Zebrafish raptor mutation inhibits the activity of mTORC1, inducing craniofacial defects due to autophagy-induced neural crest cell death.

The mechanistic target of rapamycin (mTOR) coordinates metabolism and cell growth with environmental inputs. mTOR forms two functional complexes: mTORC1 and mTORC2. Proper development requires both complexes but mTORC1 has unique roles in numerous cellular processes, including cell growth, survival and autophagy. Here, we investigate the function of mTORC1 in craniofacial development. We created a zebrafish raptor mutant via CRISPR/Cas9, to specifically disrupt mTORC1. The entire craniofacial skeleton and eyes were reduced in size in mutants; however, overall body length and developmental timing were not affected. The craniofacial phenotype associates with decreased chondrocyte size and increased neural crest cell death. We found that autophagy is elevated in raptor mutants. Chemical inhibition of autophagy reduced cell death and improved craniofacial phenotypes in raptor mutants. Genetic inhibition of autophagy, via mutation of the autophagy gene atg7, improved facial phenotypes in atg7;raptor double mutants, relative to raptor single mutants. We conclude that finely regulated levels of autophagy, via mTORC1, are crucial for craniofacial development.

Stingray Sensor System for Persistent Survey of the GEO Belt.

The Stingray sensor system is a 15-camera optical array dedicated to the nightly astrometric and photometric survey of the geosynchronous Earth orbit (GEO) belt visible above Tucson, Arizona. The primary scientific goal is to characterize GEO and near-GEO satellites based on their observable properties. This system is completely autonomous in both data acquisition and processing, with human oversight reserved for data quality assurance and system maintenance. The 15 ZWO ASI1600MM Pro cameras are mated to Sigma 135 mm f/1.8 lenses and are controlled simultaneously by four separate computers. Each camera is fixed in position and observes a 7.6-by-5.8-degree portion of the GEO belt, for a total of a 114-by-5.8-degree field of regard. The GAIA DR2 star catalog is used for image astrometric plate solution and photometric calibration to GAIA G magnitudes. There are approximately 200 near-GEO satellites on any given night that fall within the Stingray field of regard, and all those with a GAIA G magnitude brighter than approximately 15.5 are measured by the automated data reduction pipeline. Results from an initial one-month survey show an aggregate photometric uncertainty of 0.062 ± 0.008 magnitudes and astrometric accuracy consistent with theoretical sub-pixel centroid limits. Provided in this work is a discussion of the design and function of the system, along with verification of the initial survey results.

Loss of tumor protein 53 protects against alcohol-induced facial malformations in mice and zebrafish.

BACKGROUND: Alcohol exposure during the gastrulation stage of development causes the craniofacial and brain malformations that define fetal alcohol syndrome. These malformations, such as a deficient philtrum, are exemplified by a loss of midline tissue and correspond, at least in part, to regionally selective cell death in the embryo. The tumor suppressor protein Tp53 is an important mechanism for cell death, but the role of Tp53 in the consequences of alcohol exposure during the gastrulation stage has yet to be examined. The current studies used mice and zebrafish to test whether genetic loss of Tp53 is a conserved mechanism to protect against the effects of early developmental stage alcohol exposure. METHODS: Female mice, heterozygous for a mutation in the Tp53 gene, were mated with Tp53 heterozygous males, and the resulting embryos were exposed during gastrulation on gestational day 7 (GD 7) to alcohol (two maternal injections of 2.9 g/kg, i.p., 4 h apart) or a vehicle control. Zebrafish mutants or heterozygotes for the tp53zdf1  M214K mutation and their wild-type controls were exposed to alcohol (1.5% or 2%) beginning 6 h postfertilization (hpf), the onset of gastrulation. RESULTS: Examination of GD 17 mice revealed that eye defects were the most common phenotype among alcohol-exposed fetuses, occurring in nearly 75% of the alcohol-exposed wild-type fetuses. Tp53 gene deletion reduced the incidence of eye defects in both the heterozygous and mutant fetuses (to about 35% and 20% of fetuses, respectively) and completely protected against alcohol-induced facial malformations. Zebrafish (4 days postfertilization) also demonstrated alcohol-induced reductions of eye size and trabeculae length that were less common and less severe in tp53 mutants, indicating a protective effect of tp53 deletion. CONCLUSIONS: These results identify an evolutionarily conserved role of Tp53 as a pathogenic mechanism for alcohol-induced teratogenesis.

