Daniel Brian Carlin Reid

Next-generation SELEX identifies sequence and structural determinants of splicing factor binding in human pre-mRNA sequence

Many splicing factors interact with both mRNA and pre-mRNA. The identification of these interactions has been greatly improved by the development of in vivo cross-linking immunoprecipitation. However, the output carries a strong sampling bias in favor of RNPs that form on more abundant RNA species like mRNA. We have developed a novel in vitro approach for surveying binding on pre-mRNA, without cross-linking or sampling bias. Briefly, this approach entails specifically designed oligonucleotide pools that tile through a pre-mRNA sequence. The pool is then partitioned into bound and unbound fractions, which are quantified by a two-color microarray. We applied this approach to locating splicing factor binding sites in and around approximately 4000 exons. We also quantified the effect of secondary structure on binding. The method is validated by the finding that U1snRNP binds at the 5 splice site (5ss) with a specificity that is nearly identical to the splice donor motif. In agreement with prior reports, we also show that U1snRNP appears to have some affinity for intronic G triplets that are proximal to the 5ss. Both U1snRNP and the polypyrimidine tract binding protein (PTB) avoid exonic binding, and the PTB binding map shows increased enrichment at the polypyrimidine tract. For PTB, we confirm polypyrimidine specificity and are also able to identify structural determinants of PTB binding. We detect multiple binding motifs enriched in the PTB bound fraction of oligonucleotides. These motif combinations augment binding in vitro and are also enriched in the vicinity of exons that have been determined to be in vivo targets of PTB.

High-throughput mapping of protein occupancy identifies functional elements without the restriction of a candidate factor approach

There are a variety of in vivo and in vitro methods to determine the genome-wide specificity of a particular trans-acting factor. However there is an inherent limitation to these candidate approaches. Most biological studies focus on the regulation of particular genes, which are bound by numerous unknown trans-acting factors. Therefore, most biological inquiries would be better addressed by a method that maps all trans-acting factors that bind particular regions rather than identifying all regions bound by a particular trans-acting factor. Here, we present a high-throughput binding assay that returns thousands of unbiased measurements of complex formation on nucleic acid. We applied this method to identify transcriptional complexes that form on DNA regions upstream of genes involved in pluripotency in embryonic stem cells (ES cells) before and after differentiation. The raw binding scores, motif analysis and expression data are used to computationally reconstruct remodeling events returning the identity of the transcription factor(s) most likely to comprise the complex. The most significant remodeling event during ES cell differentiation occurred upstream of the REST gene, a transcriptional repressor that blocks neurogenesis. We also demonstrate how this method can be used to discover RNA elements and discuss applications of screening polymorphisms for allelic differences in binding.

Closed traumatic finger tip injuries in patients with artificial nails: removal of UV gel and acrylic nails

[1] Kim JK, Kook SH, Kim YK. Comparison of forearm rotation allowed by different types of upper extremity immobilization. J Bone Joint Surg 2012;94(5):455–60. [2] Bong MR, Egol KA, Leibman M, Koval KJ. A comparison of immediate postreduction splinting constructs for controlling initial displacement of fractures of the distal radius: a prospective randomized study of long-arm versus short-arm splinting. J Hand Surg [Am] 2006;31(5):766–70. [3] Mulford JS, Axelrod TS. Traumatic injuries of the distal radioulnar joint. Orthop Clin North Am 2007. http://dx.doi.org/10.1016/j.ocl.2007.03.007. [4] DeFroda SF, Gil JA, Bokshan S, Waryasz G. Upper extremity quad splint: indications and technique. Am J Emerg Med 2015;33(12):1818–22. [5] Denes AE, Goding R, Tamborlane J, Schwartz E. Maintenance of reduction of pediatric distal radius fractures with a sugar-tong splint. Am J Orthop 2007;36(2): 68–70. [6] Davis DI, Baratz M. Soft tissue complications of distal radius fractures. Hand Clin 2010;26:229–35. [7] Halanski M, Noonan KJ. Cast and splint immobilization: complications. J Am Acad Orthop Surg 2008;16:30–40. [8] Boyd AS, Benjamin HJ, Asplund C. Principles of casting and splinting. Am Fam Physician 2009;79:16–22. [9] Gannaway JK, Hunter JR. Thermal effects of casting materials. Clin Orthop Relat Res 1983:191–5. [10] Lavalette R, Pope MH, Dickstein H. Setting temperatures of plaster casts. The influence of technical variables. J Bone Joint Surg Am 1982;64:907–11.

