A systematic evaluation of the design and context dependencies of massively parallel reporter assays.

Massively parallel reporter assays (MPRAs) functionally screen thousands of sequences for regulatory activity in parallel. To date, there are limited studies that systematically compare differences in MPRA design. Here, we screen a library of 2,440 candidate liver enhancers and controls for regulatory activity in HepG2 cells using nine different MPRA designs. We identify subtle but significant differences that correlate with epigenetic and sequence-level features, as well as differences in dynamic range and reproducibility. We also validate that enhancer activity is largely independent of orientation, at least for our library and designs. Finally, we assemble and test the same enhancers as 192-mers, 354-mers and 678-mers and observe sizable differences. This work provides a framework for the experimental design of high-throughput reporter assays, suggesting that the extended sequence context of tested elements and to a lesser degree the precise assay, influence MPRA results.

Saturation editing of genomic regions by multiplex homology-directed repair.

Saturation mutagenesis--coupled to an appropriate biological assay--represents a fundamental means of achieving a high-resolution understanding of regulatory and protein-coding nucleic acid sequences of interest. However, mutagenized sequences introduced in trans on episomes or via random or "safe-harbour" integration fail to capture the native context of the endogenous chromosomal locus. This shortcoming markedly limits the interpretability of the resulting measurements of mutational impact. Here, we couple CRISPR/Cas9 RNA-guided cleavage with multiplex homology-directed repair using a complex library of donor templates to demonstrate saturation editing of genomic regions. In exon 18 of BRCA1, we replace a six-base-pair (bp) genomic region with all possible hexamers, or the full exon with all possible single nucleotide variants (SNVs), and measure strong effects on transcript abundance attributable to nonsense-mediated decay and exonic splicing elements. We similarly perform saturation genome editing of a well-conserved coding region of an essential gene, DBR1, and measure relative effects on growth that correlate with functional impact. Measurement of the functional consequences of large numbers of mutations with saturation genome editing will potentially facilitate high-resolution functional dissection of both cis-regulatory elements and trans-acting factors, as well as the interpretation of variants of uncertain significance observed in clinical sequencing.

The Distress and Risk Assessment Method predicts postoperative narcotic use in patients undergoing rotator cuff repair.

BACKGROUND: The Distress and Risk Assessment Method (DRAM) predicts poor outcomes in spine, hip, and knee surgery. Unlike other areas of orthopedic surgery, DRAM scores are not predictive of lower postoperative patient-reported outcomes after arthroscopic rotator cuff repair (RCR). PURPOSE: As concerns for opioid dependence and abuse grow, the purpose of this study was to analyze the correlation between preoperative DRAM scores, modified Zung scores, and postoperative narcotic use in patients who underwent arthroscopic RCR. MATERIALS AND METHODS: This prospective cohort study identified and enrolled patients >18 years of age with full-thickness rotator cuff tears at a single institution. Patients with prior shoulder surgery, greater than 1-tendon RCR, and preoperative narcotic use were excluded. One-hundred and fifty patients were enrolled, with 114 (76%) completing all preoperative and postoperative questionnaires. Preoperative DRAM scores were collected from every patient. Postoperative narcotic use was evaluated via survey and converted to total morphine equivalents. RESULTS: Increased preoperative DRAM scores predicted higher postoperative morphine equivalent units (P = .002, r = 0.29). When dividing patients into those <17 or ≥17 on the modified Zung score, 44 of 114 (39%) met criteria for "at risk or depressed." This group showed a statistically significant trend toward higher postoperative morphine equivalent unit intake (P = .004). CONCLUSION: Baseline psychological distress (DRAM) can predict narcotic requirements after RCR and serve as a powerful tool to identify patients at risk for increased narcotics requirements postoperatively. In our cohort, 39% of patients showed evidence of baseline depression, which highlights a potential role of the modified Zung score to identify patients in need of preoperative psychological counseling.

Multiplex pairwise assembly of array-derived DNA oligonucleotides.

While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synthesis lengths, high synthesis error rates, low yield and the challenges of assembling long constructs from complex pools. Toward addressing these issues, we developed a protocol for multiplex pairwise assembly of oligos from array-synthesized oligonucleotide pools. To evaluate the method, we attempted to assemble up to 2271 targets ranging in length from 192-252 bases using pairs of array-synthesized oligos. Within sets of complexity ranging from 131-250 targets, we observed error-free assemblies for 90.5% of all targets. When all 2271 targets were assembled in one reaction, we observed error-free constructs for 70.6%. While the assembly method intrinsically increased accuracy to a small degree, we further increased accuracy by using a high throughput 'Dial-Out PCR' protocol, which combines Illumina sequencing with an in-house set of unique PCR tags to selectively amplify perfect assemblies from complex synthetic pools. This approach has broad applicability to DNA assembly and high-throughput functional screens.