Prepectoral Versus Subpectoral Breast Reconstruction in High-Body Mass Index Patients.

BACKGROUND: The effect of body mass index (BMI) on complication rates in prepectoral implant-based breast reconstruction is not well established. The purpose of this study was to compare complication rates between different BMI groups in subpectoral and prepectoral reconstruction. METHODS: A single-surgeon, 4-year, retrospective review was performed of consecutive prosthetic breast reconstructions. During this time, the senior author's practice shifted from a subpectoral to prepectoral technique. Patients were stratified into BMI subgroups (<25, 25-35, and >35 kg/m2) and complication rates were analyzed. A survey was administered to blinded medical personnel and patients comparing esthetic results. RESULTS: Implant-based reconstructions were performed in 195 patients (103 subpectoral and 92 prepectoral). No significant difference in major complication rate was observed between techniques. Among patients with BMI greater than 35 kg/m2, implant exposure occurred at a significantly higher rate in the prepectoral group (P = 0.04). In patients with BMI greater than 25 kg/m2, minor asymmetry was more prevalent with prepectoral reconstruction (12.3% vs 0%; P = 0.02). Regardless of technique, the odds of reoperation increased by 7% per point increase in BMI, although this did not reach statistical significance (P = 0.07; odds ratio, 1.07; 95% confidence interval, 0.99-1.15).A total of 66 survey responses were received. Physicians rated esthetic results more positively than patients did. Patients with a BMI of less than 25 kg/m2 were rated better than other BMI groups in nearly all categories. The position of submuscular reconstruction was rated significantly better than prepectoral. CONCLUSIONS: There is a trend toward higher complication rates in prepectoral versus subpectoral breast reconstruction with increasing BMI. Nonetheless, the technique appears to be safe, with comparable clinical and cosmetic results.

An unsupervised machine learning method for discovering patient clusters based on genetic signatures.

INTRODUCTION: Many chronic disorders have genomic etiology, disease progression, clinical presentation, and response to treatment that vary on a patient-to-patient basis. Such variability creates a need to identify characteristics within patient populations that have clinically relevant predictive value in order to advance personalized medicine. Unsupervised machine learning methods are suitable to address this type of problem, in which no a priori class label information is available to guide this search. However, it is challenging for existing methods to identify cluster memberships that are not just a result of natural sampling variation. Moreover, most of the current methods require researchers to provide specific input parameters a priori. METHOD: This work presents an unsupervised machine learning method to cluster patients based on their genomic makeup without providing input parameters a priori. The method implements internal validity metrics to algorithmically identify the number of clusters, as well as statistical analyses to test for the significance of the results. Furthermore, the method takes advantage of the high degree of linkage disequilibrium between single nucleotide polymorphisms. Finally, a gene pathway analysis is performed to identify potential relationships between the clusters in the context of known biological knowledge. DATASETS AND RESULTS: The method is tested with a cluster validation and a genomic dataset previously used in the literature. Benchmark results indicate that the proposed method provides the greatest performance out of the methods tested. Furthermore, the method is implemented on a sample genome-wide study dataset of 191 multiple sclerosis patients. The results indicate that the method was able to identify genetically distinct patient clusters without the need to select parameters a priori. Additionally, variants identified as significantly different between clusters are shown to be enriched for protein-protein interactions, especially in immune processes and cell adhesion pathways, via Gene Ontology term analysis. CONCLUSION: Once links are drawn between clusters and clinically relevant outcomes, Immunochip data can be used to classify high-risk and newly diagnosed chronic disease patients into known clusters for predictive value. Further investigation can extend beyond pathway analysis to evaluate these clusters for clinical significance of genetically related characteristics such as age of onset, disease course, heritability, and response to treatment.

Comminuted olecranon fractures: biomechanical testing of locked versus minifragment non-locked plate fixation.

