Mechanistic analysis of enhancer sequences in the estrogen receptor transcriptional program.

Estrogen Receptor α (ERα) is a major lineage determining transcription factor (TF) in mammary gland development. Dysregulation of ERα-mediated transcriptional program results in cancer. Transcriptomic and epigenomic profiling of breast cancer cell lines has revealed large numbers of enhancers involved in this regulatory program, but how these enhancers encode function in their sequence remains poorly understood. A subset of ERα-bound enhancers are transcribed into short bidirectional RNA (enhancer RNA or eRNA), and this property is believed to be a reliable marker of active enhancers. We therefore analyze thousands of ERα-bound enhancers and build quantitative, mechanism-aware models to discriminate eRNAs from non-transcribing enhancers based on their sequence. Our thermodynamics-based models provide insights into the roles of specific TFs in ERα-mediated transcriptional program, many of which are supported by the literature. We use in silico perturbations to predict TF-enhancer regulatory relationships and integrate these findings with experimentally determined enhancer-promoter interactions to construct a gene regulatory network. We also demonstrate that the model can prioritize breast cancer-related sequence variants while providing mechanistic explanations for their function. Finally, we experimentally validate the model-proposed mechanisms underlying three such variants.

Towards the understanding of molecular motors and its relationship with local unfolding.

Molecular motors are machines essential for life since they convert chemical energy into mechanical work. However, the precise mechanism by which nucleotide binding, catalysis, or release of products is coupled to the work performed by the molecular motor is still not entirely clear. This is due, in part, to a lack of understanding of the role of force in the mechanical-structural processes involved in enzyme catalysis. From a mechanical perspective, one promising hypothesis is the Haldane-Pauling hypothesis which considers the idea that part of the enzymatic catalysis is strain-induced. It suggests that enzymes cannot be efficient catalysts if they are fully complementary to the substrates. Instead, they must exert strain on the substrate upon binding, using enzyme-substrate energy interaction (binding energy) to accelerate the reaction rate. A novel idea suggests that during catalysis, significant strain energy is built up, which is then released by a local unfolding/refolding event known as 'cracking'. Recent evidence has also shown that in catalytic reactions involving conformational changes, part of the heat released results in a center-of-mass acceleration of the enzyme, raising the possibility that the heat released by the reaction itself could affect the enzyme's integrity. Thus, it has been suggested that this released heat could promote or be linked to the cracking seen in proteins such as adenylate kinase (AK). We propose that the energy released as a consequence of ligand binding/catalysis is associated with the local unfolding/refolding events (cracking), and that this energy is capable of driving the mechanical work.

Interaction between hydrogen peroxide and sodium nitroprusside following chemical priming of Ocimum basilicum L. against salt stress.

Sodium nitroprusside (SNP) and hydrogen peroxide (H2 O2 ), as priming agents, have the well-recorded property to increase plant tolerance against a range of different abiotic stresses such as salinity. In this regard, the present study was conducted to evaluate the effect of different levels of SNP (100 and 200 µM) and H2 O2 (2.5 and 5 mM) as well as their combinations under salt stress (0 and 50 mM NaCl) on key physiological and biochemical attributes of the economically important aromatic plant basil (Ocimum basilicum L.) grown under hydroponic culture. Results revealed that morphological parameters such as plant height, root length, leaf fresh and dry weights (FW and DW) were significantly decreased by salinity stress, while SNP and H2 O2 treatments, alone or combined, increased FW and DW thus enhancing plant tolerance to salt stress. Furthermore, 200 µM SNP + 2.5 mM H2 O2 appeared to be the most effective treatment by causing significant increase in chlorophyll a and b, anthocyanin content and guaiacol peroxidase and ascorbate peroxidase enzymes activities under saline condition. In addition, analytical measurements showed that essential oil profile (concentration of main components) under salt stress was mostly affected by SNP and H2 O2 treatments. The highest increase was observed for methyl chavicol (43.09-69.91%), linalool (4.8-17.9%), cadinol (1.5-3.2%) and epi-α-cadinol (0.18-10.75%) compounds. In conclusion, current findings demonstrated a positive crosstalk between SNP and H2 O2 toward improved basil plant tolerance to salt stress, linked with regulation of essential oil composition.

