Use of a collagen-platelet rich plasma scaffold to stimulate healing of a central defect in the canine ACL.

The anterior cruciate ligament (ACL) of the knee fails to heal after primary repair. Here we hypothesize that a beneficial biologic repair response can be induced by placing a collagen-platelet rich plasma (collagen-PRP) material into a central ACL defect. A collagen-PRP scaffold was used to treat a central ACL defect in vivo. In the first experiment, the histologic response in treated and untreated defects was evaluated at 3 (n = 5) and 6 weeks (n = 5). In the second experiment, biomechanical testing of the treated ligaments (n = 8) was performed at 6 weeks and compared with the results of biomechanical testing of untreated defects at the same time-point (n = 6). The percentage filling of the defects in the treated ACLs was significantly higher at both the 3- and 6-week time-points when compared with the untreated contralateral control defects (50 +/- 21% vs. 2 +/- 2% at 3 weeks, and 43 +/- 11% vs. 23 +/- 11 at 6 weeks; all values mean +/- SEM. Biomechanically, the treated ACL defects had a 40% increase in strength at 6 weeks, which was significantly higher than the 14% increase in strength previously reported for untreated defects (p < 0.02). Placement of a collagen-PRP bridging scaffold in a central ACL defect can stimulate healing of the ACL histologically and biomechanically.     

A Canadian Perspective on the Challenges for Delivery of Curative-Intent Therapy in Stage III Unresectable Non-Small Cell Lung Cancer

Stage III non-small cell lung cancer (NSCLC) comprises a highly heterogenous group of patients with regards to patient fitness and tumour size and distribution, resulting in a wide range of treatment goals and therapy options. Curative-intent multimodality treatment should be considered in all patients with stage III NSCLC. For patients with unresectable disease who are fit, have adequate lung function, and have a disease that can be encompassed within a radical radiation volume, concurrent chemoradiation therapy (cCRT) is the standard of care and can produce cure rates of 20–30%. Recently, consolidation immunotherapy with durvalumab has been recognized as the standard of care following cCRT based on significant improvement rates in overall survival at 4 years. The large heterogeneity of the stage III NSCLC population, along with the need for extensive staging procedures, multidisciplinary care, intensive cCRT, and now consolidation therapy makes the delivery of timely and optimal treatment for these patients complex. Several logistical, communication, and education factors hinder the delivery of guideline-recommended care to patients with stage III unresectable NSCLC. This commentary discusses the potential challenges patients may encounter at different points along their care pathway that can interfere with delivery of curative-intent therapy and suggests strategies for improving care delivery.     