INTRODUCTION: Open reduction and internal fixation has long been accepted as optimal treatment for displaced olecranon fractures based on poor results seen with conservative management. With the presence of comminution, tension-band wiring constructs are contraindicated due to tendency to compress through fragments, thereby shortening the articular segment. Therefore, plate fixation is typically employed. Our hypothesis was that in a comminuted fracture model, 2.7 mm reconstruction plating without locking screws will perform equally to 3.5 mm locked plating in terms of fracture displacement and rotation (shear). MATERIALS AND METHODS: A three-part comminuted olecranon fracture pattern was created in nine matched pairs of cadaveric specimen using an oscillating saw in standardized, reproducible fashion. Each matched pair was then randomized to receive either 2.7 mm reconstruction plating or 3.5 mm proximal ulna locked plating. Random allocation software was used to assign the 2.7 mm plate construct to either the right or left side of each pair with the contralateral receiving the 3.5 mm plate construct. Specimens were cyclically loaded simulating passive range of motion exercises commonly performed during rehabilitation. Displacement and rotation in relation to the long axis of the ulna were measured through motion capture. Fragment gapping and rotation was quantified following 100 cycles at 10 N and again following 100 cycles at 500 N. RESULTS: No significant differences were detected between the 2.7 and 3.5 mm plates in fracture rotation or gapping following loads at 10 N (0.5° and 0.7°; 0.6 and 1.2 mm; respectively; p > 0.05) or 500 N (2.3° and 1.6°; 3.8 and 3.1 mm; respectively; p > 0.05) loading. Fragment rotation and gapping were positively correlated within each plate construct (R 2 > 0.445; p < 0.05). CONCLUSIONS: 2.7 mm plating is an alternative to 3.5 mm locked plating with decreased plate prominence without significantly sacrificing displacement and rotational control. This is beneficial in fracture patterns where the traditional dorsal plating does not offer optimal screw trajectory.

Outcomes of Bankart Repairs Using Modern Arthroscopic Technique in an Athletic Population.

PURPOSE: To report a large number of highly active patients who underwent arthroscopic Bankart repair at our institution over the last decade. METHODS: A retrospective analysis of patients who underwent primary and revision arthroscopic Bankart repairs using bioabsorbable anchors was performed. Outcome measures included recurrence of dislocation, American Shoulder and Elbow Scores (ASES), Rowe, visual analog scale (VAS), return to sports, and satisfaction scores. RESULTS: A total of 94 shoulders met the inclusion criteria. The recurrence rate was 6/94 (6.4%) at a mean follow-up of 5 years (range, 3 to 8.3). The mean postoperative scores were as follows: ASES = 91.5/100; Rowe = 84.3/100; VAS = 0.8/10; satisfaction = 8.8/10. In those who attempted to return to sports, 82.5% were able to return to the same level of competition. Statistical analyses revealed a significant increase in risk of recurrence among high school and recreational athletes. No recurrences were observed among professional or college-level athletes. No significant difference in recurrence rates was observed in regards to age, time to surgery, type of athlete (collision v limited contact), repair of SLAP lesion, number of anchors, or revision surgery. CONCLUSIONS: Although several repair techniques exist for traumatic anterior shoulder instability, arthroscopic repair remains a viable option even in a highly active patient population. This study uniquely identified high school and recreational athletes at higher risk for recurrence. This is perhaps due to inferior shoulder development and technique as well as to limited access to postoperative physical therapy. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Frontal plane stability following UKA in a biomechanical study.

INTRODUCTION: Function and kinematics following unicondylar knee arthroplasty (UKA) have been reported to be close to the native knee. Gait, stair climbing and activities of daily living expose the knee joint to a combination of varus and valgus moments. Replacement of the medial compartment via UKA is likely to change the physiologic knee stability and its ability to respond to varus and valgus moments. It was hypothesized that UKA implantation would stiffen the knee and decrease range of motion in the frontal plane. MATERIALS AND METHODS: Six fresh frozen cadaver knees were prepared and mounted in a six-degrees-of-freedom robot. An axial load of 200 N was applied with the knee in 15°, 45° and 90° of flexion. Varus and valgus moments were added, respectively, before and after implantation of medial UKA. Tests were than redone with a thicker polyethylene inlay to simulate overstuffing of the medial compartment. Range of motion in the frontal plane and the tibial response to moments were recorded via the industrial robot. RESULTS: The range of motion in the frontal plane was decreased with both, balanced and overstuffed UKA and shifted towards valgus. When exposed to valgus moments, knees following UKA were stiffer in comparison with the native knee. The effect was even more pronounced with medial overstuffing. CONCLUSION: In UKA, the compressive anatomy is replaced by much stiffer components. This lack of medial compression and relative overstuffing leads to a tighter medial collateral ligament. This drives the trend towards a stiffer joint as documented by a decrease in frontal plane range of motion. Overstuffing should strictly be avoided when performing UKA.

