in NSCLC cell lines H661 and H1299. Protein extracts were subjected to immunoblot analysis using antibodies specific for the indicated integrin subunits; (B) Detection of lunasin interactions with specific integrin subunits in situ. Left-hand panels show representative PLA analyses of lunasin and the indicated integrin subunit; Right-hand panels show the quantitation of fluorescence for each interaction. Fluorescence is expressed as relative fluorescence per cell. Data shown are the mean ± SD obtained in three independent experiments; asterisks indicate statistically significant difference (p < 0.05) from the vehicle-treated control. Full article ">Figure 5
Detection of integrin β subunit interactions pFAK, Kindlin and ILK. Left-hand panels show representative PLA analyses of lunasin and the indicated integrin subunit. Right-hand panels show the quantitation of fluorescence for each interaction. Fluorescence is expressed as relative fluorescence per cell. Data shown are the mean ± SD obtained in three independent experiments; asterisks indicate statistically significant difference (p < 0.05) from the vehicle-treated control. Full article ">Figure 6
(A) Immunoblot analyses of integrin signaling pathway components in lunasin-treated NSCLC cells. Analyses were repeated in three independent experiments and representative data from one experiment is shown; (B) Integrin signaling pathway showing effects of lunasin treatment in H661 cells. RTKs, Receptor Tyrosine Kinases; SFKs, Src Family Kinases. Proteins shown in red indicate signaling steps negatively affected specifically in H661 cells by lunasin treatment; (C) Relative expression levels of integrin signaling proteins. Immunoblots shown in (A) were analyzed using ImageJ software v1.45 (National Institutes of Health, Bethesda, MD, USA). Data represent the mean ± SD for three independent experiments. The asterisk (*) indicates a significant (p < 0.05) difference in expression levels relative to the vehicle control. Full article ">Figure 7
Effects of silencing the αv integrin subunit in NSCLC H661and H1299 cells. (A) Immunoblot analyses of αv subunit levels in H661 and H1299 cells treated with either vehicle or lunasin. Cells were treated for 24 h with 100 µM lunasin or 50 mM NaPO4, pH 7.4; (B) Immunoblot analysis of αv expression in H661 cells transfected with a control siRNA or three different siRNAs designed to silence αv expression individually or in combination. β-actin was used as a loading control; (C) Proliferation of control siRNA transfected H661 cells and H661 cells transfected with a combination of three αv-specific siRNAs; (D) Immunoblot analysis of αv expression in H1299 cells transfected with a combination of three αv-specific siRNAs; (E) Proliferation of control siRNA transfected H1299 cells and H1299 cells transfected with a combination of three αv-specific siRNAs. Proliferation assays were initiated 48 h after transfection with siRNAs; cells were treated with either 50 mM NaPO4, pH 7.4 (vehicle) or 100 µM lunasin. Data shown are the mean ± SD obtained in three independent experiments. Full article ">

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Open AccessReview

P2X and P2Y Receptors—Role in the Pathophysiology of the Nervous System

by Kamila Puchałowicz, Maciej Tarnowski, Irena Baranowska-Bosiacka, Dariusz Chlubek and Violetta Dziedziejko

Int. J. Mol. Sci. 2014, 15(12), 23672-23704; https://doi.org/10.3390/ijms151223672 - 18 Dec 2014

Cited by 79 | Viewed by 10429

Abstract

Purinergic signalling plays a crucial role in proper functioning of the nervous system. Mechanisms depending on extracellular nucleotides and their P2 receptors also underlie a number of nervous system dysfunctions. This review aims to present the role of purinergic signalling, with particular focus [...] Read more.

Purinergic signalling plays a crucial role in proper functioning of the nervous system. Mechanisms depending on extracellular nucleotides and their P2 receptors also underlie a number of nervous system dysfunctions. This review aims to present the role of purinergic signalling, with particular focus devoted to role of P2 family receptors, in epilepsy, depression, neuropathic pain, nervous system neoplasms, such as glioma and neuroblastoma, neurodegenerative diseases like Parkinson’s disease, Alzheimer’s disease and multiple sclerosis. The above-mentioned conditions are associated with changes in expression of extracellular ectonucleotidases, P2X and P2Y receptors in neurons and glial cells, as well as releasing considerable amounts of nucleotides from activated or damaged nervous tissue cells into the extracellular space, which contributes to disturbance in purinergic signalling. The numerous studies indicate a potential possibility of using synthetic agonists/antagonists of P2 receptors in treatment of selected nervous system diseases. This is of particular significance, since numerous available agents reveal a low effectiveness and often produce side effects. Full article

(This article belongs to the Special Issue Molecular Research in Neurotoxicology)

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Open AccessArticle

Refolded scFv Antibody Fragment against Myoglobin Shows Rapid Reaction Kinetics

by Hyung-Nam Song, Jun-Hyuck Jang, Young-Wan Kim, Dong-Hyung Kim, Sung-Goo Park, Myung Kyu Lee, Se-Hwan Paek and Eui-Jeon Woo

Int. J. Mol. Sci. 2014, 15(12), 23658-23671; https://doi.org/10.3390/ijms151223658 - 18 Dec 2014

Cited by 12 | Viewed by 10644

Abstract

Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage [...] Read more.

Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage of in vivo immunization. We produced a recombinant scFv fragment for the premature antibody from Escherichia coli using refolding technology. The scFv gene was constructed by connection of the V_H–V_L sequence with a (Gly₄Ser)₃ linker. The scFv fragment without the pelB leader sequence was expressed at a high level, but the solubility was extremely low. A high concentration of 8 M urea was used for denaturation. The dilution refolding process in the presence of arginine and the redox reagents GSH and GSSH successfully produced a soluble scFv protein. The resultant refolded scFv protein showed association and dissociation values of 9.32 × 10⁻⁴ M⁻¹·s⁻¹ and 6.29 × 10⁻³ s⁻¹, respectively, with an affinity value exceeding 10⁷ M⁻¹ (k_on/k_off), maintaining the original rapid reaction kinetics of the premature antibody. The refolded scFv could provide a platform for protein engineering for the clinical application for diagnosis of heart disease and the development of a continuous biosensor. Full article

(This article belongs to the Special Issue Protein Folding)