Does an Algorithmic Approach to Using Brachytherapy and External Beam Radiation Result in Good Function, Local Control Rates, and Low Morbidity in Patients With Extremity Soft Tissue Sarcoma?

BACKGROUND: High-dose-rate brachytherapy (HDR-BT) and external-beam radiation therapy (EBRT) are two modalities used in the treatment of soft tissue sarcoma. Previous work at our institution showed early complications and outcomes for patients treated with HDR-BT, EBRT, or a combination of both radiation therapy modalities. As the general indications for each of these approaches to radiation therapy differ, it is important to evaluate the use of each in an algorithmic way, reflecting how they are used in contemporary practice at sites that use these treatments. QUESTION/PURPOSES: (1) To determine the proportions of intermediate- and long-term complications associated with the use of brachytherapy in the treatment of primary high-grade extremity soft tissue sarcomas; (2), to characterize the long-term morbidity of the three radiation treatment groups using the Radiation Therapy Oncology Group/ European Organization for Research and Treatment of Cancer (RTOG/EORTC) Late Radiation Morbidity Scoring Scheme; (3) to determine whether treatment with HDR-BT, EBRT, and HDR-BT+EBRT therapy, in combination with limb-salvage surgery, results in acceptable local control in this high-risk group of sarcomas. METHODS: We retrospectively studied data from 171 patients with a diagnosis of high-grade extremity soft tissue sarcoma treated with limb-sparing surgery and radiation therapy between 1990 and 2012 at our institution, with a mean followup of 72 months. Of the 171 patients, 33 (20%) were treated with HDR-BT, 128 (75%) with EBRT, and 10 (6%) with HDR-BT+EBRT. We excluded 265 patients with soft tissue sarcomas owing to axial tumor location, previous radiation to the affected extremity, incomplete patient records, patients receiving primary amputation, recurrent tumors, pediatric patients, low- and intermediate-grade tumors, and rhabdoid histology. Fifteen patients (9%) were lost to followup for any reason including died of disease or other causes during the first 12 months postoperatively. This included four patients who received HDR-BT (12%), 11 who received EBRT (9%), and none who received HDR-BT+EBRT (0%) with less than 12 months followup. Determination of radiation therapy technique for each patient was individualized in a multidisciplinary forum of sarcoma specialists. Anticipated close or positive surgical margins and a low likelihood of complex soft tissue procedures were factors that encouraged use of brachytherapy, whereas the anticipated need for secondary procedures and/or soft tissue coverage encouraged use of EBRT alone. Combination therapy was used when the treatment volume exceeded the treatment field of the brachytherapy catheters or when the catheters were used to boost a close or positive surgical margin. Local recurrence, complications, and morbidity outcomes scores (RTOG) were calculated based on chart review. Between-group comparisons pertaining to the proportion of patients experiencing complications, morbidity outcomes scores, and local recurrence rates were not performed because of dissimilarities among the patients in each group at baseline. RESULTS: The HDR-BT treatment group showed a high incidence of intermediate-term complications, with the three most common being: deep infection (33%, 11 of 33); dehiscence and delayed wound healing (24%, eight of 33); and seroma and hematoma (21%, seven of 33). The EBRT group showed a high incidence of intermediate- and long-term complications with the three most common being: chronic radiation dermatitis (35%, 45 of 128); fibrosis (27%, 35 of 128); and chronic pain and neuritis (13%, 16 of 128). The RTOG scores for each treatment group were: HDR-BT 0.8 ± SD 1.2; EBRT 1.9 ± 2.0; and HDR-BT+EBRT 1.7 ± 1.7. Overall, 142 of 169 (84%) patients were free from local recurrence: 27 (82%) in the HDR-BT group, 108 (86%) in the EBRT group, and seven (70%) in the combination therapy group. CONCLUSIONS: In this single-institution study, an algorithmic approach to using HDR-BT and EBRT in the treatment of patients with high-grade soft tissue sarcomas can yield acceptable complication rates, good morbidity outcome scores, and a high degree of local control. Based on these results, we believe HDR-BT is best for patients with an anticipated close margin, a positive surgical margin, and for patients who are unlikely to receive a complex soft tissue procedure. Conversely, if a secondary procedure and/or soft tissue coverage are likely to be used, EBRT alone may be reasonable. Finally, combination therapy might be considered when the treatment volume exceeded the treatment field capacity for HDR-BT or when the catheters were used to boost a close or positive surgical margin. LEVEL OF EVIDENCE: Level IV, therapeutic study.

