Quantification of tRNA m1A modification by templated-ligation qPCR.

N1-methyladenosine (m1A) is a widespread modification in all eukaryotic, many archaeal, and some bacterial tRNAs. m1A is generally located in the T loop of cytosolic tRNA and between the acceptor and D stems of mitochondrial tRNAs; it is involved in the tertiary interaction that stabilizes tRNA. Human tRNA m1A levels are dynamically regulated that fine-tune translation and can also serve as biomarkers for infectious disease. Although many methods have been used to measure m1A, a PCR method to assess m1A levels quantitatively in specific tRNAs has been lacking. Here we develop a templated-ligation followed by a qPCR method (TL-qPCR) that measures m1A levels in target tRNAs. Our method uses the SplintR ligase that efficiently ligates two tRNA complementary DNA oligonucleotides using tRNA as the template, followed by qPCR using the ligation product as the template. m1A interferes with the ligation in specific ways, allowing for the quantitative assessment of m1A levels using subnanogram amounts of total RNA. We identify the features of specificity and quantitation for m1A-modified model RNAs and apply these to total RNA samples from human cells. Our method enables easy access to study the dynamics and function of this pervasive tRNA modification.

Structural basis for substrate binding and catalysis by a self-alkylating ribozyme.

Ribozymes that react with small-molecule probes have important applications in transcriptomics and chemical biology, such as RNA labeling and imaging. Understanding the structural basis for these RNA-modifying reactions will enable the development of better tools for studying RNA. Nevertheless, high-resolution structures and underlying catalytic mechanisms for members of this ribozyme class remain elusive. Here, we focus on a self-alkylating ribozyme that catalyzes nitrogen-carbon bond formation between a specific guanine and a 2,3-disubstituted epoxide substrate and report the crystal structures of a self-alkylating ribozyme, including both alkylated and apo forms, at 1.71-Å and 2.49-Å resolution, respectively. The ribozyme assumes an elongated hairpin-like architecture preorganized to accommodate the epoxide substrate in a hook-shaped conformation. Observed reactivity of substrate analogs together with an inverse, log-linear pH dependence of the reaction rate suggests a requirement for epoxide protonation, possibly assisted by the ether oxygens within the substrate.

Gluteal Vein Anatomy: Location, Caliber, Impact of Patient Positioning, and Implications for Fat Grafting.

BACKGROUND: Deaths in gluteal autografting occur due to gluteal vein injuries, but data are lacking on the precise location and caliber of these veins. OBJECTIVES: The authors sought to present the first in vivo study of gluteal vein anatomy utilizing magnetic resonance imaging. METHODS: Magnetic resonance imaging venography of 16 volunteer hemi-sections was conducted in the supine, prone, prone with a bump (jack-knife), and left and right decubitus positions in 1 session after a single contrast administration. Caliber and course of the superior and inferior gluteal veins (SGV/IGV) were analyzed vs bony landmarks and position changes. RESULTS: The SGV has a very short submuscular course before splitting into 2 smaller branches superolaterally. The IGV runs immediately deep to the gluteus maximus in the center of the buttock as a single large trunk, on average 56 mm deep (mean 27 mm of muscle belly and 30 mm subcutaneous fat). No intramuscular or subcutaneous branches greater than 2 mm were found. In the prone position, the IGV and SGV have an average caliber of 5.96 mm and 5.63 mm. Vessel caliber decreased by 21% and 27%, respectively, in the jack-knife position and by 14% and 15% in lateral decubitus. CONCLUSIONS: The SGV and IGV are immediately deep to gluteus maximus approximately 6 cm deep with a caliber on the order of 6 mm in the prone position. The distribution of these vessels suggests there is no "safe zone" in the intramuscular or submuscular planes. The jackknife or lateral decubitus positions can decrease vein caliber by up to 27%, possibly reducing the risk of injury due to either traction or direct cannula impact.

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) RNAs in the Porphyromonas gingivalis CRISPR-Cas I-C System.

The CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated protein) system is unique to prokaryotes and provides the majority of bacteria and archaea with immunity against nucleic acids of foreign origin. CRISPR RNAs (crRNAs) are the key element of this system, since they are responsible for its selectivity and effectiveness. Typical crRNAs consist of a spacer sequence flanked with 5' and 3' handles originating from repeat sequences that are important for recognition of these small RNAs by the Cas machinery. In this investigation, we studied the type I-C CRISPR-Cas system in Porphyromonas gingivalis, a human pathogen associated with periodontitis, rheumatoid arthritis, cardiovascular disease, and aspiration pneumonia. We demonstrated the importance of the 5' handle for crRNA recognition by the effector complex and consequently activity, as well as secondary trimming of the 3' handle, which was not affected by modifications of the repeat sequence.IMPORTANCEPorphyromonas gingivalis, a clinically relevant Gram-negative, anaerobic bacterium, is one of the major etiologic agents of periodontitis and has been linked with the development of other clinical conditions, including rheumatoid arthritis, cardiovascular disease, and aspiration pneumonia. The presented results on the biogenesis and functions of crRNAs expand our understanding of CRISPR-Cas cellular defenses in P. gingivalis and of horizontal gene transfer in bacteria.

Rivaroxaban for Venous Thromboembolism Prophylaxis in Abdominoplasty: A Multicenter Experience.

BACKGROUND: Abdominoplasty, a commonly performed aesthetic procedure, is considered to have an increased risk of venous thromboembolism (VTE) events. At present, routine VTE chemoprophylaxis following abdominoplasty remains controversial. OBJECTIVES: This study evaluates the authors' experience with rivaroxaban, an oral Factor Xa inhibitor, for VTE prophylaxis in abdominoplasty patients. METHODS: A retrospective case series was conducted. All patients who underwent abdominoplasty and received rivaroxaban were included. The prophylactic dose was 10 mg daily for 7 days, beginning 12 hours postoperatively. Patient demographics, comorbidities, and type of surgery were recorded. The primary outcome measured was hematologic complication, including VTE, hematoma requiring operative evacuation, and need for blood transfusion. RESULTS: From September 2012 until July 2014, 132 patients (122 women and 10 men) underwent abdominoplasty surgery and received rivaroxaban postoperatively. Mean patient age was 43.7 years, and mean body mass index was 27.1. One hundred twenty-five patients also underwent abdominal muscle plication. Eleven patients underwent a fleur de lis vertical skin resection component. One hundred patients underwent concomitant abdominal liposuction, while 79 patients also had back liposuction. Only 1 patient had a symptomatic VTE event. Three patients had a hematoma requiring operative evacuation, and all went on to heal without sequelae. Two patients received a blood transfusion for anemia during their course of rivaroxaban. CONCLUSIONS: Oral rivaroxaban administration for chemoprophylaxis in abdominoplasty patients is safe, with low rates of symptomatic VTE and hematoma formation. The authors continue routine use of the medication for patients at increased risk for VTE events. LEVEL OF EVIDENCE 4: Risk.

Osteoinduction of umbilical cord and palate periosteum-derived mesenchymal stem cells on poly(lactic-co-glycolic) acid nanomicrofibers.

The need for tissue-engineered bone to treat complex craniofacial bone defects secondary to congenital anomalies, trauma, and cancer extirpation is sizeable. Traditional strategies for treatment have focused on autologous bone in younger patients and bone substitutes in older patients. However, the capacity for merging new technologies, including the creation of nanofiber and microfiber scaffolds with advances in natal sources of stem cells, is crucial to improving our treatment options. The advantages of using smaller diameter fibers for scaffolding are 2-fold: the similar fiber diameters mimic the in vivo extracellular matrix construct and smaller fibers also provide a dramatically increased surface area for cell-scaffold interactions. In this study, we compare the capacity for a polymer with Federal Drug Administration approval for use in humans, poly(lactic-co-glycolic) acid (PLGA) from Delta polymer, to support osteoinduction of mesenchymal stem cells (MSCs) harvested from the umbilical cord (UC) and palate periosteum (PP). Proliferation of both UC- and PP-derived MSCs was improved on PLGA scaffolds. The PLGA scaffolds promoted UC MSC differentiation (indicated by earlier gene expression and higher calcium deposition), but not in PP-derived MSCs. Umbilical cord-derived MSCs on the PLGA nanomicrofiber scaffolds have potential clinical utility in providing solutions for craniofacial bone defects, with the added benefit of earlier availability.

Effects of connective tissue growth factor on the regulation of elastogenesis in human umbilical cord-derived mesenchymal stem cells.

