An antibody raised against a pathogenic serpin variant induces mutant-like behaviour in the wild-type protein.

A monoclonal antibody (mAb) that binds to a transient intermediate may act as a catalyst for the corresponding reaction; here we show this principle can extend on a macro-molecular scale to the induction of mutant-like oligomerisation in a wild-type protein. Using the common, pathogenic Glu342Lys (Z) variant of α1-antitrypsin as antigen - whose native state is susceptible to the formation of a proto-oligomeric intermediate - we have produced a mAb (5E3) that increases the rate of oligomerisation of the wild-type (M) variant. Employing ELISA, gel shift, thermal stability and FRET time-course experiments, we show that mAb5E3 does not bind to the native state of α1-antitrypsin, but recognises a cryptic epitope in the vicinity of the post-helix A loop and strand 4C that is revealed upon transition to the polymerisation intermediate, and which persists in the ensuing oligomer. This epitope is not shared by loop-inserted monomeric conformations. We show the increased amenity to polymerisation by either the pathogenic Glu342Lys mutation or the binding of mAb5E3 occurs without affecting energetic barrier to polymerisation. As mAb5E3 also does not alter the relative stability of the monomer to intermediate, it acts in a manner similar to the Glu342Lys mutation, by facilitating the conformational interchange between these two states

Enhancing Coronal Adaptation of Root Canal Fillings with a Modified Single-Cone Obturation Technique: Two Case Reports

The single-cone technique, also known as the hydraulic condensation technique, is widely employed in endodontics. However, the aforementioned method is presented with certain limitations; specifically concerning the coronal seal and the adaptation of the coronal third of a master gutta-percha (GP) with a round cross-section to the coronal dentinal walls of root canals with semi-round or oval cross-sections. Through two case reports, the current article introduces the coronal vertical condensation (CVC) technique; aiming to enhance GP adaptation to canal walls in similar scenarios. In fact, the coronal vertical condensation technique amalgamates the different aspects of warm vertical condensation and single-cone techniques. In CVC, following the placement of the master GP cone, an electrical heat carrier is inserted immediately a few millimeters apical from the canal orifice to remove the coronal portion of the master GP cone. Subsequently, a hand plugger is used to condense GP in the vertical dimension, and the coronal space is backfilled using melted GP. The implementation of CVC technique has demonstrated an improved coronal adaptation of GP with canal walls. The stated technique seems beneficial; especially in the obturation of severely curved canals or root canals with a final preparation shape of variable taper

An antibody raised against a pathogenic serpin variant induces mutant-like behaviour in the wild-type protein

A monoclonal antibody (mAb) that binds to a transient intermediate may act as a catalyst for the corresponding reaction; here we show this principle can extend on a macro molecular scale to the induction of mutant-like oligomerization in a wild-type protein. Using the common pathogenic E342K (Z) variant of α1-antitrypsin as antigen-whose native state is susceptible to the formation of a proto-oligomeric intermediate-we have produced a mAb (5E3) that increases the rate of oligomerization of the wild-type (M) variant. Employing ELISA, gel shift, thermal stability and FRET time-course experiments, we show that mAb5E3 does not bind to the native state of α1-antitrypsin, but recognizes a cryptic epitope in the vicinity of the post-helix A loop and strand 4C that is revealed upon transition to the polymerization intermediate, and which persists in the ensuing oligomer. This epitope is not shared by loop-inserted monomeric conformations. We show the increased amenity to polymerization by either the pathogenic E342K mutation or the binding of mAb5E3 occurs without affecting the energetic barrier to polymerization. As mAb5E3 also does not alter the relative stability of the monomer to intermediate, it acts in a manner similar to the E342K mutant, by facilitating the conformational interchange between these two states

Protection of hippocampal CA1 neurons against ischemia/Reperfusion injury by exercise preconditioning via modulation of Bax/Bcl-2 ratio and prevention of Caspase-3 Activation

Introduction: Ischemia leads to loss of neurons by apoptosis in specific brain regions, especially in the hippocampus. The purpose of this study was investigating the effects of exercise preconditioning on expression of Bax, Bcl-2, and caspase-3 proteins in hippocampal CA1 neurons after induction of cerebral ischemia. Methods: Male rats weighing 260-300 g were randomly allocated into three groups (sham, exercise, and ischemia). The rats in exercise group were trained to run on atreadmill 5 days a week for 4 weeks. Ischemia was induced by the occlusion of both common carotid arteries (CCAs) for 20 min. Levels of expression of Bax, Bcl-2, and caspase-3 proteins in CA1 area of hippocampus were determined by immunohistochemical staining . Results: The number of active caspase-3-positive neurons in CA1 area were significantly increased in ischemia group, compared to sham-operated group (P<0.001), and exercise preconditioning significantly reduced the ischemia/reperfusion-induced caspase-3 activation, compared to the ischemia group (P<0.05). Also, results indicated a significant increase in Bax/Bcl-2 ratio in ischemia group, compared to sham-operated group (P<0.001). Discussion: This study indicated that exercise has a neuroprotective effects against cerebral ischemia when used as preconditioning stimuli

