High incidence of dural tears with 3-column osteotomies: a systematic review of adult spinal deformity surgery literature for the past decade.

PURPOSE: Dural tear (DT) is a well-known complication of spinal surgery. We aimed to systematically review the literature from the past decade and determine the incidence and risk factors for DT in the adult spinal deformity (ASD) population to improve both the surgical strategy and counseling of patients undergoing ASD correction. METHODS: A systematic review from 2013 to 2023 utilizing PRISMA guidelines was performed. The MEDLINE database was used to collect primary English language articles. The inclusion criterion for patients was degenerative ASD. Pediatric studies, animal studies, review articles, case reports, studies investigating minimally invasive surgery (MIS), studies lacking data on DT incidence, and articles pertaining to infectious, metastatic or neoplastic, traumatic, or posttraumatic etiologies of ASD were excluded. RESULTS: Our results demonstrate that the incidence of DT in ASD surgery ranges from 2.0% to 35.7%, which is a much broader range than the reported incidence for non deformity surgery. Moreover, the average rate of DT during ASD surgery stratified by surgical technique was greater for osteotomy overall (19.5% +/- 7.9%), especially for 3-column osteotomy (3CO), and lower for interbody fusion (14.3% +/- 9.9%). Risk factors for DT in the ASD surgery cohort included older age, revision surgery, chronic severe compression, higher-grade osteotomy, complexity of surgery, rheumatoid arthritis (RA), and higher Anesthesiology Society of America (ASA) grade. CONCLUSION: To our knowledge, this is the first systematic review discussing the incidence of and risk factors for DT in the ASD population. We found that the risk factors for DT in ASD patients were older age, revision surgery, chronic severe compression, a greater degree of osteotomy, complexity of surgery, RA, and a higher ASA grade. These findings will help guide spine surgeons in patient counseling as well as surgical planning.

Thermal Damage in Orthopaedics.

There are numerous potential sources of thermal damage encountered in orthopaedic surgery. An understanding of the preclinical mechanisms of thermal damage in tissues is necessary to minimize iatrogenic injuries and use these mechanisms therapeutically. Heat generation is a phenomenon that can be used to a surgeon's benefit, most commonly for hemostasis and local control of tumors. It is simultaneously one of the most dangerous by-products of orthopaedic techniques as a result of burring, drilling, cementation, and electrocautery and can severely damage tissues if used improperly. Similarly, cooling can be used to a surgeon's advantage in some orthopaedic subspecialties, but the potential for harm to tissues is also great. Understanding the potential of a given technique to rapidly alter local temperature-and the range of temperatures tolerated by a given tissue-is imperative to harness the power of heat and cold. In all subspecialties of orthopaedic surgery, thermal damage is a relevant topic that represents a direct connection between preclinical and clinical practice.

The sustained expression of Cas9 targeting toxic RNAs reverses disease phenotypes in mouse models of myotonic dystrophy type 1.

Myotonic dystrophy type I (DM1) is a multisystemic autosomal-dominant inherited human disorder that is caused by CTG microsatellite repeat expansions (MREs) in the 3' untranslated region of DMPK. Toxic RNAs expressed from such repetitive sequences can be eliminated using CRISPR-mediated RNA targeting, yet evidence of its in vivo efficacy and durability is lacking. Here, using adult and neonatal mouse models of DM1, we show that intramuscular or systemic injections of adeno-associated virus (AAV) vectors encoding nuclease-dead Cas9 and a single-guide RNA targeting CUG repeats results in the expression of the RNA-targeting Cas9 for up to three months, redistribution of the RNA-splicing protein muscleblind-like splicing regulator 1, elimination of foci of toxic RNA, reversal of splicing biomarkers and amelioration of myotonia. The sustained reversal of DM1 phenotypes provides further support that RNA-targeting Cas9 is a viable strategy for treating DM1 and other MRE-associated diseases.

Peptide Brush Polymers for Efficient Delivery of a Gene Editing Protein to Stem Cells.

The scarcity of effective means to deliver functional proteins to living cells is a central problem in biotechnology and medicine. Herein, we report the efficient delivery of an active DNA-modifying enzyme to human stem cells through high-density cell penetrating peptide brush polymers. Cre recombinase is mixed with a fluorophore-tagged polymer carrier and then applied directly to induced pluripotent stem cells or HEK293T cells. This results in efficient delivery of Cre protein as measured by activation of a genomically integrated Cre-mediated recombination reporter. We observed that brush polymer formulations utilizing cell penetrating peptides promoted Cre delivery but oligopeptides alone or oligopeptides displayed on nanoparticles did not. Overall, we report the efficient delivery of a genome-modifying enzyme to stem cells that may be generalizable to other, difficult-to-transduce cell types.

