Design and evaluation of novel fluorescent molecular probes targeting cathepsin B

Lysosomal cysteine proteinase cathepsin B (CTB) is a member of the cysteine protease family known to participate in intracellular degradation processes and protein turnover in the lysosomes of healthy cells. Cathepsin B plays a crucial role in tumor invasion and progression by controlling extracellular degradation and participating in a proteolytic cascade activation (Gondi and Rao 2013). Its role in tumor invasion and progression makes CTB a promising biomarker and target for antibody-directed prodrug therapy (Dheer, Nicolas et al. 2019). The development of novel CTB-specific molecular probes opens new possibilities for image-based diagnostic methods for different types of cancers (Podgorski and Sloane 2003, Tan, Peng et al. 2013). Since aberrant expression of this protein has been an indicator of cancer development, detecting CTB expression and activity might be beneficial for the early detection of cancer or revealing aggressive lesions (Gondi and Rao 2013). Developing probes capable of binding with CTB is challenging due to binding site homology to other members of the cysteine cathepsin family (Turk, Stoka et al. 2012). In this study, to identify unique residues in human CTB compare to other members of the cathepsin family, amino acid sequences of these proteins were exposed to. multiple sequence alignments. Cathepsin B in humans has three active site residues critical for catalysis: Cys108; His278, and Asp298 (Ruan, Hao et al. 2015) were confirmed with multiple sequence alignment as fully conserved residues. The initial step in the development of a detection assay for CTB is finding appropriate fluorescent small molecules for enzyme binding. In this study, two ligand candidates CID8795 and CID535684 were identified and successfully conjugated to the dye ATTO680 and were tested for binding affinity and specificity to CTB. For CID535684ATTO680, no binding interaction was observed in the fluorescence polarization (FP) assay. CID8795ATTO680 demonstrated increases in fluorescence polarization assays in the presence of CTB with the half-maximal effective concentration (EC50) at 3.27 ± 1.27 nM. A third probe, Benzyloxycarbonyl (Cbz)-Lys-Lys-p-Aminobenzyl alcohol (PABA)-2’, 7’-dichloro-6’-methoxy-fluorescein (DCMF), was designed based on a known substrate scaffold for CTB. This novel substrate-based fluorescent probe was shown to be hydrolyzed by CTB having a specificity constant kcat/KM = 41.9 ± 0.07 mM-1 × s-1 . Finally, we investigated single nucleotide polymorphisms (SNPs) within the coding region of the CTSB gene within the general population (random data from 2,504 samples) included in the 1,000 Genomes project. The mapping of SNPs onto the 3D structure of cathepsin B indicates that the active site of CTB is fully conserved among humans – as no SNPs were identified within the binding pocket of CTB. According to these results, probes that bind to the enzyme’s active site should be generally useful for detecting CTB in all populations studied in the 1,000 Genomes project

Relationship between bridging and dimensions of sella turcica with classification of craniofacial skeleton

Purpose: In orthodontics, it is essential to determine the craniofacial skeleton pattern (class I, II, III) for planning treatment. Sella turcica bridging that is seen on lateral cephalometric radiographs is considered as a normal finding. This study aimed to compare sella turcica bridging and its dimensions in patients with various craniofacial patterns. Material and methods: A total of 105 lateral cephalometric radiographs (53 men and 52 women), aged 14-26 years, were randomly and equally assigned to three groups of class I, II, and III, respectively. The length, diameter, and depth of the sella turcica as well as sella turcica bridging were determined on radiographs. The chi-squared test was used for assessing the relationship between sella turcica bridging and craniofacial skeleton classification. ANOVA was used for assessing the relationship between the dimensions of the sella turcica and craniofacial skeleton classification. The Pearson's correlation coefficient was used for assessing the relationship between age and the dimensions of the sella turcica. Results: The sella turcica had a normal shape in 64.76% of patients, whereas 35.33% of patients had sella turcica bridging. In total, 11.42% of patients belonged to class I, 34.28% to class II, and 66.62% to class III. The diameter of the sella turcica had a significant relationship with age; the diameter of the sella turcica increased with age (p < 0.001). Conclusions: There is a significant relationship between craniofacial skeleton patterns and sella turcica bridging, i.e., the incidence of sella turcica bridging is higher in class III patients. The sella turcica had a greater diameter in older patients