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Graphical abstract

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Construction of the premature scFv for protein expression in E. coli. The sequence of the premature scFv with a hexa-histidine tag and a TEV sequence in the N-terminus was shown with a (Gly4Ser)3 linker between the variable heavy (VH) and variable light (VL) regions. (A) The insert of the scFv genes was amplified using PCR; (B) The NheI (5') and XhoI (3') restriction enzymes were used for the subcloning; and (C) Schematic diagram of the vector plasmid for E. coli expression. Full article ">Figure 2
The expression, isolation and solubilization of the scFv protein from E. coli. (A) The recombinant scFv showed high expression after IPTG induction (AI), corresponding to 30 kDa in SDS-PAGE (top) and Western blot analysis (bottom) compared with pre-induction (BI); (B) SDS-PAGE analysis of the inclusion bodies (IB). The pellet and the solubilized scFv using 8 M urea were separated by centrifugation; and (C) Schematic diagram of the scFv refolding steps. Full article ">Figure 3
Purification and refolding of the scFv antibody fragment. (A) Solubilized scFv was applied to a Ni-column and eluted by a gradient of 250 mM imidazole (lane 1, before column; lane 2, unbound; lane 3, wash); (B) The scFv fragment was refolded in the presence of 440 mM l-arginine and the redox reagents, 2 mM GSH and 0.2 mM GSSH (lanes 1 and 4, before refolding sample; lanes 2 and 5, the supernatant after refolding; lanes 3 and 6, the pellet after refolding); and (C) The final refolded scFv antibody fragment. Full article ">Figure 3 Cont.
Purification and refolding of the scFv antibody fragment. (A) Solubilized scFv was applied to a Ni-column and eluted by a gradient of 250 mM imidazole (lane 1, before column; lane 2, unbound; lane 3, wash); (B) The scFv fragment was refolded in the presence of 440 mM l-arginine and the redox reagents, 2 mM GSH and 0.2 mM GSSH (lanes 1 and 4, before refolding sample; lanes 2 and 5, the supernatant after refolding; lanes 3 and 6, the pellet after refolding); and (C) The final refolded scFv antibody fragment. Full article ">Figure 4
Molecular mass of scFv using a gel filtration column. Molecular weights for standard proteins are as follows: Conalbumin, 75 kDa, 9.55 mL; ovalbumin, 43 kDa, 10.31 mL; Carbonic anhydrase, 29 kDa, 11.65 mL; Aprotinin, 6.5 kDa, 15.33 mL; (gel filtration calibration kit LMW and HMW, GE Healthcare, Piscataway, NJ, USA) The scFv fragment, indicated by a black arrow, with the expected molecular weight of 29 kDa was eluted at the elution volume of 12.04 mL close to the carbonic anhydrase. The gel-phase distribution coefficient (Kav) was calculated from: Kav = (Ve − Vo)/(Vc − Vo) where Ve is elution volume, Vo (7.61 mL) is void volume (determined using blue dextran), and Vc (24 mL) is column volume. Full article ">Figure 5
Far-UV circular dichroism (CD) spectra for the refolded scFv. The scFv fragment (0.5 μg/mL) dissolved in 50 mM Tris pH 7.5, 100 mM NaCl and 5% glycerol was analyzed at room temperature. Full article ">Figure 6
Binding analysis of the refolded scFv fragment. (A) Enzyme-linked immunosorbent assay (ELISA) analysis for the binding of scFv on the human myoglobin coated surface. Serial dilutions (×1/10) were made for the premature antibody and the refolded fragment with an initial concentration of 0.1 and 30 μg/mL, respectively; (B) Comparative ELISA analysis of the refolded scFv and the whole antibody for human myoglobin, equine myoglobin, conalbumin and hemoglobin (scFv 1.9 μg/mL, IgG2-7Ds 0.5 μg/mL); (C) Comparative ELISA analysis of the refolded scFv and the whole antibody for synthetic peptides of human myoglobin and human cardiac troponin I (scFv 1.9 μg/mL, IgG2-7Ds 0.5 μg/mL); (D) The binding pattern was analyzed by an Octet-RED immunosensor; (E) The binding pattern was analyzed using increment of scFv in a continuous manner; and (F) The comparison of the kinetic parameter values for the parent premature antibody and the refolded scFv was performed using the Data Analysis 7.0 program. Full article ">

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Open AccessReview

Emerging Regulation and Function of Betatrophin

by Yi-Hsin Tseng, Yung-Hsin Yeh, Wei-Jan Chen and Kwang-Huei Lin

Int. J. Mol. Sci. 2014, 15(12), 23640-23657; https://doi.org/10.3390/ijms151223640 - 18 Dec 2014

Cited by 57 | Viewed by 9234

Abstract

Betatrophin, also known as TD26/RIFL/lipasin/ANGPTL8/C19orf80, is a novel protein predominantly expressed in human liver. To date, several betatrophin orthologs have been identified in mammals. Increasing evidence has revealed an association between betatrophin expression and serum lipid profiles, particularly in patients with obesity or [...] Read more.

Betatrophin, also known as TD26/RIFL/lipasin/ANGPTL8/C19orf80, is a novel protein predominantly expressed in human liver. To date, several betatrophin orthologs have been identified in mammals. Increasing evidence has revealed an association between betatrophin expression and serum lipid profiles, particularly in patients with obesity or diabetes. Stimulators of betatrophin, such as insulin, thyroid hormone, irisin and caloric intake, are usually relevant to energy expenditure or thermogenesis. In murine models, serum triglyceride levels as well as pancreatic cell proliferation are potently enhanced by betatrophin. Intriguingly, conflicting phenomena have also been reported that betatrophin suppresses hepatic triglyceride levels, suggesting that betatrophin function is mediated by complex regulatory processes. However, its precise physiological role remains unclear at present. In this review, we have summarized the current findings on betatrophin and their implications. Full article

(This article belongs to the Section Biochemistry)

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Open AccessReview

Contrast Agents for Photoacoustic and Thermoacoustic Imaging: A Review

by Dan Wu, Lin Huang, Max S. Jiang and Huabei Jiang

Int. J. Mol. Sci. 2014, 15(12), 23616-23639; https://doi.org/10.3390/ijms151223616 - 18 Dec 2014

Cited by 171 | Viewed by 11271

Abstract

Photoacoustic imaging (PAI) and thermoacoustic imaging (TAI) are two emerging biomedical imaging techniques that both utilize ultrasonic signals as an information carrier. Unique advantages of PAI and TAI are their abilities to provide high resolution functional information such as hemoglobin and blood oxygenation [...] Read more.

Photoacoustic imaging (PAI) and thermoacoustic imaging (TAI) are two emerging biomedical imaging techniques that both utilize ultrasonic signals as an information carrier. Unique advantages of PAI and TAI are their abilities to provide high resolution functional information such as hemoglobin and blood oxygenation and tissue dielectric properties relevant to physiology and pathology. These two methods, however, may have a limited detection depth and lack of endogenous contrast. An exogenous contrast agent is often needed to effectively resolve these problems. Such agents are able to greatly enhance the imaging contrast and potentially break through the imaging depth limit. Furthermore, a receptor-targeted contrast agent could trace the molecular and cellular biological processes in tissues. Thus, photoacoustic and thermoacoustic molecular imaging can be outstanding tools for early diagnosis, precise lesion localization, and molecular typing of various diseases. The agents also could be used for therapy in conjugation with drugs or in photothermal therapy, where it functions as an enhancer for the integration of diagnosis and therapy. In this article, we present a detailed review about various exogenous contrast agents for photoacoustic and thermoacoustic molecular imaging. In addition, challenges and future directions of photoacoustic and thermoacoustic molecular imaging in the field of translational medicine are also discussed. Full article

(This article belongs to the Special Issue Laser Application in Life Sciences)

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Open AccessArticle

Nano-Scale Spatial Assessment of Calcium Distribution in Coccolithophores Using Synchrotron-Based Nano-CT and STXM-NEXAFS

by Shiyong Sun, Yanchen Yao, Xiang Zou, Shenglan Fan, Qing Zhou, Qunwei Dai, Faqin Dong, Mingxue Liu, Xiaoqin Nie, Daoyong Tan and Shuai Li

Int. J. Mol. Sci. 2014, 15(12), 23604-23615; https://doi.org/10.3390/ijms151223604 - 18 Dec 2014

Cited by 9 | Viewed by 6814

Abstract

Calcified coccolithophores generate calcium carbonate scales around their cell surface. In light of predicted climate change and the global carbon cycle, the biomineralization ability of coccoliths has received growing interest. However, the underlying biomineralization mechanism is not yet well understood; the lack of [...] Read more.

Calcified coccolithophores generate calcium carbonate scales around their cell surface. In light of predicted climate change and the global carbon cycle, the biomineralization ability of coccoliths has received growing interest. However, the underlying biomineralization mechanism is not yet well understood; the lack of non-invasive characterizing tools to obtain molecular level information involving biogenic processes and biomineral components remain significant challenges. In the present study, synchrotron-based Nano-computed Tomography (Nano-CT) and Scanning Transmission X-ray Microscopy-Near-edge X-ray Absorption Fine Structure Spectromicroscopy (STXM-NEXAFS) techniques were employed to identify Ca spatial distribution and investigate the compositional chemistry and distinctive features of the association between biomacromolecules and mineral components of calcite present in coccoliths. The Nano-CT results show that the coccolith scale vesicle is similar as a continuous single channel. The mature coccoliths were intracellularly distributed and immediately ejected and located at the exterior surface to form a coccoshpere. The NEXAFS spectromicroscopy results of the Ca L edge clearly demonstrate the existence of two levels of gradients spatially, indicating two distinctive forms of Ca in coccoliths: a crystalline-poor layer surrounded by a relatively crystalline-rich layer. The results show that Sr is absorbed by the coccoliths and that Sr/Ca substitution is rather homogeneous within the coccoliths. Our findings indicate that synchrotron-based STXM-NEXAFS and Nano-CT are excellent tools for the study of biominerals and provide information to clarify biomineralization mechanism. Full article

(This article belongs to the Section Materials Science)