Self-Transfecting Micellar RNA: Modulating Nanoparticle Cell Interactions via High Density Display of Small Molecule Ligands on Micelle Coronas.

The intracellular delivery of synthetic nucleic acids represents a major challenge in biotechnology and in biomedicine. Methods to deliver short, double-stranded RNA to living cells are of particular interest because of the potential to engage the RNA interference machinery and to regulate mRNA expression. In this work, we describe novel RNA-polymer amphiphiles that assemble into spherical micellar nanoparticles with diameters of ca. 15-30 nm and efficiently enter live cells without transfection reagents. Each micelle consists of approximately 100 RNA strands forming a densely packed corona around a polymeric core. Importantly, the surface-displayed RNA remains accessible for hybridization with complementary RNA. Chemical modification of the termini of hybridized RNA strands enabled the display of small organic moieties on the outer surface of the micelle corona. We found that some of these modifications can have a tremendous impact on cellular internalization efficiencies. The display of hydrophobic dabcyl or stilbene units dramatically increased cell uptake, whereas hydrophilic neutral hydroxy or anionic phosphate residues were ineffective. Interestingly, neither of these modifications mediated noticeable uptake of free RNA oligonucleotides. We infer that their high density display on micellar nanoparticle surfaces is key for the observed effect; achieved with local effective surface concentrations in the millimolar range. We speculate that weak interactions with cell surface receptors that are amplified by the multivalent presentation of such modifications may be responsible. The installation of small molecule ligands on nanomaterial surfaces via hybridization of chemically modified oligonucleotides offers a simple and straightforward way to modulate cellular uptake of nanoparticles. Biological functionality of micellar RNA was demonstrated through the sequence-specific regulation of mRNA expression in HeLa cells.

Elimination of Toxic Microsatellite Repeat Expansion RNA by RNA-Targeting Cas9.

Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington's disease, and C9orf72-linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes. Here, we describe the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs. We observe specific targeting and efficient elimination of microsatellite repeat expansion RNAs both when exogenously expressed and in patient cells. Importantly, RNA-targeting Cas9 (RCas9) reverses hallmark features of disease including elimination of RNA foci among all conditions studied (DM1, DM2, C9-ALS, polyglutamine diseases), reduction of polyglutamine protein products, relocalization of repeat-bound proteins to resemble healthy controls, and efficient reversal of DM1-associated splicing abnormalities in patient myotubes. Finally, we report a truncated RCas9 system compatible with adeno-associated viral packaging. This effort highlights the potential of RCas9 for human therapeutics.

Programmable RNA Tracking in Live Cells with CRISPR/Cas9.

RNA-programmed genome editing using CRISPR/Cas9 from Streptococcus pyogenes has enabled rapid and accessible alteration of specific genomic loci in many organisms. A flexible means to target RNA would allow alteration and imaging of endogenous RNA transcripts analogous to CRISPR/Cas-based genomic tools, but most RNA targeting methods rely on incorporation of exogenous tags. Here, we demonstrate that nuclease-inactive S. pyogenes CRISPR/Cas9 can bind RNA in a nucleic-acid-programmed manner and allow endogenous RNA tracking in living cells. We show that nuclear-localized RNA-targeting Cas9 (RCas9) is exported to the cytoplasm only in the presence of sgRNAs targeting mRNA and observe accumulation of ACTB, CCNA2, and TFRC mRNAs in RNA granules that correlate with fluorescence in situ hybridization. We also demonstrate time-resolved measurements of ACTB mRNA trafficking to stress granules. Our results establish RCas9 as a means to track RNA in living cells in a programmable manner without genetically encoded tags.

Applications of Cas9 as an RNA-programmed RNA-binding protein.

