An in silico analysis of the impact of POLE mutations on cladribine docking.

OBJECTIVE: Polymerase ε exonuclease (POLE) is an enzyme involved in DNA replication and may be an attractive therapeutic target in various cancers. Here we sought to model the impact of specific POLE mutations on protein function. Due to the lack of a crystal structure, the tertiary structures of the wild type and four common mutants were modeled using I-Tasser server. MATERIALS AND METHODS: Molecular docking and dynamic simulation studies were performed, and the structure and function of the mutants analyzed through residue conservation analysis and protein folding energy changes. RESULTS: All mutants of POLE gene had favorable binding affinities compared with their wild type of counterpart. The P286R variant, but not the other variants, disrupted cladribine binding to the protein. Similarly, dynamics studies revealed instability of the P286R mutant, while V411L, L424V, and L424F appeared to favor cladribine binding. CONCLUSIONS: Since P286R is a hotspot mutation in endometrioid carcinomas, patients with this variant may not respond to cladribine. Population-based pharmacogenomics studies will be required to validate our results.

Structural and magnetic properties of hard-soft BaFe12O19/(Zn0.5Co0.5)Fe2O4ferrites.

Hard-soft nanocomposites of (1 -x) BaFe12O19/x(Zn0.5Co0.5)Fe2O4, forx= 0.00, 0.25, 0.50, 0.75 and 1.00, were prepared via co-precipitation and high-speed ball milling techniques, respectively. The synthesized samples were characterized via x-ray diffraction, transmission electron microscope, Fourier transform infrared (FTIR), and vibrating sample magnetometer. XRD revealed the formation of hard-soft nanocomposites. TEM indicated that the two phases are well distributed and the particle size distribution is narrower for low content of soft phase, leading to better exchange coupling between the grains. Magnetic measurements were performed at 300 K and 77 K. The results showed a good single-phase magnetic behavior, verifying the good exchange coupling between hard and soft phases. For low (Zn0.5Co0.5)Fe2O4content, the dipolar interactions were dominated by the exchange-coupling interactions. Additionally, the optimum values of saturation and remanent magnetizations, coercivity, and squareness ratio were obtained forx= 0.5. This was attributed to the dominance of exchange-coupling interaction. The enhancement of magnetic properties and energy product (BH)maxfor nanocomposites at low temperature is skilled in the reduction of the thermal fluxes of magnetic moments at the surface. The maximum energy product (BH)maxwas observed in C2 at both temperatures with a smaller value than that of pure BaFe12O19.

A novel homozygous TPM1 mutation in familial pediatric hypertrophic cardiomyopathy and in silico screening of potential targeting drugs.

OBJECTIVE: Familial hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease. While sarcomeric gene mutations explain many HCM cases, the genetic basis of about half of HCM cases remains elusive. Here we aimed to identify the gene causing HCM in a non-consanguineous Saudi Arabian family with affected family members and a history of sudden death. The impact of the identified mutation on protein structure and potential drug targets were evaluated in silico. MATERIALS AND METHODS: Triplets (two HCM subjects and one patent ductus arteriosus (PDA) case) and unaffected parents were screened by targeted next-generation sequencing (NGS) for 181 candidate cardiomyopathy genes. In silico structural and functional analyses, including protein modeling, structure prediction, drug screening, drug binding, and dynamic simulations were performed to explore the potential pathogenicity of the variant and to identify candidate drugs. RESULTS: A homozygous missense mutation in exon 1 of TMP1 (assembly GRCh37-chr15: 63340781; G>A) was identified in the triplets [two HCM and one patent ductus arteriosus (PDA)] that substituted glycine for arginine at codon 3 (p.Gly3Arg). The parents were heterozygous for the variant. The mutation was predicted to cause a significant and deleterious change in the TPM1 protein structure that slightly affected drug binding, stability, and conformation. In addition, we identified several putative TPM1-targeting drugs through structure-based in silico screening. CONCLUSIONS: TPM1 mutations are a common cause of HCM and other congenital heart defects. To date, TPM1 has not been associated with isolated PDA; to our knowledge, this is the first report of the homozygous missense variation p.Gly3Arg in TPM1 associated with familial autosomal recessive pediatric HCM and PDA. The identified candidate TPM1 inhibitors warrant further prospective investigation.

Two-stage distalization of the mental foramen to manage posterior mandibular vertical bone deficiency-a prospective observational study.