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Scanning electron microscopyimages of typical morphology features of coccolithophores. (A) Image of a whole cell of E. huxleyi; (B) Image of a whole cell of P. carterae. The complete coccoliths in distal shield and proximal shield views are also presented in (A). Scale bars: 2 μm Full article ">Figure 2
Different images of a single cell of E. huxleyi. (A) TXM 2D projection image; (B,C) representative two slices of reconstructed volume data showing different regions of cells; (D,E) representative two images of 3D rendering of reconstructed volume data with different rotation angles. Red color circles in (B,C) show the possible pore of the channel of the coccolith vesicle used for excreting coccolith scales. Scale bar in (A): 4 μm. Full article ">Figure 3
High-resolution (50 nm) spatial distribution of total Ca in E. huxleyi determined by STXM-NEXAFS spectromicroscopy. (A) Contrast tomography image; (B) Cluster 1 of background with blue color; (C) Cluster 2 of Ca form with yellow color; (D) Cluster 3 of Ca form with red color; (E) Cluster 4 of Ca form with green color; (F) Merged images of (B–E), Scale bars: 1 μm. Full article ">Figure 4
NEXAFS spectra of Ca L2,3 edge of total Ca in E. huxleyi by cluster measurements of images as showed in <a href="#ijms-15-23604-f003" class="html-fig">Figure 3</a>. Full article ">Figure 5
High-resolution (50 nm) spatial distribution of total Ca in P. carterae determined by STXM-NEXAFS spectromicroscopy. (A) Coccoliths image using FE-SEM; (B) Contrast tomography image; (C) Cluster 1 of background with blue color; (D) Cluster 2 of Ca form with yellow color; (E) Cluster 3 of Ca form with red color; (F) Merged images of (C–E). Scale bars: 2 μm. Full article ">Figure 6
NEXAFS spectra of total Ca in P. carterae by cluster measurements of images as showed in <a href="#ijms-15-23604-f005" class="html-fig">Figure 5</a>. Full article ">Figure 7
Dual energy (347.7 and 345 eV) contrast images of total Ca in coccoliths of E. huxleyi in the presence of different Sr concentrations using STXM. (A,B) and (B) 0 ppm Sr in original culture medium; (C,D) 50 ppm Sr in original culture medium; (E,F) 100 ppm Sr in original culture medium; (A,C,E) Ca mapping at 347.7 eV; (B,D,F) Color overlay from the difference of 347.7 and 345 eV images showing the specific distribution of Ca with gradients. Scale bars: 1 μm. Full article ">

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Open AccessArticle

The Sesquiterpene Biosynthesis and Vessel-Occlusion Formation in Stems of Aquilaria sinensis (Lour.) Gilg Trees Induced by Wounding Treatments without Variation of Microbial Communities

by Zheng Zhang, Jianhe Wei, Xiaomin Han, Liang Liang, Yun Yang, Hui Meng, Yanhong Xu and Zhihui Gao

Int. J. Mol. Sci. 2014, 15(12), 23589-23603; https://doi.org/10.3390/ijms151223589 - 18 Dec 2014

Cited by 27 | Viewed by 7381

Abstract

As widely recognized, agarwood formation in Aquilaria trees is induced by external wounding. Because agarwood usually harbors specific microbes, the function of microbes in agarwood formation has been debated for almost a century. In this study, two wounding methods, the burning-chisel-drilling method (BCD) [...] Read more.

As widely recognized, agarwood formation in Aquilaria trees is induced by external wounding. Because agarwood usually harbors specific microbes, the function of microbes in agarwood formation has been debated for almost a century. In this study, two wounding methods, the burning-chisel-drilling method (BCD) and the whole-tree agarwood-inducing method (Agar-Wit), were used under the non-contamination of environmental microorganisms. After pyrosequencing the small rRNA subunits of the wounds induced by the BCD and Agar-Wit, no substantial variation was observed either in fungal and bacterial enrichment and diversity or in the relative abundances of taxa. By contrast, significant variations in fungal and bacterial communities were detected following the partial tree pruning (PTP)-wounding. The wound-induced sesquiterpene biosynthesis and vessel-occlusion formation, however, were found to be similar in all types of wounded trunks. We thus infer that wounding in the absence of variations in microbial communities may induce agarwood formation. This result does not support the long-standing notion that agarwood formation depends on microbes. Full article

(This article belongs to the Section Biochemistry)

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Figure 1
Distribution of fungal OUTs at the species level in the wounded wood induced by three different wounding methods, BCD, Agar-Wit and PTP. The wounded wood obtained by the partial-trunk-pruning (PTP) method was divided into four zones, i.e., the decay (D), agarwood (A), transition (T), and white (W) zones. Samples obtained from the untreated healthy trees and the trees treated by the Agar-wit with ddH2O not inducer are referred to as CK1 and CK2, respectively. Full article ">Figure 2
Distribution of bacterial OUTs at the ordinal level in the agarwood induced by three different wounding methods, BCD, Agar-Wit and PTP. The wounded wood obtained by the partial-trunk-pruning (PTP) method was divided into four zones, the decay (D), agarwood (A), transition (T), and white (W) zones. The samples obtained from the untreated healthy trees and the trees treated by the Agar-wit with ddH2O not inducer are referred to as CK1 and CK2, respectively. Full article ">Figure 3
Type and content of essential oils in the agarwood samples obtained by the three different wounding methods, BCD, Agar-Wit and PTP. The wounded wood obtained by the partial-trunk-pruning (PTP) method was divided into four zones, the decay (D), agarwood (A), transition (T), and white (W) zones. The samples obtained from the untreated healthy trees and the trees treated by the Agar-wit with ddH2O not inducers are referred to as CK1 and CK2, respectively. Relative amount (%) was defined to be the peak area relative to the total peak area. Full article ">Figure 4
Wound-induced vessel occlusions detected (in the decay, agarwood, transition, and healthy zones) at least 10 months after the PTP treatment and 30 days after the Agar-wit and BCD treatments. No tyloses were detected in any vessel lumen of an unwounded control sample. PVO means the percentage of vessels with occlusions per mm2. Error bars represent the SE, n = 5. The wounded wood obtained by the PTP method was divided into four zones, the decay (D), agarwood (A), transition (T), and white (W) zones. The samples obtained from the untreated healthy trees and the trees treated by the Agar-wit with ddH2O not inducers are referred to as CK1 and CK2, respectively. Full article ">

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Open AccessArticle

Docetaxel-Encapsulating Small-Sized Polymeric Micelles with Higher Permeability and Its Efficacy on the Orthotopic Transplantation Model of Pancreatic Ductal Adenocarcinoma

by Yunfei Li, Peiran Li, Mingji Jin, Changgao Jiang and Zhonggao Gao

Int. J. Mol. Sci. 2014, 15(12), 23571-23588; https://doi.org/10.3390/ijms151223571 - 17 Dec 2014

Cited by 8 | Viewed by 6653

Abstract

Pancreatic ductal adenocarcinoma (PDAC) elicits a dense stromal response that blocks vascular access because of pericyte coverage of vascular fenestrations. In this way, the PDAC stroma contributes to chemotherapy resistance, and the small-sized nanocarrier loaded with platinum has been adopted to address this [...] Read more.

Pancreatic ductal adenocarcinoma (PDAC) elicits a dense stromal response that blocks vascular access because of pericyte coverage of vascular fenestrations. In this way, the PDAC stroma contributes to chemotherapy resistance, and the small-sized nanocarrier loaded with platinum has been adopted to address this problem which is not suitable for loading docetaxel (DTX). In the present study, we used the poly(d,l-lactide)-b-polyethylene glycol-methoxy (mPEG-b-PDLLA) to encapsulate DTX and got a small-sized polymeric micelle (SPM); meanwhile we functionalized the SPM’s surface with TAT peptide (TAT-PM) for a higher permeability. The diameters of both SPM and TAT-PM were in the range of 15–26 nm. In vitro experiments demonstrated that TAT-PM inhibited Capan-2 Luc PDAC cells growth more efficiently and induced more apoptosis compared to SPM and Duopafei. The in vivo therapeutic efficiencies of SPM and TAT-PM compared to free DTX was investigated on the orthotopic transplantation model of Capan-2 Luc. SPM exerted better therapeutic efficiency than free DTX, however, TAT-PM didn’t outperformed SPM. Overall, these results disclosed that SPM could represent a new therapeutic approach against pancreatic cancer, but its permeability to PDAC was not the only decisive factor. Full article

(This article belongs to the Section Materials Science)

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Open AccessArticle

Isolation and Structural Elucidation of Antiproliferative Compounds of Lipidic Fractions from White Shrimp Muscle (Litopenaeus vannamei)

by Carmen-María López-Saiz, Carlos Velázquez, Javier Hernández, Francisco-Javier Cinco-Moroyoqui, Maribel Plascencia-Jatomea, Maribel Robles-Sánchez, Lorena Machi-Lara and Armando Burgos-Hernández

Int. J. Mol. Sci. 2014, 15(12), 23555-23570; https://doi.org/10.3390/ijms151223555 - 17 Dec 2014

Cited by 16 | Viewed by 6998

Abstract

Shrimp is one of the most popular seafood items worldwide, and has been reported as a source of chemopreventive compounds. In this study, shrimp lipids were separated by solvent partition and further fractionated by semi-preparative RP-HPLC and finally by open column chromatography in [...] Read more.