Comparison of in vitro motion and stability between techniques for index metacarpophalangeal joint radial collateral ligament reconstruction.

PURPOSE: To evaluate a technique using interference screws to secure a tendon graft for reconstruction of the radial collateral ligament (RCL) of the index finger metacarpophalangeal (MCP) joint. We hypothesized that this technique would provide equivalent stability and flexion as a 4-tunnel reconstruction. METHODS: We isolated the RCL in 17 cadaveric index fingers. A cyclic load was applied to the intact RCL across the MCP joint to assess flexion, ulnar deviation at neutral (UD 0), and ulnar deviation at 90° of MCP joint flexion (UD 90). The RCL was excised from its bony origin and insertion. We performed each reconstruction (4-tunnel and interference screw) sequentially on each specimen in a randomized order using a palmaris longus tendon graft. We repeated testing after each reconstruction and compared differences from the intact state between techniques using paired t-tests for all joint positions (flexion/UD 0/UD 90). RESULTS: There was no statistically significant difference in UD 0 or UD 90 between the intact state and after interference screw reconstruction. Compared with the intact state, there was significantly less UD 0 and significantly more UD 90 after 4-tunnel reconstruction. There was no statistically significant difference between techniques when we compared changes in -UD 0 or UD 90. Change in flexion was statistically significantly different, which indicates that the interference screw technique better replicated intact MCP joint flexion compared with the 4-tunnel technique. CONCLUSIONS: Interference screw reconstruction of the index RCL provides stability comparable to 4-tunnel reconstruction and is less technically challenging. These results substantiate our clinical experience that the interference screw technique provides an optimal combination of stability and flexion at the index MCP joint. CLINICAL RELEVANCE: Using an interference screw to reconstruct the index RCL is less challenging than 4-tunnel reconstruction and provides stability and range of motion that closely resemble the native MCP joint.

Anatomy of the radial collateral ligament of the index metacarpophalangeal joint.

PURPOSE: To describe the origin and insertion of the radial collateral ligament (RCL) of the index metacarpophalangeal (MP) joint, relative to the MP joint line and other landmarks readily discernible intraoperatively. METHODS: We dissected 17 fresh-frozen human cadaveric index fingers. We removed all overlying soft tissue from the MP joint except for the proper RCL. We dissected the RCL from its original insertion under loupe magnification while concurrently marking the ligamentous origin and insertion points. We measured distances of these points in relation to the bony landmarks (dorsal, articular, and volar surfaces) using digital photo analysis. The same observer recorded all measurements to reduce systematic error. RESULTS: The center of the metacarpal attachment of the RCL was located 5.4 ± 1.1 mm from the dorsal border of the metacarpal, 8.0 ± 2.2mm from the volar border of the metacarpal, and 10.3 ± 3.2mm from the articular surface of the MP joint. The total width and height of the metacarpal origin site were 5.8 ± 1.6 and 6.4 ± 1.4 mm, respectively. The center of the proximal phalanx attachment of the RCL was located 6.8 ± 1.4 mm from the dorsal border of the proximal phalanx, 5.7 ± 0.9 mm from the volar border of the proximal phalanx, and 4.4 ± 0.8mm from the articular surface of the MP joint. The total width and height of the phalangeal origin site were 5.0 ± 1.1 and 5.7 ± 0.9 mm, respectively. CONCLUSIONS: Our study defines the anatomic origin and insertion of the RCL of the index MP joint in relation to landmarks that are identifiable during surgery. CLINICAL RELEVANCE: We believe this information will be useful to surgeons when repairing or reconstructing the RCL, allowing for recreation of normal RCL anatomy.