The Effect of Tricine Compound Extracted from Allium atroviolaceum Boiss. On growth and apoptosis in PC3 cell line

BACKGROUND AND OBJECTIVE: Tricine, as a flavonoid compound in many food sources, has anti-inflammatory and anti-proliferative effects in some cancer cell lines. Considering the importance of using natural anti-cancer drugs in therapy-resistant cancers such as prostate cancer, the aim of this study is to investigate the effect of tricine on cell growth and proliferation and induction of apoptosis in PC3 human prostate cancer cell line. METHODS: In this experimental study, the PC3 cell line was prepared and cultured from the Pasteur Institute of Iran. Extraction and purification of tricine were done by column chromatography and recrystallization of Allium atroviolaceum extract. The apoptotic effect of tricine at concentrations of 60, 80, 100, 120 and 140 μM was evaluated by MTT method. The apoptotic effect was evaluated in the cell group treated with IC50 concentration of tricine and untreated cells (control group) using Annexin-V kit and flow cytometry. FINDINGS: The viability of cells at different tricine concentrations were 85.66±1.52, 76±3.60, 66.33±4.16, 44±3.60, and 36.66±3.21, respectively (p<0.01). The IC50 concentration of tricine was 117.5±4.4 μM for PC3 cell line. The apoptosis rate in PC3 cells after 48 hours of treatment with IC50 concentration of tricine was 24.3±0.58%, which was not significant in comparison with control cells (23.3±0.58%). CONCLUSION: The results of this study showed that tricine resulted in cell death in the PC3 cell line, but the cell death mechanism was not apoptotic

Effect of Isolated Tricin from Arial Part of Allium atrovoilaceum Boiss. on Proliferation and Apoptosis in PC3 Cell Line

BACKGROUND AND OBJECTIVE: Tricin as a flavone compound, in many food sources, has anti-inflammatory and anti-proliferative effects in several cancer cell lines. Considering the importance of using natural anti-cancer drugs in treatment-resistant cancers such as prostate cancer, the aim of this study was to investigate the effect of tricin on cell growth and proliferation and induction of apoptosis in human PC3 prostate cancer cell line. METHODS: In this experimental study, the PC3 cell line was prepared from the Pasteur Institute of Iran and cultivated. Extraction and purification of Tricin were performed using column chromatography and re-crystallization of Alliuum atrovoilaceum extract. The cytotoxic effect of Tricin in concentrations of 60,80,100,120, and 140 μM was evaluated by MTT method. Apoptotic effect was evaluated in the treated cell group with the IC50 concentration of Tricin and untreated cells (control group), using an Annexin-V kit and flow cytometry. FINDINGS: The viability of cells at different tricin concentrations were 85.66±1.52, 76±3.60, 66.33±4.16, 44±3.60, and 36.66±3.21, respectively (p<0.01). The IC50 concentration of tricin was 117.5±4.4 μM for PC3 cell line. The apoptosis rate in PC3 cells after 48 hours of treatment with IC50 concentration of tricin was 24.3±0.58%, which was not significant in comparison with control cells (23.3±0.58%). CONCLUSION: The results of this study showed that Tricin resulted in cell death in the PC3 cell line, but the cell death mechanism was not apoptosis

Temporal visual resolution and disease severity in MS

OBJECTIVE: To examine temporal visual resolution assessed as critical flicker frequency (CFF) in patients with MS and to investigate associations with visual system damage and general disability and cognitive function. METHODS: Thirty-nine patients with MS and 31 healthy controls (HCs) were enrolled in this cross-sectional study and underwent CFF testing, high- and low-contrast visual acuity, alertness and information processing speed using the paced auditory serial addition task (PASAT), and retinal optical coherence tomography (OCT). In patients with MS, visual evoked potentials (VEPs) and Expanded Disability Status Scale (EDSS) scores were assessed. RESULTS: CFF in patients with MS (mean ± SD: 40.9 ± 4.4 Hz) was lower than in HCs (44.8 ± 4.4 Hz, p < 0.001). There was no significant CFF difference between eyes with and without previous optic neuritis (ON). CFF was not associated with visual acuity, VEP latency, the peripapillary retinal nerve fiber layer thickness, and the combined ganglion cell and inner plexiform layer volume. Instead, reduced CFF was associated with worse EDSS scores (r(2) = 0.26, p < 0.001) and alertness (r(2) = 0.42, p = 0.00042) but not with PASAT (p = 0.33). CONCLUSION: CFF reduction in MS occurs independently of ON and structural visual system damage. Its association with the EDSS score and alertness suggests that CFF reflects global disease processes and higher cortical processing rather than focal optic nerve or retinal damage

An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behaviour.

Serpins are important regulators of proteolytic pathways with an anti-protease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an inter-molecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of a number of pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39, and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A - on the opposite face of the molecule - more liable to adopt an 'open' state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of pre-formed serpin-enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the 'open' state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for rational design of ligands that is able to dynamically influence α1-AT polymerisation