Functional characterization and crystal structure of thermostable amylase from Thermotoga petrophila, reveals high thermostability and an unusual form of dimerization.

Thermostable α-amylases have many industrial applications and are therefore continuously explored from novel sources. We present the characterization of a novel putative α-amylase gene product (Tp-AmyS) cloned from Thermotoga petrophila. The purified recombinant enzyme is highly thermostable and able to hydrolyze starch into dextrin between 90 and 100°C, with optimum activity at 98°C and pH8.5. The activity increased in the presence of Rb1+, K1+ and Ca2+ ions, whereas other ions inhibited activity. The crystal structure of Tp-AmyS at 1.7Å resolution showed common features of the GH-13 family, however was apparently found to be a dimer. Several residues from one monomer interacted with a docked acarbose, an inhibitor of Tp-AmyS, in the other monomer, suggesting catalytic cooperativity within the dimer. The most striking feature of the dimer was that it resembled the dimerization of salivary amylase from a previous crystal structure, and thus could be a functional feature of some amylases.

Does team-based learning affect test scores of the basic medical sciences students in a modular curriculum?

Objectives: The aim of the study was to determine the effectiveness of team-based learning (TBL) sessions as a learning tool and to assess the satisfaction level of medical students towards TBL in modular curriculum. Methods: Using the quasi-experimental study design, TBL sessions were conducted, involving students of 1st and 2nd year of Bachelor of Medicine and Bachelor of Surgery. The TBL infrastructure comprised of pre-class preparation, in-class individual readiness assurance pre-test and post-test, before and after group discussion, respectively. The responses of the students regarding TBL satisfaction were recorded through a structured questionnaire (5-point Likert-type scale) while Wilcoxon signed rank test was applied to measure the effectiveness of TBL sessions. Results: Out of 192 students, 85% agreed or strongly agreed that TBL helped them think critically, identify their knowledge gaps, boosted their confidence, and motivated them in group participation. Significantly better post-test scores were found in all modules where TBL was used as a teaching tool (Z range = -5.33 to -11.81, P < 0.00). Conclusion: TBL increases the post-test score in majority of the students, indicating improved learning process. It not only keeps students engaged throughout the learning process but incites critical thinking, problem solving skills, and confidence. Further studies are required to see long-term benefit of TBL in students' learning.

CFTR mutations causing congenital unilateral absence of the vas deferens (CUAVD) and congenital absence of the uterus (CAU) in a consanguineous family.

Cystic fibrosis (CF) is one of the most common recessive genetic diseases, with a wide spectrum of phenotypes, ranging from infertility to severe pulmonary disease. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are considered the main genetic cause for CF. In this study, we recruited a consanguineous Iranian pedigree with four male patients diagnosed with congenital unilateral absence of the vas deferens (CUAVD), and one female patient diagnosed with congenital absence of the uterus (CAU). Testicular biopsy of one patient was performed, and hematoxylin and eosin (H and E) staining of testis sections displayed the presence of germ cell types ranging from spermatogonia to mature spermatids, indicating obstructive azoospermia. To explore the underlying genetic factor in this familial disorder, we therefore performed whole-exome sequencing (WES) on all available family members. WES data filtration and CFTR haplotype analysis identified compound heterozygous mutations in CFTR among four patients (two CUAVD patients carried p.H949Y and p.L997F, and one CUAVD and the female CAU patient carried p.H949Y and p.I148T). All these mutations were predicted to be deleterious by at least half of the prediction software programs and were confirmed by Sanger sequencing. Our study reported that CFTR compound heterozygous mutations in a consanguineous Iranian family cause infertility in both sexes.

A recurrent homozygous missense mutation in CCDC103 causes asthenoteratozoospermia due to disorganized dynein arms.

Asthenoteratozoospermia is one of the most severe types of qualitative sperm defects. Most cases are due to mutations in genes encoding the components of sperm flagella, which have an ultrastructure similar to that of motile cilia. Coiled-coil domain containing 103 (CCDC103) is an outer dynein arm assembly factor, and pathogenic variants of CCDC103 cause primary ciliary dyskinesia (PCD). However, whether CCDC103 pathogenic variants cause severe asthenoteratozoospermia has yet to be determined. Whole-exome sequencing (WES) was performed for two individuals with nonsyndromic asthenoteratozoospermia in a consanguineous family. A homozygous CCDC103 variant segregating recessively with an infertility phenotype was identified (ENST00000035776.2, c.461A>C, p.His154Pro). CCDC103 p.His154Pro was previously reported as a high prevalence mutation causing PCD, though the reproductive phenotype of these PCD individuals is unknown. Transmission electron microscopy (TEM) of affected individuals' spermatozoa showed that the mid-piece was severely damaged with disorganized dynein arms, similar to the abnormal ultrastructure of respiratory ciliary of PCD individuals with the same mutation. Thus, our findings expand the phenotype spectrum of CCDC103 p.His154Pro as a novel pathogenic gene for nonsyndromic asthenospermia.

Role of Lipid Metabolism and Signaling in Mammalian Oocyte Maturation, Quality, and Acquisition of Competence.

It has been found that the quality of oocytes from obese women has been compromised and subsequent embryos displayed arrested development. The compromised quality may be either due to the poor or rich metabolic conditions such as imbalance or excession of lipids during oocyte development. Generally, lipids are mainly stored in the form of lipid droplets and are an important source of energy metabolism. Similarly, lipids are also essential signaling molecules involved in various biological cascades of oocyte maturation, growth and oocyte competence acquisition. To understand the role of lipids in controlling the oocyte development, we have comprehensively and concisely reviewed the literature and described the role of lipid metabolism in oocyte quality and maturation. Moreover, we have also presented a simplified model of fatty acid metabolism along with its implication on determining the oocyte quality and cryopreservation for fertilization.