Shrimp is one of the most popular seafood items worldwide, and has been reported as a source of chemopreventive compounds. In this study, shrimp lipids were separated by solvent partition and further fractionated by semi-preparative RP-HPLC and finally by open column chromatography in order to obtain isolated antiproliferative compounds. Antiproliferative activity was assessed by inhibition of M12.C3.F6 murine cell growth using the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay. The methanolic fraction showed the highest antiproliferative activity; this fraction was separated into 15 different sub-fractions (M1–M15). Fractions M8, M9, M10, M12, and M13 were antiproliferative at 100 µg/mL and they were further tested at lower concentrations. Fractions M12 and M13 exerted the highest growth inhibition with an IC₅₀ of 19.5 ± 8.6 and 34.9 ± 7.3 µg/mL, respectively. Fraction M12 was further fractionated in three sub-fractions M12a, M12b, and M12c. Fraction M12a was identified as di-ethyl-hexyl-phthalate, fraction M12b as a triglyceride substituted by at least two fatty acids (predominantly oleic acid accompanied with eicosapentaenoic acid) and fraction M12c as another triglyceride substituted with eicosapentaenoic acid and saturated fatty acids. Bioactive triglyceride contained in M12c exerted the highest antiproliferative activity with an IC₅₀ of 11.33 ± 5.6 µg/mL. Biological activity in shrimp had been previously attributed to astaxanthin; this study demonstrated that polyunsaturated fatty acids are the main compounds responsible for antiproliferative activity. Full article

(This article belongs to the Section Biochemistry)

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Open AccessArticle

The Dynamics of DNA Methylation in Maize Roots under Pb Stress

by Haiping Ding, Jian Gao, Cheng Qin, Haixia Ma, Hong Huang, Pan Song, Xirong Luo, Haijian Lin, Ya'ou Shen, Guangtang Pan and Zhiming Zhang

Int. J. Mol. Sci. 2014, 15(12), 23537-23554; https://doi.org/10.3390/ijms151223537 - 17 Dec 2014

Cited by 39 | Viewed by 8048

Abstract

Plants adapt to adverse conditions through a series of physiological, cellular, and molecular processes, culminating in stress tolerance. However, little is known about the associated regulatory mechanisms at the epigenetic level in maize under lead (Pb) stress. Therefore, in this study, we aimed [...] Read more.

Plants adapt to adverse conditions through a series of physiological, cellular, and molecular processes, culminating in stress tolerance. However, little is known about the associated regulatory mechanisms at the epigenetic level in maize under lead (Pb) stress. Therefore, in this study, we aimed to compare DNA methylation profiles during the dynamic development of maize roots following Pb treatment to identify candidate genes involved in the response to Pb stress. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) was used to investigate the genome-wide DNA methylation patterns in maize roots under normal condition (A1) and 3 mM Pb(NO₃)₂ stress for 12 h (K2), 24 h (K3) and 48 h (K4). The results showed that the average methylation density was the highest in CpG islands (CGIs), followed by the intergenic regions. Within the gene body, the methylation density of the introns was higher than those of the UTRs and exons. In total, 3857 methylated genes were found in 4 tested samples, including 1805 differentially methylated genes for K2 versus A1, 1508 for K3 versus A1, and 1660 for K4 versus A1. Further analysis showed that 140 genes exhibited altered DNA methylation in all three comparisons, including some well-known stress-responsive transcription factors and proteins, such as MYB, AP2/ERF, bZIP, serine-threonine/tyrosine-proteins, pentatricopeptide repeat proteins, RING zinc finger proteins, F-box proteins, leucine-rich repeat proteins and tetratricopeptide repeat proteins. This study revealed the genome-scale DNA methylation patterns of maize roots in response to Pb exposure and identified candidate genes that potentially regulate root dynamic development under Pb stress at the methylation level. Full article

(This article belongs to the Special Issue Regulation of Plant Mineral Nutrition: Transport, Sensing and Signalling)

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Graphical abstract
Full article ">Figure 1
Unique reads mapped in different components of the maize genome (such as promoters, 5' UTRs, 3' UTRs, exons, introns, intergenic regions, CGIs, and downstream regions). Full article ">Figure 2
Methylation distributions in different genomic regions. Methylation density within promoter, gene body and intergenic regions was calculated with the ratio of methylated peaks in a particular component to the total area of that region. Full article ">Figure 3
Identification and functional classification of the differentially methylated genes. (A) Identification of the differentially methylated genes in components of the genome in all three comparisons, including 1805 differentially methylated genes of K2 versus A1, 1508 of K3 versus A1, and 1660 of K4 versus A1; (B) Functional classification of the differentially methylated genes in three comparisons. Full article ">Figure 4
qRT-PCR validation of the Medip-seq data. log2-fold change determined from the relative Ct values of 8 genes were compared with those detected by Medip-seq. Three replicates for each sample were run and the Ct values averaged. All Ct values were normalized to 18s RNA. Full article ">Figure 5
Relationship between DNA methylation in the proximal promoter regions and gene expression level in maize roots responsive to Pb stress for K2 (A); K3 (B); and K4 (C). Genes were assessed according to differential expression levels. DNA methylation level was measured by the log ratio of the p value of the methylation peaks, with each point representing the mean expression level and the relative methylation level. Full article ">

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Open AccessArticle

Lidocaine Sensitizes the Cytotoxicity of Cisplatin in Breast Cancer Cells via Up-Regulation of RARβ2 and RASSF1A Demethylation

by Kehan Li, Jianxue Yang and Xuechang Han

Int. J. Mol. Sci. 2014, 15(12), 23519-23536; https://doi.org/10.3390/ijms151223519 - 17 Dec 2014

Cited by 55 | Viewed by 7798

Abstract

It has been reported that lidocaine is toxic to various types of cells. And a recent study has confirmed that lidocaine exerts a demethylation effect and regulates the proliferation of human breast cancer cell lines. To recognize a potential anti-tumor effect of lidocaine, [...] Read more.

It has been reported that lidocaine is toxic to various types of cells. And a recent study has confirmed that lidocaine exerts a demethylation effect and regulates the proliferation of human breast cancer cell lines. To recognize a potential anti-tumor effect of lidocaine, we evaluated the DNA demethylation by lidocaine in human breast cancer lines, MCF-7 and MDA-MB-231 cells, and determined the influence of demethylation on the toxicity to these cells of cisplatin, which is a commonly utilized anti-tumor agent for breast cancer. Results demonstrated that lidocaine promoted a significant global genomic demethylation, and particularly in the promoters of tumor suppressive genes (TSGs), RARβ2 and RASSF1A. Further, the lidocaine treatment increased cisplatin-induced apoptosis and enhanced cisplatin-induced cytotoxicity. The combined treatment with both lidocaine and cisplatin promoted a significantly higher level of MCF-7 cell apoptosis than singular lidocaine or cisplatin treatment. Moreover, the abrogation of RARβ2 or RASSF1A expression inhibited such apoptosis. In conclusion, the present study confirms the demethylation effect of lidocaine in breast cancer cells, and found that the demethylation of RARβ2 and RASSF1A sensitized the cytotoxicity of cisplatin in breast cancer cells. Full article

(This article belongs to the Collection Programmed Cell Death and Apoptosis)