Shoulder Range of Motion Deficits in Youth Throwers Presenting With Elbow Pain.

BACKGROUND: Glenohumeral internal rotation deficit (GIRD) and total arc of motion difference (TAMD) have been associated with elbow injuries in throwing athletes. HYPOTHESIS: Youth pitchers with elbow pain will have greater GIRD and TAMD compared with youth pitchers without elbow pain. STUDY DESIGN: Cross-sectional study. LEVEL OF EVIDENCE: Level 3. METHODS: Glenohumeral range of motion of 25 consecutive throwing athletes presenting with elbow pain and that of a matched control group of 18 asymptomatic throwing athletes were compared. Bilateral glenohumeral internal rotation, external rotation, and horizontal adduction at 90° were measured and GIRD and TAMD were then calculated. An analysis of variance was performed to compare range of motion between throwers with and without elbow pain. RESULTS: The average GIRD of the elbow pain group was 32.7° compared with 14.5° in the control group (P < 0.05). The average TAMD in the elbow pain group was 28.3° compared with 6.7° in the control group (P < 0.05). GIRD and TAMD were present in 88% (22 of 25) and 96% (24 of 25) of the elbow pain group versus 33.3% (6 of 18) and 55.6% (10 of 18) of the control group, respectively. CONCLUSION: Compared with asymptomatic youth pitchers, those presenting with elbow pain have a statistically significant GIRD and TAMD. CLINICAL RELEVANCE: This study suggests that a GIRD and TAMD may predispose youth pitchers to present with symptomatic elbow pain.

Effect of Bone Coverage on Acetabular Implant Stresses in Standard and Dual-Mobility Total Hip Arthroplasty Constructs: A Finite Element Model.

Although mechanical stress in total hip arthroplasty modular head-neck junctions is thought to contribute to the risk of trunnionosis and related metal ion disease in total hip arthroplasty, little is known about mechanical stress in the modular acetabular components. Recent retrieval analyses of dual-mobility constructs have demonstrated corrosion between liner and shell in some dual-mobility acetabular components. The objective of this study was to evaluate acetabular stress as a function of acetabular bone coverage, component modularity, and femoral head diameter. A parametric finite element model was created. The acetabulum was set at 40° of abduction and 15° of anteversion; superolateral bone loss up to 50° was modeled; and 28-mm, 32-mm, 36-mm, and 40-mm head sizes were simulated in stance phase of gait. Fixed polyethylene-bearing, monoblock and modular dual-mobility (MDM) acetabular components were evaluated. For traditional fixed-bearing components, the largest peak stress, 49.5 MPa, was observed with 50° of bone loss and a 28-mm head. The lowest peak stress, 6.3 MPa, occurred with complete bone coverage and a 36-mm head. Peak stress in the MDM construct, 25.1 MPa, concentrated in the chromium-cobalt portion of the construct. Larger head diameters are associated with decreased stress in the acetabular component when bone loss is present. An MDM construct with a stiff inner liner may decrease overall stress in the acetabular construct, but focally increased stress near the rim of uncovered acetabular components may increase the risk of metal-on-metal corrosion. [Orthopedics. 2021;44(5):280-284.].

Comparison of actual surgical outcomes and 3-dimensional surgical simulations.

PURPOSE: The advent of imaging software programs has proved to be useful for diagnosis, treatment planning, and outcome measurement, but precision of 3-dimensional (3D) surgical simulation still needs to be tested. This study was conducted to determine whether the virtual surgery performed on 3D models constructed from cone-beam computed tomography (CBCT) can correctly simulate the actual surgical outcome and to validate the ability of this emerging technology to recreate the orthognathic surgery hard tissue movements in 3 translational and 3 rotational planes of space. MATERIALS AND METHODS: Construction of pre- and postsurgery 3D models from CBCTs of 14 patients who had combined maxillary advancement and mandibular setback surgery and 6 patients who had 1-piece maxillary advancement surgery was performed. The postsurgery and virtually simulated surgery 3D models were registered at the cranial base to quantify differences between simulated and actual surgery models. Hotelling t tests were used to assess the differences between simulated and actual surgical outcomes. RESULTS: For all anatomic regions of interest, there was no statistically significant difference between the simulated and the actual surgical models. The right lateral ramus was the only region that showed a statistically significant, but small difference when comparing 2- and 1-jaw surgeries. CONCLUSIONS: Virtual surgical methods were reliably reproduced. Oral surgery residents could benefit from virtual surgical training. Computer simulation has the potential to increase predictability in the operating room.