Shear Bond Strength of the Metal Bracket to Zirconium Ceramic Restoration Treated by the Nd: YAG Laser and Other Methods: An In Vitro Microscopic Study

Introduction: Providing reliable bonding of the bracket base and the zirconia surface is required to apply orthodontic force. The purpose of this scientific experiment was to evaluate the efficacy of three different methods of surface preparation for Zirconia, including surface roughening, sandblasting and the Nd: YAG laser, in the shear bond strength (SBS) of the orthodontic brackets.Methods: Fifty-four discs of zirconia were divided into three groups of 18: A) Hydrofluoric acid etching, B) sandblasting, and C) Nd: irradiation using the power of 1.5 W for 10 seconds. After bonding the brackets, the samples were slowly thermo-cycled (1000 times) for 24 hours. The SBS test was performed by a universal testing machine at a head speed of 0.5 mm/min. The adhesive remnant index (ARI) was scored at a magnification of 10 in the stereo microscope. All data were collected and analyzed using the variance, Kruskal-Wallis, Tukey, Don, and Weibull tests (α = 0.05).Results: The HF acid etching group (6.11± 0.94 MPa) had the highest SBS, which was followed by the laser group (6 ± 0.61 MPa) and the sandblast group (3.1080 ± 0.82 MPa). There was a significant statistical difference between the laser and HF groups and the sandblast group (P &lt; 0.05) and no significant difference between the HF group and the laser group (P = 0.03).Conclusion: Based on the obtained bond strength, the Nd: YAG laser with a power of 1.5 W could be a substitute treatment method for the HF acid-etching

Clinical feature and Genetics in Rett syndrome; A report on Iranian patients

Background: Rett Syndrome is characterized by normal development for the first 6–18 months of life followed by the loss of fine and gross motor skills and the ability to engage in social interaction. Mutations methyl CpG-binding protein 2 gene (MECP2) have been found in the majority of patients. This study was performed to investigate the relation of Rett clinical diagnosis and its relation with mutations in MECP2. Materials and Methods: children suspected to Rett syndrome were invited to take part in this study. Those who had met classic Rett syndrome diagnostic criteria were enrolled. Severity of symptoms was assessed for all patients. Of peripheral blood samples collected in EDTA tubes, the genomic DNA was extracted by standard salting out method. MEPC2 gene mutation was studied by DNA sequencing method. Results and conclusion: 23 patients accepted to participate in the study. 11(47.8%) patients had MECP2 gene mutation meanwhile 12 ones (52.2%) had no mutation. change in genetics was in association with phenotypical manifestations. The most prevalent mutation was p.v288 which is mostly in association with partially or uncontrolled seizures. This was the first time that Rett syndrome patients were studied in both clinical manifestations and genetic changes in Iran

Comparison of shear bond strength of rebonded brackets prepared with different methods of resin removal: an invitro study