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Figure 1
Global genomic demethylation of CpG islands promoted by lidocaine. (A) DNA methylation levels in MCF-7 breast cancer cells treated for 72 h with 0.01, 0.1 or 1 mM lidocaine treatment, or with 10 μM DAC treatment, respectively; (B) DNA methylation levels in MCF-7 cells treated with or without 0.1 mM lidocaine, for 48, 72 or 96 h; (C,D) DNA methylation levels in MDA-MB-231 breast cancer cells post above-mentioned treatment respectively. Results were expressed as mean ± SD for three independent experiments. Statistical significance was assessed by the unpaired Student’s t-test (* p ˂ 0.05, ** p ˂ 0.01, or *** p ˂ 0.001, ns: no significance). Full article ">Figure 1 Cont.
Global genomic demethylation of CpG islands promoted by lidocaine. (A) DNA methylation levels in MCF-7 breast cancer cells treated for 72 h with 0.01, 0.1 or 1 mM lidocaine treatment, or with 10 μM DAC treatment, respectively; (B) DNA methylation levels in MCF-7 cells treated with or without 0.1 mM lidocaine, for 48, 72 or 96 h; (C,D) DNA methylation levels in MDA-MB-231 breast cancer cells post above-mentioned treatment respectively. Results were expressed as mean ± SD for three independent experiments. Statistical significance was assessed by the unpaired Student’s t-test (* p ˂ 0.05, ** p ˂ 0.01, or *** p ˂ 0.001, ns: no significance). Full article ">Figure 2
Lidocaine-promoted RARβ2 and RASSF1A demethylation up-regulates the expression of both genes. Methylation levels of RARβ2 (A) or RASSF1A (B) in MCF-7 cells treated for 72 h with 0.01, 0.1 or 1 mM lidocaine, or with 10 μM DAC, respectively; (C) mRNA level of RARβ2 or RASSF1A in MCF-7 cells post treatment with 0.1 mM lidocaine or with 10 μM DAC for 72 h; Western blot assay for protein level of RARβ2 (D) or RASSF1A (E) in MCF-7 cells with or without 0.1 mM lidocaine treatment for 96 h. Each value was expressed as mean ± SD for three independent tests. Statistical significance was assessed by the unpaired Student’s t-test (* p ˂ 0.05, ** p ˂ 0.01, or *** p ˂ 0.001). Full article ">Figure 3
Lidocaine reduces the viability of MCF-7 and MDA-MB-231 cells in the presence of cisplatin. (A) Relative viability of MCF-7 and MDA-MB-231 cells treated with 0.2 μM cisplatin or (and) 0.1 mM lidocaine for 72 h; (B) Influence of 0.02, 0.1 or 0.5 mM lidocaine on the viability of 0.2 μM cisplatin-treated MCF-7 cells for 72 h; Relative viability of MCF-7 (C) or MDA-MB-231 (D) cells treated with 0.2 μM cisplatin or (and) 0.1 mM for 36, 48 or 72 h. All results were averaged for triple independent experiments. And each statistical significance was considered when p ˂ 0.05 or less. * p ˂ 0.05, ** p ˂ 0.01, or *** p ˂ 0.001, ns: no significance. Full article ">Figure 4
Colony formation of MCF-7 cells treated with lidocaine and/or cisplatin. (A) Colony formation of MCF-7 cells post the transfection with 0.2 μM cisplatin, 0.1 mM lidocaine or with both agents for 96 h; (B) The colony number in groups with or without the treatment of 0.2 μM cisplatin and/or 0.1 mM lidocaine. All experiments were performed independently in triplicate. Statistical significance was shown as * p ˂ 0.05, ** p ˂ 0.01, or *** p ˂ 0.001, ns: no significance. Full article ">Figure 5
Lidocaine enhances cisplatin-induced apoptosis in MCF-7 cells. (A) Apoptotic cells induced by 0.2 μM cisplatin, or (and) 0.1 mM lidocaine for 36, 48 or 60 h; (B) Western blot analysis of activated caspase 3, cleaved PARP by activated caspase 3 and cytochrome c released from mitochondria in MCF-7 cells treated with 0.2 μM cisplatin, and/or 0.1 mM lidocaine for 60 h; (C–E) Relative levels of activated caspase 3, PARP cleavage and cytochrome c release to β-actin in MCF-7 cells treated with lidocaine, with cisplatin or with both agents; (F) Caspase 3 activity in lidocaine-, cisplatin- or both agents-treated MCF-7 cells, revealing by AMC Caspase Profiling Kit. Each value was averaged for three independent experiment results, and statistical significance was considered when p ˂ 0.05 or less, * p ˂ 0.05, ** p ˂ 0.01, *** p ˂ 0.001, **** p ˂ 0.0001, ns: no significance. Full article ">Figure 6
Transfection of RARβ2- or RASSF1A-specific siRNA inhibits the lidocaine and cisplatin-induced apoptosis in MCF-7 cells. (A–D) Western blot analysis of RARβ2 (A,C) or RASSF1A (B,D) expression in MCF-7 cells post-transfection of the RARβ2- or RASSF1A-specific siRNA, without (A,B) or with (C,D) the lidocane and/or cisplatin treatment; (E,F) Apoptotic cells induced by the combined treatment with 0.1 mM lidocaine and 0.2 μM cisplatin for 48 h, post-transfection of RARβ2- (E) or RASSF1A-specific siRNA (F) * p ˂ 0.05, ** p ˂ 0.01, ns: no significance. Full article ">

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Open AccessReview

TXNDC5, a Newly Discovered Disulfide Isomerase with a Key Role in Cell Physiology and Pathology

by Elena Horna-Terrón, Alberto Pradilla-Dieste, Cristina Sánchez-de-Diego and Jesús Osada

Int. J. Mol. Sci. 2014, 15(12), 23501-23518; https://doi.org/10.3390/ijms151223501 - 17 Dec 2014

Cited by 67 | Viewed by 10141

Abstract

Thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family, acting as a chaperone of endoplasmic reticulum under not fully characterized conditions As a result, TXNDC5 interacts with many cell proteins, contributing to their proper folding and correct formation of [...] Read more.

Thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family, acting as a chaperone of endoplasmic reticulum under not fully characterized conditions As a result, TXNDC5 interacts with many cell proteins, contributing to their proper folding and correct formation of disulfide bonds through its thioredoxin domains. Moreover, it can also work as an electron transfer reaction, recovering the functional isoform of other protein disulfide isomerases, replacing reduced glutathione in its role. Finally, it also acts as a cellular adapter, interacting with the N-terminal domain of adiponectin receptor. As can be inferred from all these functions, TXNDC5 plays an important role in cell physiology; therefore, dysregulation of its expression is associated with oxidative stress, cell ageing and a large range of pathologies such as arthritis, cancer, diabetes, neurodegenerative diseases, vitiligo and virus infections. Its implication in all these important diseases has made TXNDC5 a susceptible biomarker or even a potential pharmacological target. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Human Liver Diseases)

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Full article ">Figure 1
Flow chart displaying the information collection process. Two different sources of data were used: Data from online bioinformatics databases and a search in PubMed. EndNote X1 (Thomson Reuters, New York, NY, USA). * Some references may appear in more than one section of the review. Full article ">Figure 2
Scheme showing TXNDC5 domains. The graphic shows the three Trx-like domains, placed between amino acids 14–95, 118–221 and 251–355 [<a href="#B26-ijms-15-23501" class="html-bibr">26</a>]. Full article ">Figure 3
Current model of TXNDC5 interactions with different compounds. Panels (A) and (B) represent different angles of the docking assay using protein structure from Protein Data Bank [<a href="#B24-ijms-15-23501" class="html-bibr">24</a>]. First, many binding modes were generated by local and blind docking on the surface and in the cavities of the protein. Then, CHARMM (Chemistry at Harvard Macromolecules Mechanics) energies were estimated and most favorable energies were evaluated with FACTS (Fast Analytical Continuum Treatment of Solvation), clustered and visualized. Images show molecular coupling between TXNDC5 protein and the compounds cyclosporine A (blue), diazoside (red) and paracetamol (green). The interaction is mainly established within two domains of the protein involved in the action and its regulation [<a href="#B59-ijms-15-23501" class="html-bibr">59</a>,<a href="#B60-ijms-15-23501" class="html-bibr">60</a>]. Full article ">

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Open AccessReview

Protecting the Melatonin Rhythm through Circadian Healthy Light Exposure

by Maria Angeles Bonmati-Carrion, Raquel Arguelles-Prieto, Maria Jose Martinez-Madrid, Russel Reiter, Ruediger Hardeland, Maria Angeles Rol and Juan Antonio Madrid

Int. J. Mol. Sci. 2014, 15(12), 23448-23500; https://doi.org/10.3390/ijms151223448 - 17 Dec 2014

Cited by 167 | Viewed by 30918

Abstract

Currently, in developed countries, nights are excessively illuminated (light at night), whereas daytime is mainly spent indoors, and thus people are exposed to much lower light intensities than under natural conditions. In spite of the positive impact of artificial light, we pay a [...] Read more.