Baseline Cartilage Thickness and Meniscus Extrusion Predict Longitudinal Cartilage Loss by Quantitative Magnetic Resonance Imaging: Data From the Osteoarthritis Initiative.

OBJECTIVE: This study aimed to assess how meniscus damage and baseline cartilage thickness influence the rate of cartilage loss and knee pain. METHODS: Of 4796 participants in the Osteoarthritis Initiative, 86 had baseline and 48-month follow-up quantitative magnetic resonance imaging data for medial compartment cartilage thickness. Baseline meniscus pathology was scored by a musculoskeletal radiologist using Whole-Organ Magnetic Resonance Imaging Score. Findings were correlated with 72-month Knee injury and Osteoarthritis Outcome Score. RESULTS: Univariate analysis showed cartilage change was not influenced by demographic variables. Multivariable regression revealed that initial cartilage thickness (-1.07 mm at 48 months for every 1 mm decrease at baseline, P < 0.001) and meniscus extrusion (-0.33 mm if present at baseline, P < 0.001) were the strongest predictors of medial compartment cartilage thickness at 48 months. Knee injury and Osteoarthritis Outcome Score pain scores did not correlate with cartilage loss. CONCLUSIONS: Baseline cartilage thickness and meniscus extrusion are important and independent predictors for accelerated cartilage loss. However, the degree of cartilage loss did not correlate with midterm change in clinical outcome scores.

Hepatocyte-targeted RNAi therapeutics for the treatment of chronic hepatitis B virus infection.

RNA interference (RNAi)-based therapeutics have the potential to treat chronic hepatitis B virus (HBV) infection in a fundamentally different manner than current therapies. Using RNAi, it is possible to knock down expression of viral RNAs including the pregenomic RNA from which the replicative intermediates are derived, thus reducing viral load, and the viral proteins that result in disease and impact the immune system's ability to eliminate the virus. We previously described the use of polymer-based Dynamic PolyConjugate (DPC) for the targeted delivery of siRNAs to hepatocytes. Here, we first show in proof-of-concept studies that simple coinjection of a hepatocyte-targeted, N-acetylgalactosamine-conjugated melittin-like peptide (NAG-MLP) with a liver-tropic cholesterol-conjugated siRNA (chol-siRNA) targeting coagulation factor VII (F7) results in efficient F7 knockdown in mice and nonhuman primates without changes in clinical chemistry or induction of cytokines. Using transient and transgenic mouse models of HBV infection, we show that a single coinjection of NAG-MLP with potent chol-siRNAs targeting conserved HBV sequences resulted in multilog repression of viral RNA, proteins, and viral DNA with long duration of effect. These results suggest that coinjection of NAG-MLP and chol-siHBVs holds great promise as a new therapeutic for patients chronically infected with HBV.

Benzene-contaminated toluene and acute myeloid leukemia: a case series and review of literature.

We report seven cases of acute myeloid leukemia (AML) with occupational exposure to a toluene-based hydrocarbon solvent. The cases were employed at a facility, which manufactured rubber belts and hoses, between 1950 and 2005 for periods ranging from 21 to 37 total years. Detailed histories were obtained for three workers who were diagnosed with AML within a 3-year period (2003-2005). Death certificates, medical records, and accounts by workers were reviewed. Benzene, a known cause of AML, is typically a contaminant of toluene. Benzene contamination in toluene and other widely used solvents and the potential for concurrent benzene exposure during usage of these solvents in occupational settings are discussed.

Post-operative Urinary Dysfunction Following Shoulder Surgery: Rates and Risk Factors.