Frailty and Health-Related Quality of Life Improvement Following Adult Spinal Deformity Surgery.

BACKGROUNDnAlthough the Adult Spinal Deformity Frailty Index (ASD-FI) predicts major complications and prolonged hospital length of stay after adult spinal deformity surgery, the impact of frailty on postoperative changes in health-related quality of life (HRQoL) is unknown.nnnMETHODSnPatients who underwent instrumented fusion of ≥4 levels for adult spinal deformity with minimum 2-year follow-up were stratified by Adult Spinal Deformity Frailty Index score into 3 groups: nonfrail, frail, and severely frail. Baseline and follow-up demographics, HRQoL measures, and radiographic parameters were analyzed. Primary outcome measures included proportion of patients who achieved substantial clinical benefit (SCB) in terms of Oswestry Disability Index, 36-Item Short Form Health Survey Physical Component Summary, and numeric back and leg pain scores.nnnRESULTSnInclusion criteria were met by 332 patients (135 nonfrail, 175 frail, 22 severely frail). Frail and severely frail patients were older and had more comorbidities, worse baseline HRQoL and pain scores, and worse radiographic deformity than nonfrail patients (P < 0.05). At 2-year follow-up, all outcome scores were worse in frail and severely frail patients compared with nonfrail patients. Frail patients improved more than nonfrail patients and were more likely to reach SCB for Oswestry Disability Index (43.7% vs. 29.3%; Pxa0= 0.025), 36-Item Short Form Health Survey Physical Component Summary (56.9% vs. 51.2%; Pxa0= 0.03), and leg pain (45.8% vs. 23.0%; Pxa0= 0.03) scores, but not back pain (57.5% vs. 63.4%; Pxa0= 0.045) score.nnnCONCLUSIONSnDespite higher risk stratification and worse baseline HRQoL, frail patients were more likely to reach SCB for most HRQoL measures compared with nonfrail patients. Severely frail patients were the least likely to reach SCB for most HRQoL measures.

A Polymorphic Microdeletion in the RGS9 Gene Suppresses PTB Binding and Associates with Obesity

Objective: RGS9 is a member of the family of Regulators of G-Protein Signaling (RGS) proteins defined by the presence of an RGS domain which can accelerate the GTPase-activity of G protein Gα subunits. An insertion/deletion (I/D) polymorphism of the nucleotide sequence TTTCT (rs3215227) has been identified in the human RGS9 gene, which matches the consensus high affinity binding motif for the ubiquitously expressed RNA binding Polypyrimidine Tract Binding Protein (PTB). In this study, we evaluate the genetic association and functional relevance of this polymorphism in type 2 diabetes and obesity. nSubjects and methods: We genotyped a larger population of 9272 Chinese and Malaysian individuals for the RGS9 I/D polymorphism using Taq Man allelic discrimination protocols. We found that the D allele of the RGS9 polymorphism was associated with a decreased prevalence of obesity in women (P=0.003, OR=0.753 95%CI 0.625-0.906) and girls (P=0.002, OR=0.604 95%CI 0.437-0.835). The association was moderate in boys (P=0.038, OR=0.724 95%CI 0.533-0.983) and not significant in men. Furthermore, we found that the transcript deletion variant exhibited a 10-fold reduction in PTB binding in vitro and that the splicing of the deletion variant was less affected by PTB co-expression. nConclusions: We provide genetic and biochemical data to support a genetic role of RGS9 in obesity but unlikely in T2D. The RGS9 I/D polymorphism influence the post-transcriptional processing of the gene through an altered affinity for the splicing factor PTB and are associated with obesity.

Rod Fracture After Apparently Solid Radiographic Fusion in Adult Spinal Deformity Patients.