Inferior alveolar nerve (IAN) transpositioning is a modality utilized to manage posterior mandibular vertical deficiency. Several complications have been reported including improper implant positioning, mandibular body fracture, and neurosensory disturbance. The aim of this prospective observational study was to introduce a two-stage mental foramen distalization technique to minimize the complications associated with IAN transpositioning. Ten patients with severely atrophied mandibular ridges were included. Cone beam computed tomography was ordered to accurately locate the position of the IAN and its incisive terminal branch before designing the outline of two cortical osteotomies anterior and posterior to the mental foramen. The osteotomies were created using a piezoelectric device, followed by separation and identification of the nerve. The incisive branch was severed to freely transpose the IAN. A new foramen was created far distally and the cortical windows were repositioned and fixed with two screws. Healing was uneventful for all patients. Neurosensory recovery was assessed by MRC scale. All cases showed full recovery within 6 weeks, except for two patients who showed complete recovery after 16 weeks. Four months postoperative, all patients showed complete consolidation of the cortical windows without any signs of failure implants were placed at the pre-planned surgical sites. Histomorphometric analysis of core biopsies from seven surgical sites showed bone area percentages ranging from 46% to 63%. The two-stage mental foramen distalization technique is a predictable and safer technique for IAN transpositioning specifically in cases of vertical bone deficiency associated with limited inter-arch space.

Next-generation sequencing identifies a homozygous mutation in ACADVL associated with pediatric familial dilated cardiomyopathy.

OBJECTIVE: Pediatric familial dilated cardiomyopathy (DCM) is a rare and severe heart disease. The genetics of familial DCM are complex and include over 100 known disease-causing genes, but many causative genes are unknown. We aimed to identify the causative gene for DCM in a consanguineous Saudi Arabian family with affected family members and a history of sudden death. PATIENTS AND METHODS: Affected (two children) and unaffected (one sibling and the mother) family members were screened by next-generation sequencing (NGS) for 181 candidate DCM genes and underwent metabolic screening. Fifty-seven clinically annotated controls and 46 DCM cases were then tested for the identified mutation. In silico structural and functional analyses including protein modeling, structure prediction and dynamic simulations were performed. RESULTS: A homozygous missense mutation in exon 15 of the acyl-CoA dehydrogenase very long chain gene (ACADVL; chr17:7127303; G>A) was identified in affected subjects that substituted histidine for arginine at codon 450 (p.R450H). The variant was heterozygous in the mother and unaffected sister. The mutation was absent in 57 clinically annotated controls and 48 pediatric DCM cases. The mutation was predicted to cause a significant and deleterious change in the ACADVL protein structure that affected drug binding, stability, and conformation. Metabolic screening confirmed VLCAD deficiency in affected individuals. CONCLUSIONS: The ACADVL R450H mutation is an uncommon cause of the DCM phenotype that appears to be autosomal recessive. Targeted NGS is useful for identifying the causative mutation(s) in familial DCM of unknown genetic cause.

The MTHFR C677T polymorphism is associated with mitral valve rheumatic heart disease.

OBJECTIVE: Rheumatic heart disease (RHD) is a serious complication of rheumatic fever (RF). Plasma homocysteine (Hcy) levels are increased in RHD patients. MTHFR catalyzes the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate and plays a vital role in Hcy metabolism. We hypothesize that the MTHFR C677T polymorphism is associated with a risk of RHD. PATIENTS AND METHODS: Eighty-six patients with RHD and 130 matched controls without a history of RHD were eligible for the study. The diagnosis of RHD was made according to modified Jones' criteria and echocardiography. Using echocardiography, RHD patients were further divided into mitral valve lesion (MVL) and combined valve lesion (CVL) groups. MTHFR C677T polymorphisms were genotyped by DNA sequencing. The chi-squared test was used to evaluate differences in genotypes. RESULTS: Control genotypes were in Hardy-Weinberg equilibrium. The C677T homozygous genotype (OR = 4.09; 95% CIs 1.16-14.44; p = 0.020) and recessive model (TT vs. CC+CT; OR = 4.05; 95% CIs 1.17-14.04; p = 0.019) were significantly associated with MVL RHD. CONCLUSIONS: This is the first study to investigate the association between the MTHFR C677T polymorphism and risk of RHD. The MTHFR C677T polymorphism is associated with RHD in patients with MVLs, perhaps via an Hcy-mediated cytokine effect.

Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates.