Three-dimensional surgical simulation.

In this article, we discuss the development of methods for computer-aided jaw surgery, which allows us to incorporate the high level of precision necessary for transferring virtual plans into the operating room. We also present a complete computer-aided surgery system developed in close collaboration with surgeons. Surgery planning and simulation include construction of 3-dimensional surface models from cone-beam computed tomography, dynamic cephalometry, semiautomatic mirroring, interactive cutting of bone, and bony segment repositioning. A virtual setup can be used to manufacture positioning splints for intraoperative guidance. The system provides further intraoperative assistance with a computer display showing jaw positions and 3-dimensional positioning guides updated in real time during the surgical procedure. The computer-aided surgery system aids in dealing with complex cases with benefits for the patient, with surgical practice, and for orthodontic finishing. Advanced software tools for diagnosis and treatment planning allow preparation of detailed operative plans, osteotomy repositioning, bone reconstructions, surgical resident training, and assessing the difficulties of the surgical procedures before the surgery. Computer-aided surgery can make the elaboration of the surgical plan a more flexible process, increase the level of detail and accuracy of the plan, yield higher operative precision and control, and enhance documentation of cases.

Protective Effects of Controlled Mechanical Loading of Bone in C57BL6/J Mice Subject to Disuse.

Prolonged reduction in weightbearing causes bone loss. Disuse of bone is associated with recovery from common musculoskeletal injury and trauma, bed rest resulting from various medical conditions, and spaceflight. The hindlimb-suspension rodent model is popular for simulating unloading and disuse. We hypothesized that controlled mechanical loading of the tibia would protect against bone loss occurring from concurrent disuse. Additionally, we hypothesized that areas of high mechanical peak strains (midshaft) would provide more protection than areas of lower strain (distal shaft). Adult C57BL6/J mice were suspended for 3 weeks, with one limb subjected to tibial compression four times per week. µCT imaging was completed at days 0, 11, and 21, in addition to serum analysis. Significant bone loss caused by hindlimb suspension was detected in trabecular bone by day 11 and worsened by day 21 (p < 0.05). Bone loss was also detected in cortical thickness and area fraction by day 21. However, four short bouts per week of compressive loading protected the loaded limb from much of this bone loss. At day 21, we observed a 50% loss in trabecular bone volume/total volume and a 6% loss in midshaft cortical thickness in unloaded limbs, but only 15% and 2% corresponding losses in contralateral loaded limbs (p = 0.001 and p = 0.02). Many bone geometry parameters of the loaded limbs of suspended animals did not significantly differ from non-suspended control limbs. Conversely, this protective effect of loading was not detected in cortical bone at the lower-strained distal shaft. Analysis of bone metabolism markers suggested that the benefits of loading occurred through increased formation instead of decreased resorption. This study uniquely isolates the role of externally applied mechanical loading of the mouse tibia, in the absence of muscle stimulation, in protecting bone from concurrent disuse-related loss, and demonstrates that limited bouts of loading may be highly effective during prolonged disuse. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Parametric Finite Element Analysis of Intramedullary Nail Fixation of Proximal Femur Fractures.