BACKGROUNDnRod fracture occurs with delayed fusion or pseudarthrosis after adult spinal deformity (ASD) surgery. Rod fracture after apparent radiographic fusion has not been previously investigated.nnnMETHODSnPatients with ASD in a multicenter database were assessed for radiographic fusion by a committee of 3 spinal deformity surgeons. Fusions were rated as bilaterally fused (A), unilaterally fused (B), partially fused (C), or not fused (D). Patients with grade A or B fusion and 2-year follow-up were included. Patients with radiographic fusion were evaluated for subsequent rod fracture. Adjusted analyses were conducted with multiple logistic regression, using backwards-variable selection to a threshold of Pxa0<xa00.2, to assess for associated factors.nnnRESULTSnOf 402 patients with radiographically apparent solid fusion, 9.5% (38) subsequently suffered a broken rod. On multivariate analysis, greater rates of rod fracture were seen among patients of age group 60-69 years (vs. 18-49), body mass index 30-34 and 35+ (vs. <25), stainless-steel rods (vs. titanium), patients with rods ≤5.5 mm (vs. 6.35 mm), and patients with Charlson score 0 (vs. 3+). Of the 38 patients with rod fractures, 18 (47.4%) presented with worsened pain, and 8 (21.1%) required revision at minimum 2-year follow-up.nnnCONCLUSIONSnRod fracture occurred in 9.5% of patients with apparently solid radiographic fusion after ASD surgery. Advanced age, obesity, small diameter rods (5.5 mm), osteotomy, and lower comorbidity burden were significantly associated with rod fracture. Nearly one-half of these patients noted worsening pain, and 21.1% required revision surgery. Instrumentation failure may occur and may be symptomatic even in the setting of apparent fusion on plain radiographs.

Peak Timing for Complications After Adult Spinal Deformity Surgery

BACKGROUNDnOverall complication rates for adult spinal deformity (ASD) surgery have been reported; however, little data exist on the peak timing associated with specific complications. This study quantifies the peak timing for multiple complication types in an ASD cohort at minimum 2-year follow-up.nnnMETHODSnMulticenter, prospective analysis of all complications after ASD surgery in a consecutively enrolled cohort was performed. Inclusion criteria were ASD, age ≥18 years, spinal fusion ≥4 levels, and minimum 2-year follow-up. Complications included major and minor and specific complication types. Peak timing of specific complications was identified and described. Regression analysis was performed to assess correlation between patient/surgical factors and complication timing.nnnRESULTSnThere were 280 patients who met the inclusion criteria. Mean follow-up time was 2.9 years (range, 2-5 years). Of the patients, 209 (74.6%) had at least 1 complication, accounting for 529 total complications (258 minor and 271 major). Both major and minor complications peaked at <3 months. Infection and neurologic complications peaked at <3 months. Proximal junctional kyphosis had bimodal peaks at <3 and >24 months. Implant failure peaked at 12-24 and >24 months. There was a significant positive correlation between preoperative sagittal vertical axis and total complications at 6-12 months, major complications at 24 months, and reoperation. Body mass index was associated with total complications and implant failure at 12-24 and >24 months.nnnCONCLUSIONSnThe peak timing of specific complications after ASD surgery is identifiable. Understanding when these complications are likely to occur may improve patient counseling, early diagnosis, and prophylactic interventions and may help inform future reimbursement models.

Variability in Treatment for Patients with Cervical Spine Fracture and Dislocation: An Analysis of 107,152 Patients

BACKGROUNDnCervical spine injuries are a common cause of morbidity and mortality; however, the optimal treatment of many of these injuries is debated, and previous studies have shown substantial variation in treatment. We sought to examined treatment variation in arthrodesis and halo/tong placement in cervical spine injury patients over a 12-year period.nnnMETHODSnData from the Healthcare Cost and Utilization Project National Inpatient Sample, from 2000 to 2011, were used for this study. Patients were identified with a cervical vertebral facture or dislocation based on the International Classification of Diseases, 9th Revision codes. Using χ2 analysis, spinal arthrodesis rates and halo/tong placement rates were compared between hospitals based on teaching status for patients with and without spinal cord injury (SCI).nnnRESULTSnThe records of 107,152 patients with cervical fractures were examined. From 2000 to 2011, the overall arthrodesis rates fell from 25.2% to 20.6% (P < 0.001), and halo/tong placement rates fell from 13.2% to 3.6% (Pxa0<xa00.001). In patients with cervical fracture without SCI, arthrodesis rates fell from 17.6% to 13.9% (P < 0.001), in cervical fracture patients with SCI, arthrodesis rates rose from 50.0% to 58.9% (P < 0.001), and in cervical dislocation patients, arthrodesis rates rose from 47.6% to 57.5% (Pxa0<xa00.001). During the 12-year period, teaching hospitals had higher arthrodesis rates compared with nonteaching hospitals for patients with cervical fractures with SCI (57.3% vs. 53.4%, Pxa0= 0.001) and higher halo/tong placement rates for patients with cervical dislocations (2.7% vs. 1.7%, Pxa0= 0.004). Individual hospital variation showed a 3.5-fold variation in arthrodesis rates in 2000 to 2002, which fell to 3.0-fold by 2009 toxa02011.nnnCONCLUSIONSnArthrodesis rates for cervical fracture patients significantly decreased, and arthrodesis rates for cervical dislocation and SCI patients increased from 2000 to 2011, with variability in treatment based on hospital teaching status. Rates of halo/tong placement rapidly decreased for cervical spine trauma at both teaching and nonteaching hospitals. Individual hospital treatment variation also decreased over the study period. Further clinical studies examining the optimal treatment for spine trauma may lead to continued decreases in treatment variability.