BACKGROUND: Whole genome sequencing has revolutionised the interrogation of mycobacterial genomes. Recent studies have reported conflicting findings on the genomic stability of Mycobacterium tuberculosis during the evolution of drug resistance. In an age where whole genome sequencing is increasingly relied upon for defining the structure of bacterial genomes, it is important to investigate the reliability of next generation sequencing to identify clonal variants present in a minor percentage of the population. This study aimed to define a reliable cut-off for identification of low frequency sequence variants and to subsequently investigate genetic heterogeneity and the evolution of drug resistance in M. tuberculosis. METHODS: Genomic DNA was isolated from single colonies from 14 rifampicin mono-resistant M. tuberculosis isolates, as well as the primary cultures and follow up MDR cultures from two of these patients. The whole genomes of the M. tuberculosis isolates were sequenced using either the Illumina MiSeq or Illumina HiSeq platforms. Sequences were analysed with an in-house pipeline. RESULTS: Using next-generation sequencing in combination with Sanger sequencing and statistical analysis we defined a read frequency cut-off of 30% to identify low frequency M. tuberculosis variants with high confidence. Using this cut-off we demonstrated a high rate of genetic diversity between single colonies isolated from one population, showing that by using the current sequencing technology, single colonies are not a true reflection of the genetic diversity within a whole population and vice versa. We further showed that numerous heterogeneous variants emerge and then disappear during the evolution of isoniazid resistance within individual patients. Our findings allowed us to formulate a model for the selective bottleneck which occurs during the course of infection, acting as a genomic purification event. CONCLUSIONS: Our study demonstrated true levels of genetic diversity within an M. tuberculosis population and showed that genetic diversity may be re-defined when a selective pressure, such as drug exposure, is imposed on M. tuberculosis populations during the course of infection. This suggests that the genome of M. tuberculosis is more dynamic than previously thought, suggesting preparedness to respond to a changing environment.

Clinical performance of ormocer, nanofilled, and nanoceramic resin composites in Class I and Class II restorations: a three-year evaluation.

PURPOSE: This prospective long-term clinical trial evaluated and compared the three-year clinical performance of an ormocer, a nanofilled, and a nanoceramic resin composite with that of a microhybrid composite placed in Class I and Class II cavities. METHODS: Forty patients, each with four Class I and II restorations under occlusion, were enrolled in this study. A total of 160 restorations were placed, 25% for each material, as follows: an ormocer-based composite, Admira; a nanofilled resin composite, Filtek Supreme XT; a nanoceramic resin composite, Ceram X; and a microhybrid resin composite, Tetric Ceram. A single operator placed all restorations according to the manufacturers' instructions. Immediately after placement the restorations were finished/polished. Clinical evaluation was performed at baseline and at yearly intervals after placement by two other independent examiners using modified US Public Health Service (USPHS) criteria. The changes in the USPHS parameters during the three-year period were analyzed with the Friedman test. Comparison of the baseline scores with those at the recall visits was made using the Wilcoxon signed rank test. The level of significance was set at p < 0.05. RESULTS: All materials showed only minor changes, and no differences were detected between their performance at baseline and after three years. Only two ormocer, one nanofilled, and one microhybrid restorations in molars failed because of loss of retention. Regarding the clinical performance, there were no statistically significant differences among the materials used (p>0.05). CONCLUSIONS: The ormocer, nanofilled, and nanoceramic composites provided acceptable clinical performance over a three-year period.

Activity size distribution of some natural radionuclides.

In this study, the results concerning the activity size distribution of the long-lived ((210)Pb) radon decay product aerosols and the thoron decay product aerosols ((212)Pb) and ((7)Be) of the outdoor atmosphere are presented. Also, the mass size distribution of the aerosol particles is determined. The low-pressure Berner cascade impactor Model 20/0.015 was used as a sampling device. The activity size distribution of these radionuclides was determined by one log-normal distribution (accumulation mode) whereas the mass size distribution was by two log-normal distributions (accumulation and coarse mode). The activity median aerodynamic diameter (AMAD) of (212)Pb was found to be 305 nm with a geometric standard deviation (σg) of 2.41. The specific air activity concentration of (212)Pb was found to be 0.14 ± 0.012 Bq m(-3). An AMAD of (210)Pb of 610 nm with σg of 1.8 was determined, whereas that of 550 nm with σg of 1.97 was determined for (7)Be. The specific air activity concentration of (210)Pb and (7)Be was found to be 0.0016±2.5×10(-4) and 0.00348 ± 4×10(-4) Bq m(-3), respectively. Using a dosimetric model, the total deposition fraction as well as the total equivalent dose has been evaluated considering the observed parameters of the activity size distribution of (212)Pb. At a total deposition fraction of ∼21 %, the total equivalent dose was found to be 0.41 µSv.

Radon decay products in realistic living rooms and their activity distributions in human respiratory system.