Next-generation sequencing reveals a novel pathological mutation in the TMC1 gene causing autosomal recessive non-syndromic hearing loss in an Iranian kindred.

OBJECTIVES: Hearing loss (HL) is the most common sensory-neural disorder with excessive clinical and genetic heterogeneity, which negatively affects life quality. Autosomal recessive non-syndromic hearing loss (ARNSHL) is the most common form of the disease with no specific genotype-phenotype correlation in most of the cases. Whole exome sequencing (WES) is a powerful tool to overcome the problem of finding mutations in heterogeneous disorders. METHODS: A comprehensive clinical and pedigree examination was performed on a multiplex family from Khuzestan province suffering from hereditary HL. Direct sequencing of GJB2 and genetic linkage analysis of DFNB1A/B was accomplished. WES was utilized to find possible genetic etiology of the disease. Co-segregation analysis of the candidate variant was done. High resolution melting analysis was applied to detect variant status in 50 healthy matched controls. RESULTS: Clinical investigations suggested ARNSHL in the pedigree. The family was negative for DFNB1A/B. WES revealed a novel nonsense mutation, c.256G > T (p.Glu86*), in TMC1 segregating with the phenotype in the pedigree. The variant was absent in the controls. CONCLUSION: Here, we report successful application of WES to identify the molecular pathogenesis of ARNSHL in a large family. The novel nonsense TMC1 variant meets the criteria of being pathogenic according to the ACMG-AMP variant interpretation guideline.

Dissipation at the angstrom scale: Probing the surface and interior of an enzyme.

Pursuing a materials science approach to understanding the deformability of enzymes, we introduce measurements of the phase of the mechanical response function within the nanorheology paradigm. Driven conformational motion of the enzyme is dissipative as characterized by the phase measurements. The dissipation originates both from the surface hydration layer and the interior of the molecule, probed by examining the effect of point mutations on the mechanics. We also document changes in the mechanics of the enzyme examined, guanylate kinase, upon binding its four substrates. GMP binding stiffens the molecule, ATP and ADP binding softens it, while there is no clear mechanical signature of GDP binding. A hyperactive two-Gly mutant is found to possibly trade specificity for speed. Global deformations of enzymes are shown to be dependent on both hydration layer and polypeptide chain dynamics.

Mechanical properties of BiP protein determined by nano-rheology.

Immunoglobulin Binding Protein (BiP) is a chaperone and molecular motor belonging to the Hsp70 family, involved in the regulation of important biological processes such as synthesis, folding and translocation of proteins in the Endoplasmic Reticulum. BiP has two highly conserved domains: the N-terminal Nucleotide-Binding Domain (NBD), and the C-terminal Substrate-Binding Domain (SBD), connected by a hydrophobic linker. ATP binds and it is hydrolyzed to ADP in the NBD, and BiP's extended polypeptide substrates bind in the SBD. Like many molecular motors, BiP function depends on both structural and catalytic properties that may contribute to its performance. One novel approach to study the mechanical properties of BiP considers exploring the changes in the viscoelastic behavior upon ligand binding, using a technique called nano-rheology. This technique is essentially a traditional rheology experiment, in which an oscillatory force is directly applied to the protein under study, and the resulting average deformation is measured. Our results show that the folded state of the protein behaves like a viscoelastic material, getting softer when it binds nucleotides- ATP, ADP, and AMP-PNP-, but stiffer when binding HTFPAVL peptide substrate. Also, we observed that peptide binding dramatically increases the affinity for ADP, decreasing it dissociation constant (KD ) around 1000 times, demonstrating allosteric coupling between SBD and NBD domains.

Identifying Chaotropic and Kosmotropic Agents by Nanorheology.

