Catastrophic Trunnion Failure in an Anatomic Titanium Alloy Stem.

Introduction: Certain titanium alloy stems have been shown to be susceptible to failure at the neck with catastrophic trunnion failure. Failure has been particularly noted in the single wedge Accolade 1 stem design. Other stems also used this alloy including the anatomic designed Citation stem. Methods: This case series details 3 catastrophic failures of the TMZF version of the Citation femoral stem. Results: Each of these failures appear to be attributed to cyclical wear of the TMZF trunnion against the cobalt chromium femoral head. Wear resulted in ultimate implant failure and significant metal debris in the joint capsule at the time of revision surgery. Discussion: While surgeons are aware of the risk of catastrophic failure for the Accolade 1 stem, failure may similarly happen in the TMZF Citation stem. Surgeons should monitor these implants with care and discuss the potential for trunnion failure with their patients.

Precise Cas9 targeting enables genomic mutation prevention.

Here, we present a generalized method of guide RNA "tuning" that enables Cas9 to discriminate between two target sites that differ by a single-nucleotide polymorphism. We employ our methodology to generate an in vivo mutation prevention system in which Cas9 actively restricts the occurrence of undesired gain-of-function mutations within a population of engineered organisms. We further demonstrate that the system is scalable to a multitude of targets and that the general tuning and prevention concepts are portable across engineered Cas9 variants and Cas9 orthologs. Finally, we show that the mutation prevention system maintains robust activity even when placed within the complex environment of the mouse gastrointestinal tract.

Transcriptional Profiling of the Oral Pathogen Streptococcus mutans in Response to Competence Signaling Peptide XIP.

In the cariogenic Streptococcus mutans, competence development is regulated by the ComRS signaling system comprised of the ComR regulator and the ComS prepeptide to the competence signaling peptide XIP (ComX-inducing peptide). Aside from competence development, XIP signaling has been demonstrated to regulate cell lysis, and recently, the expression of bacteriocins, small antimicrobial peptides used by bacteria to inhibit closely related species. Our study further explores the effect of XIP signaling on the S. mutans transcriptome. RNA sequencing revealed that XIP induction resulted in a global change in gene expression that was consistent with a stress response. An increase in several membrane-bound regulators, including HdrRM and BrsRM, involved in bacteriocin production, and the VicRKX system, involved in acid tolerance and biofilm formation, was observed. Furthermore, global changes in gene expression corresponded to changes observed during the stringent response to amino acid starvation. Effects were also observed on genes involved in sugar transport and carbon catabolite repression and included the levQRST and levDEFG operons. Finally, our work identified a novel heat shock-responsive intergenic region, encoding a small RNA, with a potential role in competence shutoff. IMPORTANCE Genetic competence provides bacteria with an opportunity to increase genetic diversity or acquire novel traits conferring a survival advantage. In the cariogenic pathogen Streptococcus mutans, DNA transformation is regulated by the competence stimulating peptide XIP (ComX-inducing peptide). The present study utilizes high-throughput RNA sequencing (RNAseq) to provide a greater understanding of how global gene expression patterns change in response to XIP. Overall, our work demonstrates that in S. mutans, XIP signaling induces a response that resembles the stringent response to amino acid starvation. We further identify a novel heat shock-responsive intergenic region with a potential role in competence shutoff. Together, our results provide further evidence that multiple stress response mechanisms are linked through the genetic competence signaling pathway in S. mutans.

Design, synthesis, and testing toward a 57-codon genome.

Recoding--the repurposing of genetic codons--is a powerful strategy for enhancing genomes with functions not commonly found in nature. Here, we report computational design, synthesis, and progress toward assembly of a 3.97-megabase, 57-codon Escherichia coli genome in which all 62,214 instances of seven codons were replaced with synonymous alternatives across all protein-coding genes. We have validated 63% of recoded genes by individually testing 55 segments of 50 kilobases each. We observed that 91% of tested essential genes retained functionality with limited fitness effect. We demonstrate identification and correction of lethal design exceptions, only 13 of which were found in 2229 genes. This work underscores the feasibility of rewriting genomes and establishes a framework for large-scale design, assembly, troubleshooting, and phenotypic analysis of synthetic organisms.