Aim: Clinicians and researchers have always made every effort to achieve proper bonding between the surface of the tooth and the rebonded orthodontic brackets in order to prevent the re-fracture failure of orthodontic pressures throughout treatment. The aim of this study was to determine the bond strength of rebonded brackets by four adhesive removal methods.Materials and methods: Seventy-five orthodontic brackets were bonded to the extracted first premolar teeth and then debonded. Fifteen of these teeth were rebonded to new brackets after adhesive removal and were named as the control group. Sixty debonded brackets were divided into four groups by means of resin-removal: laser, burr, sandblast, and direct flame. These recycled brackets were re-bonded to the teeth with the same basic bonding methods. The shear bond strength (SBS) was measured at the speed of 0.5&amp;nbsp;mm/min and the data were analyzed using ANOVA and post hoc tests (&amp;alpha;=0.05).Results: SBS was significantly different in all four groups compared to the control group (p&amp;lt;0.05). The burr and sandblast groups had the lowest levels of SBS. Although the laser and flame groups had the highest levels of SBS, they did not show a significant difference (p=0.99).Conclusions: 5W Er:YAG irradiation and direct flame due to relatively good bond strength can be recommended as proposed methods for orthodontic brackets recycling

Four novel ARSA gene mutations with pathogenic impacts on metachromatic leukodystrophy: a bioinformatics approach to predict pathogenic mutations

Metachromatic leukodystrophy (MLD) disorder is a rare lysosomal storage disorder that leads to severe neurological symptoms and an early death. MLD occurs due to the deficiency of enzyme arylsulfatase A (ARSA) in leukocytes, and patients with MLD excrete sulfatide in their urine. In this study, the ARSA gene in 12 non-consanguineous MLD patients and 40 healthy individuals was examined using polymerase chain reaction sequencing. Furthermore, the structural and functional effects of new mutations on ARSA were analyzed using SIFT (sorting intolerant from tolerant), I-Mutant 2, and PolyPhen bioinformatics software. Here, 4 new pathogenic homozygous mutations c.585G>T, c.661T>A, c.849C>G, and c.911A>G were detected. The consequence of this study has extended the genotypic spectrum of MLD patients, paving way to a more effective method for carrier detection and genetic counseling

Stability of changes in mandibular intermolar and intercuspid distances following orthodontic treatment

Background: The aim of this study was to determine the pattern and amount of change exhibited in mandibular intercanine and intermolar width during treatment and assessing its stability1-3 years post-retention. Materials and Methods: The material consisted of 70 cases of which 20 cases were treated without extraction and 30 cases were treated with extraction, which were compared with 20 untreated cases which served as a control group. A series of three measurements were made for each case of the treated group: At the beginning of treatment, end of active treatment and 1-3 years post-retention; and for the control group: At 12, 15 and 18 years of age. The Wilcoxon signed ranks test was used to evaluate treatment changes in each group. The Kruskal-Wallis H test was used to compare the treatment changes between the 3 groups (α = 0.05). SPSS 16 software (SPSS Inc., Chicago, IL, USA) was used to evaluate the data. Results: Mean changes of intercanine width for three groups was –0.5 mm for control group, –0.26 mm for non-extraction group and +0.18 mm for extraction group. Intermolar width of the extraction group decreased significantly during treatment. In contrast to the extraction group, the control and non-extraction groups both demonstrated an increase in mean intermolar width which was 0.66 mm and 0.91 mm respectively. Conclusion: It was concluded that although mean changes of intercanine and intermolar width were statistically significant but they were not perceptible clinically

Investigation of the Mitochondrial ATPase 6/8 and tRNALys Genes Mutations in Autism

Objective: Autism results from developmental factors that affect many or all functional brain systems. Brain is one of tissues which are crucially in need of adenosine triphosphate (ATP). Autism is noticeably affected by mitochondrial dysfunction which impairs energy metabolism. Considering mutations within ATPase 6, ATPase 8 and tRNALys genes, associated with different neural diseases, and the main role of ATPase 6/8 in energy generation, we decided to investigate mutations on these mtDNA-encoded genes to reveal their roles in autism pathogenesis.Materials and Methods: In this experimental study, mutation analysis for the mentioned genes were performed in a cohort of 24 unrelated patients with idiopathic autism by employing amplicon sequencing of mtDNA fragments.Results: In this study, 12 patients (50%) showed point mutations that represent a significant correlation between autism and mtDNA variations. Most of the identified substitutions (55.55%) were observed on MT-ATP6, altering some conserved amino acids to other ones which could potentially affect ATPase 6 function. Mutations causing amino acid replacement denote involvement of mtDNA genes, especially ATPase 6 in autism pathogenesis.Conclusion: MtDNA mutations in relation with autism could be remarkable to realize an understandable mechanism of pathogenesis in order to achieve therapeutic solutions