Background: Post-operative urinary retention (POUR) and dysfunction are recognized complications following orthopaedic surgery. Recent literature has focused on urinary retention and its associated complications following hip and knee reconstruction and lower extremity surgery. There is a paucity of literature focusing on POUR and shoulder surgery. The purpose of this study is to describe the rates of urinary dysfunction in patients undergoing shoulder surgery as well as the associated risk factors. Methods: This was a single institution, prospective cohort study. Eligibility criteria included patients older than 50 years of age undergoing open or arthroscopic shoulder surgery. The primary outcome was the American Urological Association (AUA) symptom score (7 questions total scored 0-5, total 35 points max) administered before and after surgery. Higher scores reflect worse urinary dysfunction. Intra-operative data such as type of surgery, type of anesthesia, use of anticholinergics, peripheral nerve block, length of case, and amount of intravenous fluids were collected. Results: Of 194 patients, the mean age was 61.4 years (Standard Deviation (S.D.) = 13.0)) and the average BMI was 29.2 (S.D. = 5.6). The sample was 35.6% female. Overall, 46.4% reported worse AUA scores post-operatively within the first 3 to 5 days, including 4.1% of which were clinically defined as "moderately worse" (>5 point worse) or "much worse" (>11 points worse). Worse preoperative AUA scores correlated with worse postop AUA score on linear regression analysis (r=0.883, P<0.0001). Males with a history of BPH showed a statistically significant positive association with worsening urinary dysfunction postoperatively (P=0.039). Four patients (2.1%) required postoperative catheterization. A significantly higher percentage of patients with preoperative AUA scores of ≥11 experienced worsening of urinary function post-operatively (P=0.04). Conclusion: Worsening of urinary function following shoulder surgery is common. The AUA score may be used to identify at-risk patients and to track changes in urinary function post-operatively. Men with a diagnosis of BPH are at particularly high risk. Further investigation is needed to elucidate the impact of urinary dysfunction on patient outcomes, satisfaction, and cost as well as the role of prophylactic medications.

Repair of flexor digitorum profundus to distal phalanx: a biomechanical evaluation of four techniques.

PURPOSE: Many techniques for repair of the flexor digitorum profundus to the distal phalanx show excessive gapping with variable clinical results. The purpose of this study was to test the biomechanical characteristics of an anchor-button (AB) technique, as compared to 3 other techniques. METHODS: Twenty-four fresh-frozen human cadaveric fingers were randomized to 4 groups, 6 in each: group 1, 2-strand Bunnell suture button pullout technique; group 2, modified Kessler suture and 2 retrograde anchors; group 3: locking Krakow suture with 2 retrograde anchors; group 4, AB technique incorporating a 2-part repair, consisting of a locking dorsal Krakow suture with 2 retrograde anchors and a locking palmar Krakow suture fixed with a button. Tendon-to-bone gapping was measured after cyclical loading. Ultimate load to failure was measured at the end of 500 cycles. RESULTS: The AB technique resulted in significantly less gapping when compared to the other techniques. It also resulted in a significantly stronger repair compared to all the other groups with an average load to failure comparable to the native tendon-to-bone interface. CONCLUSIONS: The AB repair might allow for early active postoperative motion after repair of flexor digitorum profundus avulsion injuries and tendon reconstruction procedures; however, the soft tissue effects of this multistrand technique are unknown in clinical repairs.

Short term outcomes after transcatheter mitral valve repair.

BACKGROUND: Outcome data following transcatheter mitral valve repair (TMVR) with the MITRACLIP® device are scarce outside the pivotal randomized controlled trials. METHODS: The Nationwide Readmission Data base (NRD) was utilized for years 2013-2017 to identify the study population. Thirty-day readmission pattern, in-hospital complications, causes of readmissions, and multivariate predictors for readmission, complications and mortality were explored. RESULTS: We noted a total of 14,647 index admissions related to MITRACLIP of which 48% of procedures were performed at high volume centers (Annual hospital volume ≥ 25). A total of 15% of patients were readmitted within 30 days of discharge most frequently due to cardiac causes. Approximately 33% of patients were discharged within 24 h of the procedure. The in-hospital mortality rate was 2.8% and in-hospital complication rate was 14.6%. The most common complications were cardiac complications (8.2%), bleeding related complications (5.9%) and vascular complications (0.65%). On multivariate modeling, female sex, CHF, Atrial fibrillation, prior PCI, COPD, CKD, transfer to skilled nursing facility, length of stay ≥2 days were associated with a high risk of readmission. Additionally, coagulopathy, chronic kidney disease and lengthier hospital stays were associated with high risk of complication or death. CONCLUSION: The 30-day readmission rate following commercial treatment with the MITRACLIP device is 15%. Half of these admission were from a cardiac etiology. Heart failure, atrial arrhythmias and clip related complications round out the top 3 cardiac reasons for readmission. There was no impact of hospital size, teaching status or case volume on mortality and in hospital complication rates.