Comparison of Cas9 activators in multiple species.

Several programmable transcription factors exist based on the versatile Cas9 protein, yet their relative potency and effectiveness across various cell types and species remain unexplored. Here, we compare Cas9 activator systems and examine their ability to induce robust gene expression in several human, mouse, and fly cell lines. We also explore the potential for improved activation through the combination of the most potent activator systems, and we assess the role of cooperativity in maximizing gene expression.

Cas9 gRNA engineering for genome editing, activation and repression.

We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.

Highly efficient Cas9-mediated transcriptional programming.

The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. We demonstrate its utility in activating endogenous coding and noncoding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).

Microfluidic platforms for RNA interference screening of virus-host interactions.

RNA interference (RNAi) is a powerful tool for functional genomics with the capacity to comprehensively analyze host-pathogen interactions. High-throughput RNAi screening is used to systematically perturb cellular pathways and discover therapeutic targets, but the method can be tedious and requires extensive capital equipment and expensive reagents. To aid in the development of an inexpensive miniaturized RNAi screening platform, we have developed a two part microfluidic system for patterning and screening gene targets on-chip to examine cellular pathways involved in virus entry and infection. First, a multilayer polydimethylsiloxane (PDMS)-based spotting device was used to array siRNA molecules into 96 microwells targeting markers of endocytosis, along with siRNA controls. By using a PDMS-based spotting device, we remove the need for a microarray printer necessary to perform previously described small scale (e.g. cellular microarrays) and microchip-based RNAi screening, while still minimizing reagent usage tenfold compared to conventional screening. Second, the siRNA spotted array was transferred to a reversibly sealed PDMS-based screening platform containing microchannels designed to enable efficient cell loading and transfection of mammalian cells while preventing cross-contamination between experimental conditions. Validation of the screening platform was examined using Vesicular stomatitis virus and emerging pathogen Rift Valley fever virus, which demonstrated virus entry pathways of clathrin-mediated endocytosis and caveolae-mediated endocytosis, respectively. The techniques here are adaptable to other well-characterized infection pathways with a potential for large scale screening in high containment biosafety laboratories.

Characterization of the functional domains of the SloR metalloregulatory protein in Streptococcus mutans.

Streptococcus mutans is a commensal member of the healthy plaque biofilm and the primary causative agent of dental caries. The present study is an investigation of SloR, a 25-kDa metalloregulatory protein that modulates genes responsible for S. mutans-induced cariogenesis. Previous studies of SloR homologues in other bacterial pathogens have identified three domains critical to repressor functionality: an N-terminal DNA-binding domain, a central dimerization domain, and a C-terminal FeoA (previously SH3-like) domain. We used site-directed mutagenesis to identify critical amino acid residues within each of these domains of the SloR protein. Select residues were targeted for mutagenesis, and nonconservative amino acid substitutions were introduced by overlap extension PCR. Furthermore, three C-terminally truncated SloR variants were generated using conventional PCR. The repressor functionality and DNA-binding ability of each variant was assessed using CAT reporter gene assays, real-time semiquantitative reverse transcriptase (qRT)-PCR, and electrophoretic mobility shift assays. We identified 12 residues within SloR that cause significant derepression of sloABC promoter activity (P < 0.05) compared to the results for wild-type SloR. Derepression was particularly noteworthy in metal ion-binding site 1 mutants, consistent with the site's importance in gene repression by SloR. In addition, a hyperactive SloR(E169A/Q170A) mutant was identified as having significantly heightened repression of sloABC promoter activity, and experiments with C-terminal deletion mutants support involvement of the FeoA domain in SloR-mediated gene repression. Given these results, we describe the functional domains of the S. mutans SloR protein and propose that the hyperactive mutant could serve as a target for rational drug design aimed at repressing SloR-mediated virulence gene expression.