Currently, in developed countries, nights are excessively illuminated (light at night), whereas daytime is mainly spent indoors, and thus people are exposed to much lower light intensities than under natural conditions. In spite of the positive impact of artificial light, we pay a price for the easy access to light during the night: disorganization of our circadian system or chronodisruption (CD), including perturbations in melatonin rhythm. Epidemiological studies show that CD is associated with an increased incidence of diabetes, obesity, heart disease, cognitive and affective impairment, premature aging and some types of cancer. Knowledge of retinal photoreceptors and the discovery of melanopsin in some ganglion cells demonstrate that light intensity, timing and spectrum must be considered to keep the biological clock properly entrained. Importantly, not all wavelengths of light are equally chronodisrupting. Blue light, which is particularly beneficial during the daytime, seems to be more disruptive at night, and induces the strongest melatonin inhibition. Nocturnal blue light exposure is currently increasing, due to the proliferation of energy-efficient lighting (LEDs) and electronic devices. Thus, the development of lighting systems that preserve the melatonin rhythm could reduce the health risks induced by chronodisruption. This review addresses the state of the art regarding the crosstalk between light and the circadian system. Full article

(This article belongs to the Special Issue Advances in the Research of Melatonin 2014)

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Figure 1
General overview of the functional organization of the circadian system in mammals. Inputs: environmental periodical cues can reset the phase of the central pacemaker so that the period and phase of circadian rhythms coincide with the timing of the external cues; Central pacemakers: the suprachiasmatic nuclei (SCN) is considered to be the major pacemaker of the circadian system, driving circadian rhythmicity in other brain areas and peripheral tissues by sending them neural and humoral signals (such as melatonin, secreted by the pineal gland (P)). The SCN receives light-dark cycle information through the retinohypothalamic tract (RHT). Peripheral oscillators: most peripheral tissues and organs contain circadian oscillators. Usually, they are under the control of the SCN; however, under some circumstances (e.g., restricted feeding, jet lag and shift work), they can desynchronize from the SCN; Outputs: central pacemakers and peripheral oscillators are responsible for the daily rhythmicity observed in most physiological and behavioral functions. Some of these overt rhythms (physical exercise, core temperature, sleep-wake cycle and feeding time), in turn, provide feedback, which can modify the function of the SCN and peripheral oscillators, (redrawn from [<a href="#B11-ijms-15-23448" class="html-bibr">11</a>]). Full article ">Figure 2
Molecular clock of mammals. Circadian locomotor output cycles kaput (CLOCK)/brain and muscle aryl hydrocarbon receptor nuclear translocator-like (BMAL1) heterodimers (red and green ovals) bind the DNA of clock target genes at E-boxes or E’-boxes and permit their transcription. The resulting period (PER) and cryptochrome (CRY) proteins (blue and yellow) dimerize in the cytoplasm and translocate to the nucleus where they inhibit CLOCK/BMAL1 proteins from initiating further transcription (redrawn from [<a href="#B16-ijms-15-23448" class="html-bibr">16</a>]). Full article ">Figure 3
Absence and presence of circadian photoreception in two totally blind subjects. A and B correspond to the sleep-wake pattern and the results of melatonin suppression test in a 70-year old blind patient with congenital glaucoma who reported no conscious light perception and whose electroretinogram (ERG) and visually evoked potential (VEP) responses were not detectable. In (A), the sleep-wake pattern is double-plotted according to time of day (abscissa) and study day (ordinate). It is evident that the subject’s circadian system was not entrained to the light-dark cycle, and the core body temperature rhythm (circle) exhibited a non-24-h period; (B) shows the null effect of light (white bar) on melatonin secretion; C and D correspond to a 21-year-old woman with Leber’s congenital amaurosis, a type of retinal dystrophy. The ERG was undetectable, but an abnormal VEP was recorded. As represented in C, her circadian system was normally entrained (24-h period) and melatonin secretion was suppressed when she was exposed to light. Both results indicated that this patient, despite her lack of conscious light perception, preserved the retina-SCN-pineal pathway (reproduced from [<a href="#B122-ijms-15-23448" class="html-bibr">122</a>]). Full article ">Figure 4
Schematic view of brain regions and circuits inervated by intrinsically photosensitive retinal ganglion cells (ipRGCs). The location of their somas, axons and main targets are represented in blue. Projections of ipRGCs to the SCN (orange) allow photic entrainment of the circadian clock. The red pathway with green nuclei represents a polysynaptic circuit originating in the SCN, which photically regulates melatonin release by the pineal gland (P) through sympathetic innervation. Synaptic links in this pathway include the paraventricular nucleus (PVN) of the hypothalamus, the intermediolateral nucleus (IML) of the spinal cord and the superior cervical ganglion (SCG). The olivary pretectal nucleus (OPN) is another direct target of ipRGCs, and is a crucial link in the circuitry underlying the pupillary light reflex, shown in brown (fibers) and purple (nuclei). Synapses in this parasympathetic circuit are found at the Edinger-Westphal nucleus (EW), the ciliary ganglion (CG) and the iris muscles (I). Other targets of the ipRGCs include two components of the lateral geniculate nucleus of the thalamus, the ventral division (LGNv) and the intergeniculate leaflet (IGL) (reproduced from [<a href="#B138-ijms-15-23448" class="html-bibr">138</a>]). Full article ">Figure 5
Example of a pupillographic recording in response to a 5-s bright white light stimulus in a normal human subject. The response waveform during the constriction phase has two components. When the light is turned ON, there is transient phase characterized by a short-latency, high-velocity maximal change in pupil size. Thereafter, the pupil partly redilates, or escapes, to a state of partial pupil constriction that represents the sustained phase of the pupil light reflex. When the light stimulus ends, the pupil starts to recover its original size after a period (which does not always occur) in which some degree of contraction persists after the light stimulus (modified from [<a href="#B155-ijms-15-23448" class="html-bibr">155</a>]). Full article ">Figure 6
Spectral responses of the pupillary light reflex (PLR). Comparison of the PLR to 480 and 620 nm monochromatic long-duration (5 min) stimulations at 6 different irradiances in a single subject. After the initial and rapid pupil constriction, the steady state equilibrium and the persistent responses are present in all except the lowest irradiances, thus depending on wavelength and light intensity. The amplitude of the steady state equilibrium response is rapidly attained and particularly robust at 480 nm for the highest irradiances used. The persistent responses are also greater at 480 nm, as compared to 620 nm at equivalent irradiances. Note that for the higher irradiances, the pupil has not yet returned to the baseline within 5 min after extinction of the stimulus (reproduced from [<a href="#B134-ijms-15-23448" class="html-bibr">134</a>]). Full article ">Figure 7
Age-related losses in retinal illumination due to decreasing crystalline lens light transmission and pupil area. The percentage of loss per decade is reasonably uniform and most prominent at shorter violet (400–440 nm) and blue (440–500 nm) wavelengths (reproduced from [<a href="#B294-ijms-15-23448" class="html-bibr">294</a>]). Full article ">Figure 8
Short wavelength light sensitivity for melatonin suppression. Comparison of the effects of 460 nm (blue circles) and 555 nm (green circles) light exposure on melatonin suppression in a blind man. The graph represents the direct effects for melatonin suppression of exposure to green (555 nm) and blue (460 nm) monochromatic light on the male subject. Exposure to 555 nm light caused no suppression of melatonin as compared to the corresponding clock time the previous day, whereas exposure to 460 nm light suppressed melatonin and maintained the suppression effect throughout the entire 6.5 h exposure (reproduced from [<a href="#B140-ijms-15-23448" class="html-bibr">140</a>]). Full article ">

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Open AccessReview

Recent Developments in β-Cell Differentiation of Pluripotent Stem Cells Induced by Small and Large Molecules

by S. Suresh Kumar, Abdullah A. Alarfaj, Murugan A. Munusamy, A. J. A. Ranjith Singh, I-Chia Peng, Sivan Padma Priya, Rukman Awang Hamat and Akon Higuchi

Int. J. Mol. Sci. 2014, 15(12), 23418-23447; https://doi.org/10.3390/ijms151223418 - 17 Dec 2014

Cited by 26 | Viewed by 12703

Abstract

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold promise as novel therapeutic tools for diabetes treatment because of their self-renewal capacity and ability to differentiate into beta (β)-cells. Small and large molecules play [...] Read more.

