Structural and functional implications of the QUA2 domain on RNA recognition by GLD-1

The STAR family comprises ribonucleic acid (RNA)-binding proteins that play key roles in RNA-regulatory processes. RNA recognition is achieved by a KH domain with an additional α-helix (QUA2) that seems to extend the RNA-binding surface to six nucleotides for SF1 (Homo sapiens) and seven nucleotides for GLD-1 (Caenorhabditis elegans). To understand the structural basis of this probable difference in specificity, we determined the solution structure of GLD-1 KH-QUA2 with the complete consensus sequence identified in the tra-2 gene. Compared to SF1, the GLD-1 KH-QUA2 interface adopts a different conformation resulting indeed in an additional sequence-specific binding pocket for a uracil at the 5′end. The functional relevance of this binding pocket is emphasized by our bioinformatics analysis showing that GLD-1 binding sites with this 5′end uracil are more predictive for the functional response of the messenger RNAs to gld-1 knockout. We further reveal the importance of the KH-QUA2 interface in vitro and that its alteration in vivo affects the level of translational repression dependent on the sequence of the GLD-1 binding motif. In conclusion, we demonstrate that the QUA2 domain distinguishes GLD-1 from other members of the STAR family and contributes more generally to the modulation of RNA-binding affinity and specificity of KH domain containing proteins.     

Differential expression of centrosome regulators in Her2+ breast cancer cells versus non-tumorigenic MCF10A cells

Centrosome amplification (CA) amongst particular breast cancer subtypes (Her2+ subtype) is associated with genomic instability and aggressive tumor phenotypes. However, changes in signaling pathways associated with centrosome biology have not been fully explored in subtype specific models. Novel centrosome regulatory genes that are selectively altered in Her2+ breast cancer cells are of interest in discerning why CA is more prevalent in this subtype. To determine centrosome/cell cycle genes that are altered in Her2+ cells that display CA (HCC1954) versus non-tumorigenic cells (MCF10A), we carried out a gene microarray. Expression differences were validated by real-time PCR and Western blotting. After the microarray validation, we pursued a panel of upregulated and downregulated genes based on novelty/relevance to centrosome duplication. Functional experiments measuring CA and BrdU incorporation were completed after genetic manipulation of targets (TTK, SGOL1, MDM2 and SFRP1). Amongst genes that were downregulated in HCC1954 cells, knockdown of MDM2 and SFRP1 in MCF10A cells did not consistently induce CA or impaired BrdU incorporation. Conversely, amongst upregulated genes in HCC1954 cells, knockdown of SGOL1 and TTK decreased CA in breast cancer cells, while BrdU incorporation was only altered by SGOL1 knockdown. We also explored the Kaplan Meier Plot resource and noted that MDM2 and SFRP1 are positively associated with relapse free survival in all breast cancer subtypes, while TTK is negatively correlated with overall survival of Luminal A patients. Based on this functional screen, we conclude that SGOL1 and TTK are important modulators of centrosome function in a breast cancer specific model.     

A reference-quality,fully annotated genome from a Puerto Rican individual

Until 2019, the human genome was available in only one fully annotated version, GRCh38, which was the result of 18 years of continuous improvement and revision. Despite dramatic improvements in sequencing technology, no other genome was available as an annotated reference until 2019, when the genome of an Ashkenazi individual, Ash1, was released. In this study, we describe the assembly and annotation of a second individual genome, from a Puerto Rican individual whose DNA was collected as part of the Human Pangenome project. The new genome, called PR1, is the first true reference genome created from an individual of African descent. Due to recent improvements in both sequencing and assembly technology, and particularly to the use of the recently completed CHM13 human genome as a guide to assembly, PR1 is more complete and more contiguous than either GRCh38 or Ash1. Annotation revealed 37,755 genes (of which 19,999 are protein coding), including 12 additional gene copies that are present in PR1 and missing from CHM13. Fifty-seven genes have fewer copies in PR1 than in CHM13, 9 map only partially, and 3 genes (all noncoding) from CHM13 are entirely missing from PR1.     

Computational analysis of small RNA cloning data

Cloning and sequencing is the method of choice for small regulatory RNA identification. Using deep sequencing technologies one can now obtain up to a billion nucleotides--and tens of millions of small RNAs--from a single library. Careful computational analyses of such libraries enabled the discovery of miRNAs, rasiRNAs, piRNAs, and 21U RNAs. Given the large number of sequences that can be obtained from each individual sample, deep sequencing may soon become an alternative to oligonucleotide microarray technology for mRNA expression profiling. In this report we present the methods that we developed for the annotation and expression profiling of small RNAs obtained through large-scale sequencing. These include a fast algorithm for finding nearly perfect matches of small RNAs in sequence databases, a web-accessible software system for the annotation of small RNA libraries, and a Bayesian method for comparing small RNA expression across samples.     

Characterisation of the hydrophobic collapse of polystyrene in water using free energy techniques

We characterise the hydrophobic collapse of single polystyrene chains in water using molecular dynamics simulations. Specifically, we calculate the potential of mean force for the collapse of a single polystyrene chain in water using metadynamics, comparing the results between all atomistic with coarse-grained (CG) molecular simulation. We next explore the scaling behaviour of the collapsed globular shape at the minimum energy configuration, characterised by the radius of gyration, as a function of chain length. The exponent is close to one third, consistent with that predicted for a polymer chain in bad solvent. We also explore the scaling behaviour of the solvent accessible surface area (SASA) as a function of chain length, finding a similar exponent for both all atomistic and CG simulations. Furthermore, calculation of the local water density as a function of chain length near the minimum energy configuration suggests that intermediate chain lengths are more likely to form dewetted states, as compared to shorter or longer chain lengths.     

Hemodynamic assessment of pulmonary hypertension in mice: a model-based analysis of the disease mechanism

Biomechanics and Modeling in Mechanobiology - This study uses a one-dimensional fluid dynamics arterial network model to infer changes in hemodynamic quantities associated with pulmonary...     

Moving forward on the pathway of cell-based therapies in ischemic heart disease and heart failure-time for new recommendations?

Although substantial advances have been made in treating ischemic heart disease and subsequent heart failure, the overall morbidity and mortality from these conditions remain high. Stem cell-based therapy has emerged as a promising approach for prompting cardiac rejuvenation. Various cell types have been tested in the clinical arena, proving consistent safety results. As for efficiency outcomes,contradictory findings have been reported, partly due to inconsistency in study protocols but also due to poor survival, engraftment and differentiation of transplanted cells in the hostile milieu of the ischemic host tissue. Studies have varied in terms of route of delivery, type and dose of implanted stem cells,patient selection and randomization, and assessment of therapeutic effect.Founded on the main achievements and challenges within almost 20 years of research, a number of official documents have been published by leading experts in the field. Core recommendations have focused on developing and optimizing effective strategies to enrich cell retention and their regenerative potential. Issued consensus and position papers have stemmed from an unmet need to provide a harmonized framework for future research, resulting in improved therapeutic application of cell-based therapies for cardiac regeneration and repair.