The Streptococcus pyogenes CRISPR-Cas system has gained widespread application as a genome editing and gene regulation tool as simultaneous cellular delivery of the Cas9 protein and guide RNAs enables recognition of specific DNA sequences. The recent discovery that Cas9 can also bind and cleave RNA in an RNA-programmable manner indicates the potential utility of this system as a universal nucleic acid-recognition technology. RNA-targeted Cas9 (RCas9) could allow identification and manipulation of RNA substrates in live cells, empowering the study of cellular gene expression, and could ultimately spawn patient- and disease-specific diagnostic and therapeutic tools. Here we describe the development of RCas9 and compare it to previous methods for RNA targeting, including engineered RNA-binding proteins and other types of CRISPR-Cas systems. We discuss potential uses ranging from live imaging of transcriptional dynamics to patient-specific therapies and applications in synthetic biology.

Identification of novel long noncoding RNAs underlying vertebrate cardiovascular development.

BACKGROUND: Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators with important functions in development and disease. Here, we sought to identify and functionally characterize novel lncRNAs critical for vertebrate development. METHODS AND RESULTS: By relying on human pluripotent stem cell differentiation models, we investigated lncRNAs differentially regulated at key steps during human cardiovascular development with a special focus on vascular endothelial cells. RNA sequencing led to the generation of large data sets that serve as a gene expression roadmap highlighting gene expression changes during human pluripotent cell differentiation. Stage-specific analyses led to the identification of 3 previously uncharacterized lncRNAs, TERMINATOR, ALIEN, and PUNISHER, specifically expressed in undifferentiated pluripotent stem cells, cardiovascular progenitors, and differentiated endothelial cells, respectively. Functional characterization, including localization studies, dynamic expression analyses, epigenetic modification monitoring, and knockdown experiments in lower vertebrates, as well as murine embryos and human cells, confirmed a critical role for each lncRNA specific for each analyzed developmental stage. CONCLUSIONS: We have identified and functionally characterized 3 novel lncRNAs involved in vertebrate and human cardiovascular development, and we provide a comprehensive transcriptomic roadmap that sheds new light on the molecular mechanisms underlying human embryonic development, mesodermal commitment, and cardiovascular specification.

Intracellular mRNA regulation with self-assembled locked nucleic acid polymer nanoparticles.

We present an untemplated, single-component antisense oligonucleotide delivery system capable of regulating mRNA abundance in live human cells. While most approaches to nucleic acid delivery rely on secondary carriers and complex multicomponent charge-neutralizing formulations, we demonstrate efficient delivery using a simple locked nucleic acid (LNA)-polymer conjugate that assembles into spherical micellar nanoparticles displaying a dense shell of nucleic acid at the surface. Cellular uptake of soft LNA nanoparticles occurs rapidly within minutes as evidenced by flow cytometry and fluorescence microscopy. Importantly, these LNA nanoparticles knockdown survivin mRNA, an established target for cancer therapy, in a sequence-specific fashion as analyzed by RT-PCR.

Timing of C-arm drape contamination.

BACKGROUND: Surgical site infection remains a concern in orthopedic surgery, and contamination of C-arm covers is a potentially modifiable risk factor. METHODS: A single-cohort study was conducted using 30 consecutive patients undergoing operative fracture fixation. Cultures were obtained from the C-arm cover after initial draping and every 20 min thereafter. The total number of persons in the operating room (person-hours/h of study time) and the number of door openings were recorded. The C-arm position changes and the time to contamination were monitored. RESULTS: The median time from the start of the operation to contamination was 20 min. There was a 17% contamination rate on initial draping, 50% at 20 min, 57% at 40 min, and 80% by 80 min. The C-arms in five cases were not contaminated during the surgery. Time to contamination correlated significantly with lateral position changes (correlation [r]=0.64; p=0.003) but was not related to C-arm position changes (r=0.22; p=0.34), number of door openings (r=0.20; p=0.39), or person-hours/h (r=0.04; p=0.85). CONCLUSIONS: Contamination of the C-arm drape occurs often and early during surgery for orthopedic fractures. We recommend minimal contact with the C-arm to avoid contamination of the surgical field.

Alternative splicing in stem cell self-renewal and diferentiation.

This chapter provides a review of recent advances in understanding the importance of alternative pre-messenger RNA splicing in stem cell biology. The majority of transcribed pre-mRNAs undergo RNA splicing where introns are excised and exons are juxtaposed to form mature messenger RNA sequences. This regulated, selective removal of whole or portions of exons by alternative splicing provides avenues for control of RNA abundance and proteome diversity. We discuss several examples of key alternative splicing events in stem cell biology and provide an overview of recently developed microarray and sequencing technologies that enable systematic and genome-wide assessment of the extent of alternative splicing during stem cell differentiation.