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold promise as novel therapeutic tools for diabetes treatment because of their self-renewal capacity and ability to differentiate into beta (β)-cells. Small and large molecules play important roles in each stage of β-cell differentiation from both hESCs and hiPSCs. The small and large molecules that are described in this review have significantly advanced efforts to cure diabetic disease. Lately, effective protocols have been implemented to induce hESCs and human mesenchymal stem cells (hMSCs) to differentiate into functional β-cells. Several small molecules, proteins, and growth factors promote pancreatic differentiation from hESCs and hMSCs. These small molecules (e.g., cyclopamine, wortmannin, retinoic acid, and sodium butyrate) and large molecules (e.g. activin A, betacellulin, bone morphogentic protein (BMP4), epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), noggin, transforming growth factor (TGF-α), and WNT3A) are thought to contribute from the initial stages of definitive endoderm formation to the final stages of maturation of functional endocrine cells. We discuss the importance of such small and large molecules in uniquely optimized protocols of β-cell differentiation from stem cells. A global understanding of various small and large molecules and their functions will help to establish an efficient protocol for β-cell differentiation. Full article

(This article belongs to the Special Issue Artificial Organs)

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Open AccessArticle

PRRT2 Mutations Are Related to Febrile Seizures in Epileptic Patients

by Zheng-Wen He, Jian Qu, Ying Zhang, Chen-Xue Mao, Zhi-Bin Wang, Xiao-Yuan Mao, Zhi-Yong Deng, Bo-Ting Zhou, Ji-Ye Yin, Hong-Yu Long, Bo Xiao, Yu Zhang, Hong-Hao Zhou and Zhao-Qian Liu

Int. J. Mol. Sci. 2014, 15(12), 23408-23417; https://doi.org/10.3390/ijms151223408 - 16 Dec 2014

Cited by 16 | Viewed by 6603

Abstract

Previous studies reported that the proline-rich transmembrane protein 2 (PRRT2) gene was identified to be related to paroxysmal kinesigenic dyskinesia (PKD), infantile convulsions with PKD, PKD with migraine and benign familial infantile epilepsy (BFIE). The present study explores whether the PRRT2 [...] Read more.

Previous studies reported that the proline-rich transmembrane protein 2 (PRRT2) gene was identified to be related to paroxysmal kinesigenic dyskinesia (PKD), infantile convulsions with PKD, PKD with migraine and benign familial infantile epilepsy (BFIE). The present study explores whether the PRRT2 mutation is a potential cause of febrile seizures, including febrile seizures plus (FS+), generalized epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome (DS); thus, it may provide a new drug target for personalized medicine for febrile seizure patients. We screened PRRT2 exons in a cohort of 136 epileptic patients with febrile seizures, including FS+, GEFS+ and DS. PRRT2 genetic mutations were identified in 25 out of 136 (18.4%) febrile seizures in epileptic patients. Five loss-of-function and coding missense mutations were identified: c.649delC (p.R217Efs*12), c.649_650insC (p.R217Pfs*8), c.412C>G (p.Pro138Ala), c.439G>C (p.Asp147His) and c.623C>A (p.Ser208Tyr). PRRT2 variants were probably involved in the etiology of febrile seizures in epileptic patients. Full article

(This article belongs to the Special Issue Pharmacogenetics and Personalized Medicine)

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Open AccessArticle

Homogenized Finite Element Analysis on Effective Elastoplastic Mechanical Behaviors of Composite with Imperfect Interfaces

by Wu-Gui Jiang, Ren-Zhi Zhong, Qing H. Qin and Yong-Gang Tong

Int. J. Mol. Sci. 2014, 15(12), 23389-23407; https://doi.org/10.3390/ijms151223389 - 16 Dec 2014

Cited by 37 | Viewed by 10413

Abstract

A three-dimensional (3D) representative volume element (RVE) model was developed for analyzing effective mechanical behavior of fiber-reinforced ceramic matrix composites with imperfect interfaces. In the model, the fiber is assumed to be perfectly elastic until its tensile strength, and the ceramic material is [...] Read more.

A three-dimensional (3D) representative volume element (RVE) model was developed for analyzing effective mechanical behavior of fiber-reinforced ceramic matrix composites with imperfect interfaces. In the model, the fiber is assumed to be perfectly elastic until its tensile strength, and the ceramic material is modeled by an elasto-plastic Drucker-Prager constitutive law. The RVE model is then used to study the elastic properties and the tensile strength of composites with imperfect interfaces and validated through experiments. The imperfect interfaces between the fiber and the matrix are taken into account by introducing some cohesive contact surfaces. The influences of the interface on the elastic constants and the tensile strengths are examined through these interface models. Full article

(This article belongs to the Special Issue Advances in Anisotropic and Smart Materials)

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Open AccessReview

RNA Recognition and Stress Granule Formation by TIA Proteins

by Saboora Waris, Matthew Charles James Wilce and Jacqueline Anne Wilce

Int. J. Mol. Sci. 2014, 15(12), 23377-23388; https://doi.org/10.3390/ijms151223377 - 16 Dec 2014

Cited by 58 | Viewed by 11898

Abstract

Stress granule (SG) formation is a primary mechanism through which gene expression is rapidly modulated when the eukaryotic cell undergoes cellular stresses (including heat, oxidative, viral infection, starvation). In particular, the sequestration of specifically targeted translationally stalled mRNAs into SGs limits the expression [...] Read more.

Stress granule (SG) formation is a primary mechanism through which gene expression is rapidly modulated when the eukaryotic cell undergoes cellular stresses (including heat, oxidative, viral infection, starvation). In particular, the sequestration of specifically targeted translationally stalled mRNAs into SGs limits the expression of a subset of genes, but allows the expression of heatshock proteins that have a protective effect in the cell. The importance of SGs is seen in several disease states in which SG function is disrupted. Fundamental to SG formation are the T cell restricted intracellular antigen (TIA) proteins (TIA-1 and TIA-1 related protein (TIAR)), that both directly bind to target RNA and self-associate to seed the formation of SGs. Here a summary is provided of the current understanding of the way in which TIA proteins target specific mRNA, and how TIA self-association is triggered under conditions of cellular stress. Full article

(This article belongs to the Special Issue Post-Transcriptional Gene Regulation by Ribonucleoprotein Complexes)

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Schematic of the domain structure of T cell restricted intracellular antigen (TIA) protein isoforms. TIA proteins consist of three RNA recognition motifs (RRMs) that provide RNA/DNA binding specificity and a Q-rich prion-related domain (PRD) domain, involved in stress granule (SG) formation. The two isoforms of both proteins that form as a result of alternative splicing are indicated. Full article ">Figure 2
Schematic of SG formation formed via the self-association of TIA proteins (shown in brown with circles representing RRM domains, and a tail representing the C-terminal PRD) bound to stalled ribonucleoprotein-mRNA complexes. Upon phosphorylation of eIF2 under conditions of stress, the pre-initiation complex is stalled. Accumulation of the stalled mRNA complex results in its incorporation into SG (involving interactions between the TIA protein PRDs) until such time that heat shock proteins are able to reverse the process and translation is resumed. Full article ">Figure 3
Cartoon representation of TIA protein RNA-recognition motif (RRM) structures solved using NMR spectroscopy. RRM 1 from TIAR (PDB entry:2CQI) and RRMs 2 and 3 from TIA-1 (PDB entry:2MJN) are shown. Amino acid residues of RRMs 2 and 3 shown to be perturbed upon binding by ssRNA are highlighted in ruby showing that binding mainly occurs across the surface the β-sheet, but also involves residues outside the canonical RRM fold [<a href="#B42-ijms-15-23377" class="html-bibr">42</a>,<a href="#B44-ijms-15-23377" class="html-bibr">44</a>,<a href="#B46-ijms-15-23377" class="html-bibr">46</a>,<a href="#B49-ijms-15-23377" class="html-bibr">49</a>]. No evidence for specific binding of RNA by RRM1 has been observed. Full article ">