Proximal femur fracture fixation with intramedullary nailing relies on stability at the fracture site and integrity of the fixation construct to achieve union. The biomechanics that dictate fracture site stability and implant stress depend on fracture type as well as implant features such as nail length, nail diameter, presence of distal fixation screws, and material composition of the implant. When deciding how to fix a fracture, surgeons have choices in these implant-related design variables. This study models all combinations of a range of implant variables for nine standard AO/OTA proximal femur fractures using finite element analysis. Under simulated maximum load during gait, the maximum stress in the implant and screws as well as interfragmentary motions at the fracture site in the axial and shear directions were computed. The results were separated by fracture type to show the influence of each design variable on measured biomechanical outcomes. Filling the reamed canal with the largest fitting nail diameter reduced axial and shear interfragmentary motion for all fracture types. Nail length was less predictive of shear interfragmentary motion for most simulated fracture types than other construct variables. Furthermore, gapping at the fracture site predisposed the construct to higher implant stresses and larger interfragmentary motions. Clinical significance: Biomechanical outcomes from this computational study can aid in surgical decision-making for optimizing hip fracture fixation with IM nailing. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2358-2366, 2019.

Design and Evaluation of Synthetic Terminators for Regulating Mammalian Cell Transgene Expression.

Tuning heterologous gene expression in mammalian production hosts has predominantly relied upon engineering the promoter elements driving the transcription of the transgene. Moreover, most regulatory elements have borrowed genetic sequences from viral elements. Here, we generate a set of 10 rational and 30 synthetic terminators derived from nonviral elements and evaluate them in the HT1080 and HEK293 cell lines to demonstrate that they are comparable in terms of tuning gene expression/protein output to the viral SV40 element and often require less sequence footprint. The mode of action of these terminators is determined to be an increase in mRNA half-life. Furthermore, we demonstrate that constructs comprising completely nonviral regulatory elements ( i.e., promoters and terminators) can outperform commonly used, strong viral based elements by nearly 2-fold. Ultimately, this novel set of terminators expanded our genetic toolkit for engineering mammalian host cells.

Collagen-infilled 3D printed scaffolds loaded with miR-148b-transfected bone marrow stem cells improve calvarial bone regeneration in rats.

Differentiation of progenitors in a controlled environment improves the repair of critical-sized calvarial bone defects; however, integrating micro RNA (miRNA) therapy with 3D printed scaffolds still remains a challenge for craniofacial reconstruction. In this study, we aimed to engineer three-dimensional (3D) printed hybrid scaffolds as a new ex situ miR-148b expressing delivery system for osteogenic induction of rat bone marrow stem cells (rBMSCs) in vitro, and also in vivo in critical-sized rat calvarial defects. miR-148b-transfected rBMSCs underwent early differentiation in collagen-infilled 3D printed hybrid scaffolds, expressing significant levels of osteogenic markers compared to non-transfected rBMSCs, as confirmed by gene expression and immunohistochemical staining. Furthermore, after eight weeks of implantation, micro-computed tomography, histology and immunohistochemical staining results indicated that scaffolds loaded with miR-148b-transfected rBMSCs improved bone regeneration considerably compared to the scaffolds loaded with non-transfected rBMSCs and facilitated near-complete repair of critical-sized calvarial defects. In conclusion, our results demonstrate that collagen-infilled 3D printed scaffolds serve as an effective system for miRNA transfected progenitor cells, which has a promising potential for stimulating osteogenesis and calvarial bone repair.

Fosl1 overexpression directly activates trophoblast-specific gene expression programs in embryonic stem cells.

During early development in placental mammals, proper trophoblast lineage development is essential for implantation and placentation. Defects in this lineage can cause early pregnancy failures and other pregnancy disorders. However, transcription factors controlling trophoblast development remain poorly understood. Here, we utilize Fosl1, previously implicated in trophoblast giant cell development as a member of the AP-1 complex, to trans-differentiate embryonic stem (ES) cells to trophoblast lineage-like cells. We first show that the ectopic expression of Fosl1 is sufficient to induce trophoblast-specific gene expression programs in ES cells. Surprisingly, we find that this transcriptional reprogramming occurs independently of changes in levels of ES cell core factors during the cell fate change. This suggests that Fosl1 acts in a novel way to orchestrate the ES to trophoblast cell fate conversion compared to previously known reprogramming factors. Mapping of Fosl1 targets reveals that Fosl1 directly activates TE lineage-specific genes as a pioneer factor. Our work suggests Fosl1 may be used to reprogram ES cells into differentiated cell types in trophoblast lineage, which not only enhances our knowledge of global trophoblast gene regulation but also may provide a future therapeutic tool for generating induced trophoblast cells from patient-derived pluripotent stem cells.