Chemical agents are classified into chaotropes (disorder inducing) and kosmotropes (order inducing) based on their ability to destabilize or stabilize, respectively, the structure of hydrated macromolecules and their solutions. Here, we examine the effect of such agents on the mechanical stiffness of the hydration layer of proteins, measured by nanorheology. We examine four different agents and conclude that chaotropic substances induce the overall softening of the protein-hydration layer system, whereas the kosmotropic substances induce stiffening. Specifically, with glucose and trifluoroethanol, two known kosmotropic agents, we observe the stiffening of the hydration layer. In contrast, with guanidine hydrochloride and urea, known kaotropic agents, we observe softening. Thus, the viscoelastic mechanics of the folded, hydrated protein provides an experimental measure of the effect that chaotropes and kosmotropes have on the dynamics.

The Effect of New Model PREPARED on Obtaining Informed Consent Skill in Midwifery Students of Shahid Sadoughi University of Medical Sciences.

BACKGROUND: Professional ethics culture should be taught to students appropriately. Studies have shown that midwifery students are not entirely familiar with the skill of obtaining informed consent. Using a new and applicable model to teach this skill to midwifery students is necessary. This study was conducted to determine the effect of a new standard model, PREPARED, on the skill of obtaining informed consent in midwifery students of Shahid Sadoughi University of Medical Sciences. MATERIALS AND METHODS: This interventional study was conducted on 37 5th semester midwifery students through a census method. After determining psychometric indices, in two phases with a 4-week interval (before and after the training), the PREPARED checklist was completed by the professors of the research team in the presence of students in the delivery room while they were performing midwifery care considering their compliance to the checklist. Descriptive statistics paired t-test were used for data analysis. RESULTS: The lowest mean score before the training belonged to alternative methods (1.00) and treatment expenses (1.00). After the training, treatment plan had the highest mean score (3.54 (0.69)). The mean and standard deviation of scores before and after training the students were 9.12 (2.00) and 30.6824 (5.25), respectively. Based on the results of the paired t-test (P = 0.001), the difference was statistically significant. CONCLUSION: Results showed that the implementation of the new model of PREPARED would increase the skill of obtaining informed consent in midwifery students and could be applied for educating students of other medical majors in Iran.

MRI Phenotyping of COL9A2/Trp2 and COL9A3/Trp3 Alleles in Lumbar Disc Disease: A Case-control Study in South-Western Iranian Population Reveals a Significant Trp3-Disease Association in Males.

STUDY DESIGN: A case-control study of the Trp2/3 alleles of COL9A2/3 genes and their correlation with occurrence of Lumbar disc disease (DDD) as phenotyped by magnetic resonance imaging. OBJECTIVE: To establish a better understanding of relationship between presence of said alleles and occurrence of DDD in South-Western Iranian population. SUMMARY OF BACKGROUND DATA: A number of genetic predisposing factors have been identified in elevating the risk of developing DDD. Specifically, the Trp2 and Trp3 alleles of COL9A2 and COL9A3 genes have been suggested as DDD risk variants. METHODS: A total of 108 patients (mean age = 41±11.8 yrs, range = 20-66 yrs) with 57 controls (mean age = 35±10.0 yrs, range = 20-58 yrs) participated in the study. The frequency of G/A polymorphism in COL9A2 gene on location 326 on chromosome 1 and G/A/C/ or T polymorphism in 103 location of COL9A3 gene on chromosome 20 was assessed using a PCR short-primer technique. Outcome measure was defined as presence of DDD on MRI. Odds ratios (OR) and 95% confidence intervals (CI) were used to assess the likelihood of DDD given occurrence of Trp2(3). RESULTS: Each allele was present in both patients and controls. The Trp2 allele was positive in 28.5% of individuals (31.5% of patients; 22.8% of controls), OR 1.55 (0.71-3.56). The Trp3 allele, the frequency was 23.6% in all patients (26.9% patients; 17.5% controls), OR 1.72 (0.73-4.33). We observed a 5.8-fold increase in the odds of DDD in males when the Trp3 allele was present, OR 5.83 (1.09-9.98), P = 0.0273. CONCLUSION: Both Trp2 and Trp3 alleles occurred more frequently compared with other studied ethnicities. The sampled Iranian population exhibited a similar Trp2 frequency to a Southern Chinese population, and Trp3 occurrence to Finnish and Greek population. We found that male patient were much more likely to develop DDD when Trp 3 was present. LEVEL OF EVIDENCE: N/A.