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Open AccessArticle

Evaluation of Posterolateral Lumbar Fusion in Sheep Using Mineral Scaffolds Seeded with Cultured Bone Marrow Cells

by María D. Cuenca-López, José A. Andrades, Santiago Gómez, Plácido Zamora-Navas, Enrique Guerado, Nuria Rubio, Jerónimo Blanco and José Becerra

Int. J. Mol. Sci. 2014, 15(12), 23359-23376; https://doi.org/10.3390/ijms151223359 - 16 Dec 2014

Cited by 14 | Viewed by 6965

Abstract

The objective of this study is to investigate the efficacy of hybrid constructs in comparison to bone grafts (autograft and allograft) for posterolateral lumbar fusion (PLF) in sheep, instrumented with transpedicular screws and bars. Hybrid constructs using cultured bone marrow (BM) mesenchymal stem [...] Read more.

The objective of this study is to investigate the efficacy of hybrid constructs in comparison to bone grafts (autograft and allograft) for posterolateral lumbar fusion (PLF) in sheep, instrumented with transpedicular screws and bars. Hybrid constructs using cultured bone marrow (BM) mesenchymal stem cells (MSCs) have shown promising results in several bone healing models. In particular, hybrid constructs made by calcium phosphate-enriched cells have had similar fusion rates to bone autografts in posterolateral lumbar fusion in sheep. In our study, four experimental spinal fusions in two animal groups were compared in sheep: autograft and allograft (reference group), hydroxyapatite scaffold, and hydroxyapatite scaffold seeded with cultured and osteoinduced bone marrow MSCs (hybrid construct). During the last three days of culture, dexamethasone (dex) and beta-glycerophosphate (β-GP) were added to potentiate osteoinduction. The two experimental situations of each group were tested in the same spinal segment (L4–L5). Spinal fusion and bone formation were studied by clinical observation, X-ray, computed tomography (CT), histology, and histomorphometry. Lumbar fusion rates assessed by CT scan and histology were higher for autograft and allograft (70%) than for mineral scaffold alone (22%) and hybrid constructs (35%). The quantity of new bone formation was also higher for the reference group, quite similar in both (autograft and allograft). Although the hybrid scaffold group had a better fusion rate than the non-hybrid scaffold group, the histological analysis revealed no significant differences between them in terms of quantity of bone formation. The histology results suggested that mineral scaffolds were partly resorbed in an early phase, and included in callus tissues. Far from the callus area the hydroxyapatite alone did not generate bone around it, but the hybrid scaffold did. In nude mice, labeled cells were induced to differentiate in vivo and monitored by bioluminescence imaging (BLI). Although the cultured MSCs had osteogenic potential, their contribution to spinal fusion when seeded in mineral scaffolds, in the conditions disclosed here, remains uncertain probably due to callus interference with the scaffolds. At present, bone autografts are better than hybrid constructs for posterolateral lumbar fusion, but we should continue to seek better conditions for efficient tissue engineering. Full article

(This article belongs to the Section Biochemistry)

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Full article ">Figure 1
In vivo ectopic bone formation assay. (a) General view of the shape, size, and porosity of a single mineral scaffold (Pro Osteon 500) used as a grafting material alone and with added cells. Bar = 2 mm; (b) Scanning electron microscopy (SEM) image showing bone marrow-cultured cells attached to a mineral scaffold covered with fibronectin ready for transplantation. Bar = 20 µm; (c) Histology of a scaffold seeded with MSCs transplanted on dorsal surface of a nude mouse and recovered four weeks later. Scaffold (★) is outlined by a bone tissue layer (BT) and fibroadipose tissue (F-AT). Sirius red staining. Bar = 200 µm; (d) Bone tissue examined with polarization microscopy shows a woven-fibered matrix. Bar = 50 µm. Full article ">Figure 2
Noninvasive bioluminescence imaging (BLI) of sheep MSCs differentiation in subcutaneous implanted HA scaffolds, implanted in the back of severely immunodeficient mice and seeded with sheep MSCs previously transduced with the CMV:RLuc:mRFP reporter (a constitutively expressed reporter of cell number) and a cell differentiation reporter (OC:PLuc:eGFP), and imaged at (a) day 0; (b) 30 days; and (c) 60 days. The diagram illustrates the HA scaffold implantation sites (grey square) with seeded cells; (d) Production of new bone tissue by labeled sheep MSCs, having made possible the fusion of the two arthrodesed lumbar vertebrae (L4–L5). Picrosirius-red stain. Bar = 150 µm. Full article ">Figure 3
Computed tomography (CT) diagnosis of instrumented lumbar fusion. (a) Control X-ray radiography taken after surgery, showing screws and rods used to fix L4–L5 vertebral bodies. [L], left side; [R], right side; (b) CT-study in bone graft groups showing 3D-CT reconstruction, axial-CT, a coronal-CT showing bone formation in both sides, and correlated axial-CT & histological section. [L] left side, Allo-group; [R] right side, Auto-group; (c) CT-study in mineral scaffold groups showing 3D-CT reconstruction, some axial-CT, a coronal-CT, and correlated axial-CT and histological section, assessing fusion only in right side. [L] left side, HA group; [R] right side, HA + MSCs group. Full article ">Figure 4
Histology of the external callus found in experimental groups. In all cases, calluses were in continuity with the vertebral cortex and were composed of immature bone (IB) and mature bone (MB), which included bone grafts (★) and scaffolds (★). Howship’s lacunae (HL) in the calluses reveal that bone remodeling was active after six months. (a) Bone grafting groups. [L] left side, Allo-group; [R] right side, Auto-group; (b) Mineral scaffolding groups. [L] left side, HA group; [R] right side, HA + MSCs group. Toluidine blue/pyronin G staining. Bar = 100 µm. Full article ">Figure 5
Polarization microscopy of mineral scaffolds. (a) Mineral scaffold (Pro Osteon 500) before transplantation; (b) Mineral scaffold inside callus in left side (HA group) is partially eroded and completely osteointegrated (BT); (c) Mineral scaffold inside callus in right side (HA + MSCs group) is also partially eroded and fully osteointegrated (BT) as bone tissue; (d) Mineral scaffold outside callus in left side (HA group) is covered by fibrous tissue (FT); (e) Mineral scaffold outside callus in right side (HA + MSCs group) is covered by bone tissue (BT) formed by added cells. Bar = 20 µm. Full article ">Figure 6
Histology of mineral scaffold surfaces with deposited bone tissue. (a,b) On the left side (the HA group (a)), and on the right side (HA + MSCs group (b)), mineral scaffolds found inside the callus have an eroded contour (black arrows) that indicates previous osteoclastic resorption; (c) On the contrary, the mineral scaffold outside the callus on the right side (HA + MSCs group) does not; (a–c) von Kossa staining. Bar = 50 µm; (d,e) Inside callus, on left side; In the HA group (d), as well as on the right side (HA + MSCs group (e)), mineral scaffolds show a peripheral cement line (white arrows) that has been deposited before bone formation; (f) On the contrary, a cement line is not observed on the mineral scaffold surface when mineral scaffolds are found outside the callus on the right side (HA + MSCs group); in this case, bone tissue was formed by adding cells directly onto the mineral scaffold surface. Toluidine blue/pyronin G staining. Bar = 50 µm. Full article ">Figure 7
Bone histomorphometry. Examples of digitized images of cross-sectioned vertebrae from Auto- [R], and Allo- [L] groups (a); and HA + MSCs [R], and HA [L] groups (b), processed to measure new bone tissue area (BTA, in mm2). Original pixel size was 5.2 µm. Insets show correlated axial CT scans; in (c) a graphic representation of those measures is shown. Full article ">

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Int. J. Mol. Sci., Volume 15, Issue 12 (December 2014) – 135 articles , Pages 21603-23998

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