Intramolecular phenotypic capacitance in a modular RNA molecule

Phenotypic capacitance refers to the ability of a genome to accumulate mutations that are conditionally hidden and only reveal phenotype-altering effects after certain environmental or genetic changes. Capacitance has important implications for the evolution of novel forms and functions, but experimentally studied mechanisms behind capacitance are mostly limited to complex, multicomponent systems often involving several interacting protein molecules. Here we demonstrate phenotypic capacitance within a much simpler system, an individual RNA molecule with catalytic activity (ribozyme). This naturally occurring RNA molecule has a modular structure, where a scaffold module acts as an intramolecular chaperone that facilitates folding of a second catalytic module. Previous studies have shown that the scaffold module is not absolutely required for activity, but dramatically decreases the concentration of magnesium ions required for the formation of an active site. Here, we use an experimental perturbation of magnesium ion concentration that disrupts the folding of certain genetic variants of this ribozyme and use in vitro selection followed by deep sequencing to identify genotypes with altered phenotypes (catalytic activity). We identify multiple conditional mutations that alter the wild-type ribozyme phenotype under a stressful environmental condition of low magnesium ion concentration, but preserve the phenotype under more relaxed conditions. This conditional buffering is confined to the scaffold module, but controls the catalytic phenotype, demonstrating how modularity can enable phenotypic capacitance within a single macromolecule. RNA’s ancient role in life suggests that phenotypic capacitance may have influenced evolution since life’s origins.Phenotypic capacitance requires mutations that reversibly alternate between hidden and revealed states in response to environmental or genetic perturbations (1, 2). In the hidden or “cryptic” state the mutations can survive selection because they do not change the phenotype, and multiple such mutations can accumulate within individual genomes in a population. Perturbation after such accumulation can reveal the combined effects of multiple mutations. By exposing the phenotypic effects of mutations that may not have been beneficial individually, capacitance provides a mechanism of generating new phenotypes—from macromolecules to morphological traits—with novel functions (1–4). The term “phenotypic capacitance” is appropriate for situations where altered phenotypes with a genetic basis can be hidden and revealed, even when no adaptive potential of the revealed phenotypes is demonstrated (2). “Evolutionary capacitance,” on the other hand, is a term reserved for instances when an adaptive role is demonstrated. Phenotypic capacitance has been known in fruit flies since the 1950s (5), but demonstrations of adaptive potential (4, 6), and the various molecular mechanisms behind it (7–11) have been reported only relatively recently.A module in a biological system is a group of system parts that interact more with each other than with parts outside of the module (12). In RNA molecules, the parts are nucleotides, and modules are units of tertiary structure that fold independently, and often perform different functions, such as binding to different proteins, RNAs, or small molecules. Modularity has been described in the telomerase RNA component (13), ribosomal RNA (14), long noncoding RNA (e.g., Xist, Hotair) (15), riboswitches (16), and self-splicing introns (17, 18). A link between modularity and phenotypic capacitance could provide a mechanism for the evolution of novel functions involving modules of RNA structure, and especially for functional innovations that require multiple simultaneous mutations. It is not known how modularity might change the potential to hide and reveal the effects of mutations in RNA structures, which is a prerequisite for phenotypic capacitance.To investigate a possible link between modularity and phenotypic capacitance in RNA, we chose to study the Azoarcus group I RNA enzyme (ribozyme). Group I ribozymes such as this have two structural modules that are functionally distinct (Fig. 1). The first of them is a scaffold module (Fig. 1, yellow) that folds rapidly, and forms a nearly identical structure even when removed from the context of the rest of the ribozyme (19, 20). The scaffolding it provides facilitates the folding of the less thermodynamically stable catalytic module (Fig. 1, blue) (21). Biochemical studies have shown that the catalytic phenotype (protein-free splicing) resides in the catalytic module, which can maintain activity if the scaffold module is deleted, but only under conditions of very high magnesium (80 mM) and extended incubation times (16 h) (22). This instability caused by deleting the scaffold supports the idea that this module acts as an intramolecular chaperone.Open in a separate windowFig. 1.The modular structure of the Azoarcus group I ribozyme. (A) The scaffold module (yellow) and the catalytic module (blue) are shown in the context of the ribozyme secondary structure. The regions that show base pairing (P) are numbered sequentially from the 5′ to the 3′ end, according to group I intron standards. The substrate for the ribozyme is written in lowercase letters. The modules are also shown in the context of the 3D crystal structure (PDB ID: 1ZZN), from a “top view” (B) and “side view” (C), with respect to the scaffold module.The diversification of RNA functions has played an important role in the evolution of extant organisms, and may have been even more important at life’s beginnings when RNA enzymes (ribozymes) played a central role as catalysts in the RNA world scenario (23). In a previous publication using the Azoarcus ribozyme, we demonstrated an adaptive role for accumulated cryptic variation that was revealed when altered enzymatic activity was required (24). Here, we focus on how the functionally distinct modules of this structure might facilitate the occurrence of environmentally conditional mutations that enable phenotypic capacitance.We aimed to identify mutations that maintain the catalytic phenotype under normal conditions but alter this phenotype under stressful conditions. The ideal stressful condition for our RNA system is a low concentration of magnesium ions. The concentration of magnesium ions inside of cells is maintained at a higher concentration than any other divalent ion due to its role in many cellular functions. Importantly for our current experiments, it is known that magnesium ions are critical for stabilizing the native structure of RNA molecules (25). Low concentrations of magnesium only allow folding of the most stable RNA structures and can lead to misfolding of less stable structures. In addition, many ribozymes, including group I introns, use highly coordinated magnesium ions in their active site (26). Based on previous reports on the magnesium dependence of the Azoarcus ribozyme (19), we here studied a stressful environment with low (2 mM) MgCl₂, a relaxed environment with high (25 mM) MgCl₂, and an intermediate environment (10 mM MgCl₂). We note that magnesium availability is often limited in natural environments, a stressful condition that organisms have evolved adaptive responses to. For example, magnesium homeostasis in bacteria is maintained by the expression of ion transporters that are sometimes regulated by cis-acting RNA regulatory elements. These “magnesium riboswitch” elements control downstream mRNA expression by conformational changes induced by altered magnesium concentrations (27). This example and others (28) highlight the widespread importance of our chosen experimental stressor.     

Targeted or whole genome sequencing of formalin fixed tissue samples: potential applications in cancer genomics

Current genomic studies are limited by the poor availability of fresh-frozen tissue samples. Although formalin-fixed diagnostic samples are in abundance, they are seldom used in current genomic studies because of the concern of formalin-fixation artifacts. Better characterization of these artifacts will allow the use of archived clinical specimens in translational and clinical research studies. To provide a systematic analysis of formalin-fixation artifacts on Illumina sequencing, we generated 26 DNA sequencing data sets from 13 pairs of matched formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FF) tissue samples. The results indicate high rate of concordant calls between matched FF/FFPE pairs at reference and variant positions in three commonly used sequencing approaches (whole genome, whole exome, and targeted exon sequencing). Global mismatch rates and C·G > T·A substitutions were comparable between matched FF/FFPE samples, and discordant rates were low (<0.26%) in all samples. Finally, low-pass whole genome sequencing produces similar pattern of copy number alterations between FF/FFPE pairs. The results from our studies suggest the potential use of diagnostic FFPE samples for cancer genomic studies to characterize and catalog variations in cancer genomes.