In this study, the individual activity concentrations of attached short-lived radon decay products ((218)Po, (214)Pb and (214)Po) in aerosol particles were measured in ten poorly ventilated realistic living rooms. Using standard methodologies, the samples were collected using a filter holder technique connected with alpha-spectrometric. The mean value of air activity concentration of these radionuclides was found to be 5.3±0.8, 4.5±0.5 and 3.9±0.4 Bq m(-3), respectively. Based on the physical properties of the attached decay products and physiological parameters of light work activity for an adult human male recommended by ICRP 66 and considering the parameters of activity size distribution (AMD = 0.25 µm and σ(g) = 2.5) given by NRC, the total and regional deposition fractions in each airway generation could be evaluated. Moreover, the total and regional equivalent doses in the human respiratory tract could be estimated. In addition, the surface activity distribution per generation is calculated for the bronchial region (BB) and the bronchiolar region (bb) of the respiratory system. The maximum values of these activities were found in the upper bronchial airway generations.

A polymorphism in the promoter region of the CD86 (B7.2) gene is associated with systemic sclerosis.

Systemic sclerosis (SSc) is a connective tissue disease of unknown aetiology characterized by fibrosis of the skin and internal organs, vascular abnormalities and humoral autoimmunity. Strong T-cell-dependent autoantibody and HLA associations are found in SSc subsets. The co-stimulatory molecule, CD86, expressed by antigen-presenting cells, plays a crucial role in priming naïve lymphocytes. We hypothesized that SSc, or one of the disease subsets, could be associated with single-nucleotide polymorphisms of the CD86 gene. Using sequence specific primer-polymerase chain reaction (SSP-PCR) methodology, we assessed four CD86 polymorphisms in 221 patients with SSc and 227 healthy control subjects from the UK. Haplotypes were constructed by inference and confirmed using PHASE algorithm. We found a strong association between SSc and a specific haplotype (haplotype 5), which was more prevalent in patients than in controls (29% vs 15%, OR = 2.3, chi(2) = 12, P = 0.0005). This association could be attributed to the novel -3479 promoter polymorphism; a significant difference was observed in the distribution of the CD86 -3479 G allele in patients with SSc compared to controls (43.7% vs. 32.4%, OR = 1.7, chi(2) = 12.1, P = 0.0005). TRANSFAC analyses suggest that the CD86-3479T allele contains putative GATA and TBP sites, whereas G allele does not. We assessed the relative DNA protein-binding activity of the -3479 polymorphism in vitro using electromobility gel shift assays (EMSA), which showed that the -3479G allele has less binding affinity compared to the T allele for nuclear proteins. These findings highlight the importance of co-stimulatory pathways in SSc pathogenesis.

Mono and trinuclear complexes of alpha-oximinoacetoacetylpyridine-4-phenylthiosemicarbazone.

Complexes of several transition metal ions with alpha-oximinoacetoacetyl pyridine-4-phenylthiosemicarbazone (H3OAPT) have been prepared. Attempts were made to elucidate their geometries by elemental analysis, molar conductance, magnetic measurements and by some spectroscopic (IR, ESR and electronic) techniques. All the investigated metal ions form mononuclear complexes except for CuII, which forms mononuclear and trinuclear complexes with its chloride and acetate salts, respectively. The IR spectra show that the ligand behaves as a mono or binegative tridentate. Moreover, it acts as a trinegative hexadentate in the trinuclear CuII complex. The protonation constants (logK1H = 9.9 and log K2H = 6.0), as well as the stability constants of the metal complexes, are determined by the pH-titration of H3OAPT and its metal(II) complexes against 0.01 M NaOH. CuII complexes possess square-planar stereochemistry while CoII and NiII have an octahedral one. The crystal field parameters of CoII and NiII complexes are evaluated.

Serum strontium estimation as a diagnostic criterion of the type of drowning water.

The estimation of serum strontium in rabbits before and after death by drowning, in soft or hard water, or by barbiturate intoxication is highly promising as a method in medicolegal practice. It not only diagnoses drowning but also indicates the type of drowning water. Atomic absorption spectrophotometry is a good method for the estimation.

Characterization and elimination of the interfering effects of foreign species in the atomic-absorption determination of iron.

The inhibition-release titration method has been used to study interference effects in flame atomic-absorption determination of iron. Interferences from anions, cations and complexing agents with the atomic-absorption of iron when a stoichiometric air-acetylene flame is used, can be obviated by a preliminary treatment of the sample solution with sulphosalicylic acid to convert the iron into the same complex before aspiration, thus giving a constant environment for the iron in the flame processes.