Effect of Narcotic Prescription Limiting Legislation on Opioid Utilization Following Lumbar Spine Surgery

BACKGROUND CONTEXTnPrescription opioid abuse is a public health emergency. Opioid prescriptions for spine patients account for a large proportion of use. Some states have implemented statutory limits on prescribers, however it remains unclear whether such laws are effective.nnnPURPOSEnThis investigation compares opioid prescription patterns for patients undergoing lumbar spine surgery before and after the passage of statewide narcotic-limiting legislation in Rhode Island.nnnSTUDY DESIGN/SETTINGnRetrospective review of prospectively-collected medical and pharmacologic data.nnnPATIENT SAMPLEnTwo patient cohorts (pre-law January 1, 2016-June 31, 2016 and post-law June 1, 2017-December 31, 2017) that included all patients undergoing selected lumbar spine surgeries (lumbar discectomy, lumbar decompression without fusion, and posterior lumbar fusion).nnnMETHODSnDemographic and surgical variables were collected from the patients medical charts, and information on controlled substances was collected from the state prescription drug monitoring program database. Variables collected included the number of pills and total morphine milligram equivalents (MMEs) of the first prescription, number of prescriptions filled within 30 days of surgery, total MMEs filled in the 30-day postoperative period, and total MMEs filled from 30 to 90 days after surgery. For comparison of continuous variables, t test or Mann-Whitney U test were used as appropriate. Chi-squared analysis was utilized for comparison of categorical variables. Independent risk factors for prolonged postoperative opioid use were evaluated using logistic regression.nnnRESULTSnThere were no significant differences between pre-law (nu202f=u202f241) and post-law (nu202f=u202f311) cohorts in terms of age, sex, preoperative opioid use, or preoperative anxiolytic use (p > .05). A greater than 50% decline was observed among all patients from the pre-law to the post-law period in terms of the number of pills (51.61 vs 23.60 pills, p < .001) and MMEs (525.56 vs 218.77 MMEs, p < .001) provided in the first postoperative opioid prescription. The mean total MMEs provided in the first 30 days decreased significantly (891.26 vs 628.63 MMEs, p < .001) despite an increase in the average number of opioid prescriptions filled (1.75 vs 2.04 prescriptions, p = .002) during this time. There was no significant difference in mean MMEs filled from 30 to 90 days. Upon subgroup analysis, there was a statistically significant decline in both the mean first prescription and total 30-day MMEs regardless of preoperative opioid status (all p < .05) or specific procedure performed (all p < .05). Preoperative opioid use was strongly associated with prolonged postoperative opioid requirements throughout the study period (OR 4.71, 95% CI 3.11-7.13, p < .001). There were no significant differences between cohorts in terms of emergency department (ED) visits or unplanned hospital readmissions at 30 and 90 days following surgery (all p > .05).nnnCONCLUSIONSnThe institution of mandatory statewide opioid prescription limits has resulted in a significant reduction in initial and 30-day opioid prescriptions following lumbar spine surgery. Decreased opioid utilization was observed in all patients, regardless of preoperative opioid tolerance or procedure performed. No significant change in postoperative ED visits or unplanned hospital readmissions was seen following implementation of the legislation. This investigation provides preliminary evidence that narcotic limiting legislation may be effective in decreasing opioid prescriptions after lumbar spine surgery for both opioid-naïve and opioid-tolerant patients.

Spondylolysis and Spondylolisthesis

Spondylolysis and spondylolisthesis are caused by a stress fracture of the pars interarticularis and are especially common in athletes required to hyperextend repetitively such as football linemen and gymnasts. Bracing and physical therapy are the mainstays of initial conservative care. Operative intervention is generally reserved for failure of conservative care except in high-grade slips.