Effects of two erbium-doped yttrium aluminum garnet lasers and conventional treatments as composite surface abrasives on the shear bond strength of metal brackets bonded to composite resins

Background: Bonding brackets to dental surfaces restored with composites are increasing. No studies to date have assessed the efficacy of laser irradiation in roughening of composite and the resulted shear bond strength (SBS) of the bonded bracket. We assessed, for the 1 st time, the efficacy of two laser beams compared with conventional methods. Materials and Methods: Sixty-five discs of light-cured composite resin were stored in deionized distilled water for 7 days. They were divided into five groups of 12 plus a group of five for scanning electron microscopy (SEM): Bur-abrasion followed by phosphoric acid etching (bur-PA), hydrofluoric acid conditioning (HF), sandblasting, 3 W and 2 W erbium-doped yttrium aluminum garnet laser irradiation for 12 s. After bracket bonding, specimens were water-stored (24 h) and thermocycled (500 cycles), respectively. SBS was tested at 0.5 mm/min crosshead speed. The adhesive remnant index (ARI) was scored under ×10 magnification. SEM was carried out as well. Data were analyzed using analysis of variance (ANOVA), Kruskal-Wallis, Tukey, Dunn, one-sample t-test/Wilcoxon tests, and Weibull analysis (α =0.05). Results: The SBS values (megapascal) were bur-PA (11.07 ± 1.95), HF (19.70 ± 1.91), sandblasting (7.75 ± 1.10), laser 2 W (15.38 ± 1.38), and laser 3 W (20.74 ± 1.73) (compared to SBS = 6, all P = 0.000). These differed significantly (ANOVA P = 0.000) except HF versus 3 W laser (Tukey P > 0.05). ARI scores differed significantly (Kruskal-Wallis P = 0.000), with sandblasting and 2 W lasers having scores inclined to the higher end (safest debonding). Weibull analysis implied successful clinical outcome for all groups, except for sandblasting with borderline results. Conclusion: Considering its high efficacy and the lack of adverse effects bound with other methods, the 3 W laser irradiation is recommended for clinical usage

The effect of local injection of the human growth hormone on the mandibular condyle growth in rabbit

Background: The aim of this study was to evaluate the effect of local injection of human growth hormone (GH) in stimulating cartilage and bone formation in a rabbit model of temporomandibular joint (TMJ). Materials and Methods: In an experimental animal study, 16 male Albino New Zealand white rabbits aged 12 weeks were divided into two groups: In the first group (7 rabbits) 2 mg/kg/1 ml human GH and in the control group (9 rabbits) 1 ml normal saline was administered locally in both mandibular condyles. Injections were employed under sedation and by single experienced person. Injections were made for 6 times with 3 injections a week in the all test and control samples. Rabbits were sacrified at the 20th day from the beginning of study and TMJs were histologically examined. ANOVA (two-sided) with Dunnett post hoc test was used to compare data of bone and cartridge thickness while chi-square test was used to analyze hyperplasia and disk deformity data. P < 0.05 was considered as significant. Results: Cartilage layer thickness was greater in the GH-treated (0.413 ± 0.132) than the control group (0.287 ± 0.098) (P value = 0.02). Although bone thickness and condylar cartilage hyperplasia were greater in the GH-treated group, these differences were not statistically significant (P value = 0.189 and 0.083, respectively). There was no statistically significant difference between two groups regarding the disc deformity (P value = 0.46). Conclusion: Local injection of human GH in the TMJ is able to accelerate growth activity of condylar cartilage in rabbit