Exonuclease 5 is dispensable for meiotic progression and male fertility in mouse.

Exonuclease 5 (Exo5) belongs to a class of bi-directional, ssDNA-specific exonucleases that mainly involved in the DNA repair pathways. Exo5 has been reported to be crucial for DNA- DNA mismatch repair (MMR) in several human cell lines. However, its in vivo function in mammals still needs to be explored. Thus, to study the in vivo role of Exo5 in spermatogenesis, Exo5 knockout mice were generated using CRISPR/Cas9 technology. Unexpectedly, we found that the knockout mice are fertile despite a slight decrease in sperm count. Furthermore, Exo5-/- mice showed no detectable developmental anomalies, exhibited no remarkable differences in the epididymal histology and testis/body weight ratio. Moreover, cytological investigations on meiocytes revealed non-significant differences in chromosomal synapsis, recombination, and meiotic progression of prophase I, further demonstrating that Exo5 has no essential role in spermatogenesis in mice under normal breeding conditions. Collectively, these data indicate that Exo5 is dispensable for meiotic progression and fertility in mice.

Protective potential of Vitamin E against methylphenidate-induced male gonadal changes in albino rats.

OBJECTIVE: Attention deficit hyperactivity disorder ranks among the top neuropsychiatric disorder of childhood and adolescents. Methylphenidate (MPH) is the most frequently used pharmacologic agent to treat this condition. Its long-term use has been associated with many unwanted and adverse effects on many organs including male gonads, but so far no study has been done to find out a protective agent. This study investigated the protective potential of Vitamin E (Vit E) against the microscopic and morphometric alterations in male gonads induced by MPH, using albino rats. METHODS: Adult male albino rats were assigned into three equal groups including one control and two experimental groups. Experimental groups administered with MPH (10 mg/kg) and MPH (10 mg/kg) + Vit E orally (50 mg/kg), daily for 40 days. Testes of the sacrificed animals were removed, processed, and stained with hematoxylin and eosin for examining the microscopic and morphometric alterations and protective potential of Vit E. Data were analyzed using ANOVA. RESULTS: Experimental animals treated with MPH showed a significant decrease in the diameter of seminiferous tubules (296.86 ± 14.70 µm) and height of germinal epithelium (51.73 ± 3.15 µm) with a corresponding gain in the thickness of the interstitium (47.05 ± 4.94 µm). Animals treated with MPH + Vit E did not reveal any significant testicular microscopic changes and seminiferous tubular alterations induced by MPH. CONCLUSION: Vit E demonstrated a protective potential against the adverse changes induced by MPH in the male gonads in albino rats.

Synthesis of polyaniline nanoparticles and their application for the removal of Crystal Violet dye by ultrasonicated adsorption process based on Response Surface Methodology.

The present study focuses the synthesis of polyaniline nanoparticles (PANP) by rapid mixing polymerization method. They were recognized by FTIR and SEM techniques. Moreover they were utilized for the removal of Crystal Violet (CV) dye by ultrasonicated adsorption process. It ensures a quick alternative method compared to other conventional processes, which led to enhancement of mass transfer by ultrasound waves. The effectiveness of the process was confirmed through the effect of certain conditions like sonication time, temperature, adsorbent dosage and CV concentrations. The validity of the process was estimated by various adsorption isotherms. Kinetics and thermodynamic studies was also conducted to authenticate the process. The optimum operating parameters (OOP) were evaluated by Response Surface Methodology (RSM) based on central composite design (CCD) for the removal of CV dye. Moreover analysis of variances (ANOVA) was employed to estimate the significance of experimental variables. The predicated removal efficiency was found to be 94.29% which prove to be effectiveness of the process.

Development of a nested-PCR assay for the rapid detection of Pilidiella granati in pomegranate fruit.

Pilidiella granati, a causal agent of twig blight and crown rot of pomegranate, is an emerging threat that may cause severe risk to the pomegranate industry in the future. Development of a rapid assay for the timely and accurate detection of P. granati will be helpful in the active surveillance and management of the disease caused by this pathogen. In this study, a nested PCR method was established for the detection of P. granati. Comparative analysis of genetic diversity within 5.8S rDNA internal transcribed spacer (ITS) sequences of P. granati and 21 other selected fungal species was performed to design species-specific primers (S1 and S2). This primer pair successfully amplified a 450 bp product exclusively from the genomic DNA of P. granati. The developed method can detect 10 pg genomic DNA of the pathogen in about 6 h. This technique was successfully applied to detect the natural infection of P. granati in the pomegranate fruit. The designed protocol is rapid and precise with a high degree of sensitivity.

Spectrum of beta-thalassemia mutations in various regions of Punjab and Islamabad, Pakistan: establishment of prenatal diagnosis.

We present here an analysis of 888 unrelated beta-thal chromosomes consisting of 444 transfusion dependent children from various regions of Punjab and Islamabad Pakistan. By using Multiplex ARMS- PCR, restriction endonuclease analysis, allele specific oligonucleotide (ASO) hybridization and sequencing, 17 beta-thal mutations and 3 Hb variants were detected in 99.5 % (884/888) of the chromosomes analyzed. First trimester prenatal diagnosis by chorionic villus sampling (CVS) was also carried out in seven pregnancies at risk of beta-thalassemia. Our results indicate that three most common mutations accounted for 86.8% of the beta-thal alleles in this region. These findings have important implications for prevention of beta-thalassemia through genetic counseling and prenatal diagnosis in this part of Pakistan.