Characteristics and Outcomes of Patients Admitted With Type 2 Myocardial Infarction.

Type 2 myocardial infarction (Type 2 MI) is a common problem and carries a high diagnostic uncertainty. Large studies exploring outcomes in type 2 MI are lacking. Nationwide Readmission Database (2017) was queried using the International Classification of Diseases codes (ICD-10-CM) to identify type 2 MI patients. Characteristics, in-hospital outcomes, 30-day readmissions, and predictors of in-hospital mortality as well as 30-day readmissions were explored. We identified 21,738 patients with a diagnosis of type 2 MI. Most common primary diagnosis at presentation included infection/sepsis (27.5%), hypertensive heart disease (15.3%) and pulmonary diseases (8.5%). Overall, in-hospital mortality and 30-day readmission for patients with type 2 MI were 9.0% and 19.1% respectively. On multivariable analysis, significant predictors of increased in-hospital mortality included male gender, coexisting atrial fibrillation/flutter, peripheral vascular disease, coagulopathy, malignancy, and fluid/electrolyte abnormalities. Significant predictors of 30-day readmission were coexisting diabetes mellitus, atrial fibrillation/ flutter, carotid artery stenosis, anemia, COPD, CKD and prior history of myocardial infarction, A primary diagnosis of sepsis, pulmonary issues including respiratory failure, neurological conditions including stroke carried highest risk of mortality however readmission risk was not influenced by primary diagnosis at presentation. In conclusion, approximately 1 in 10 patients admitted for type 2 MI died during admission, and nearly 1 in 5 patients were readmitted at 30 days after discharge. In-hospital mortality varied based on associated primary diagnosis at presentation. Proposed predictive model for mortality and 30-day readmission in our study can help to target high risk patients for post-Type 2 MI care.

Osseous changes following reverse total shoulder arthroplasty combined with latissimus dorsi transfer: a case series.

BACKGROUND: This is the first report on the incidence of proximal humerus osseous changes and associated clinical consequences in reverse total shoulder arthroplasty combined with a latissimus dorsi tendon transfer (RTSA+LDT). METHODS: A multicenter, retrospective review identified all patients who had undergone a primary RTSA+LDT and had at least 3-month radiographic follow-up between 2012 and 2017. Data collection included demographics, oral steroid use, repair technique for LDT fixation, radiographic humeral osseous changes, complications, and need for revision surgery. RESULTS: Twenty-four patients were included with an average age of 70.7 ± 7.9 years and follow-up of 16.3 (3-50) months. Ten patients (41.7%) developed osseous changes at the transfer location. There was no increased risk of developing osseous changes based on the surgical fixation technique (P = .421). Average time to earliest radiographic detection of osseous changes was 2.7 ± 1.7 months, with all changes occurring at or before 6 months. Two patients developed proximal humerus fractures, of which 1 had osseous changes through which the fracture occurred. DISCUSSION: RTSA+LDT may place the proximal humeral cortex at greater risk than previously described. Using a long-stem prosthesis in the setting of RTSA+LDT may limit the consequences of this complication.

speck, First Identified in Drosophila melanogaster in 1910, Is Encoded by the Arylalkalamine N-Acetyltransferase (AANAT1) Gene.

The pigmentation mutation speck is a commonly used recombination marker characterized by a darkly pigmented region at the wing hinge. Identified in 1910 by Thomas Hunt Morgan, speck was characterized by Sturtevant as the most "workable" mutant in the rightmost region of the second chromosome and eventually localized to 2-107.0 and 60C1-2. Though the first speck mutation was isolated over 110 years ago, speck is still not associated with any gene. Here, as part of an undergraduate-led research effort, we show that speck is encoded by the Arylalkylamine N-acetyltransferase 1 (AANAT1) gene. Both alleles from the Morgan lab contain a retrotransposon in exon 1 of the RB transcript of the AANAT1 gene. We have also identified a new insertion allele and generated multiple deletion alleles in AANAT1 that all give a strong speck phenotype. In addition, expression of AANAT1 RNAi constructs either ubiquitously or in the dorsal portion of the developing wing generates a similar speck phenotype. We find that speck alleles have additional phenotypes, including ectopic pigmentation in the posterior pupal case, leg joints, cuticular sutures and overall body color. We propose that the acetylated dopamine generated by AANAT1 decreases the dopamine pool available for melanin production. When AANAT1 function is decreased, the excess dopamine enters the melanin pathway to generate the speck phenotype.