BACKGROUND: A key to clinical microtia reconstruction is construct flexibility. The most significant current limitation to engineered elastic cartilage is maintaining an elastic phenotype, which is principally dependent on elastin production (although other parameters, including maintenance of a ratio above 1 for collagens II to I, minimizing collagen X content, and presence of adequate matrix fibrillin for elastin binding, all play supporting roles). Connective tissue growth factor (CTGF), a compound secreted by chondrocytes, has been shown to promote an elastic phenotype in mature rabbit chondrocytes; however, CTGF effect on undifferentiated mesenchymal stem cells (MSCs) has not been characterized. The principal aim of this study is to analyze CTGF effect on elastin production in umbilical cord (UC)-derived MSCs and to determine optimal timing of treatment to maximize elastin production. METHODS: Human UCMSCs (hUCMSCs) were isolated from Wharton jelly using an explant technique, grown to passage 3, seeded onto nanofiber scaffolds, and chondroinduced for 21 days. Nanofiber scaffolds were electrospun using solubilized poly L-lactide/D-lactide/glycolide (PLGA). Chondrogenic media was supplemented with 25 µg/mL CTGF starting at day 0 or 7. Messenger RNA (mRNA) for Collagen I, II, X, fibrillin, and elastin was quantified by RT-PCR; glycosaminoglycan (GAG) matrix deposition was assessed and normalized by cellular DNA content. Elastin protein was assessed by Western blot analysis. All experiments were performed in triplicate with MSCs from 4 distinct cords. Multiway analysis of variance with Newman-Keuls post test was used to determine statistical significance. RESULTS: Connective tissue growth factor treatment results in increased GAG/DNA ratio; the differentiation index was maintained above 1 in all conditions, with increased collage II noted at days 7 and 14 in CTGF conditions; no difference in collagen X or fibrillin mRNA was noted. Increased elastin mRNA and protein were noted at day 14 in conditions treated with CTGF at day 7 after differentiation. CONCLUSIONS: Connective tissue growth factor leads to maximal elastin increase in UCMSCs after 7 days of chondroinduction and not in undifferentiated MSCs. With appropriately timed treatment, CTGF may be a useful adjunct in maintaining an elastic cartilage phenotype in engineered cartilage from human UCMSCs.

The assessment of velopharyngeal function using nasal ram pressure testing following palatoplasty.

Objective : To compare nasal ram pressure (NRP) data with perceptual speech assessments in young children following palatoplasty. Design and Setting : NRP monitoring, a noninvasive technique using a two-pronged nasal cannula that detects nasal airflow during speech, was performed on patients 12 months following palatoplasty in our Craniofacial Center. Patients : Eighteen English-speaking nonsyndromic patients, aged 21 to 28 months, without hearing or language deficits, underwent NRP testing. Main Outcome Measured : During NRP measurement, the number of oral stop consonants produced spontaneously or following prompts was recorded. Stops were considered produced with velopharyngeal closure if NRP demonstrated no nasal airflow. The percentage of closed stops as measured by NRP was compared to the clinical assessment by craniofacial team speech-language pathologists. Results : Eight patients achieved 100% stop closure. Four patients obtained 90% to 96% stop closure. Three patients had stop closure of 85%, 78%, and 50%. Three patients had 0% stop closure; two of these patients subsequently underwent secondary palatal surgery. No significant clinical speech deficits were noted in patients with >90% closure. No association with defect size or cleft type was identified. Conclusions : NRP monitoring is well-tolerated in young children following palatoplasty. Achieving velopharyngeal (VP) closure for >90% of stops is associated with a clinical assessment of adequate speech and VP function. Postoperative NRP testing may serve as a useful adjunct to clinical speech evaluation for early identification of children who require more intensive speech therapy or secondary palatal surgery.

Prophylactic flap reconstruction of the knee prior to total knee arthroplasty in high-risk patients.

Insufficient soft-tissue coverage following total knee arthroplasty (TKA) may threaten prosthesis retention or compromise joint function. A retrospective review was conducted of all patients who underwent prophylactic flap reconstruction of the knee prior to TKA or salvage flap reconstruction over a 6-year period. Twenty-three patients underwent prophylactic flap reconstruction. Complications at the time of flap transfer were common (48%), however, all flaps survived. All 23 successfully completed subsequent TKA with no wound complications occurring at the time of TKA. Complications in the salvage group were also frequent (44%) and 3 required above knee amputation. Postoperative range of motion was significantly better in the prophylactic group, as well as when cutaneous flaps were chosen. Prophylactic flap reconstruction of the knee prior to TKA in high-risk patients is an excellent option in this small subset of patients, many of whom would not be offered TKA without addressing the compromised soft-tissue envelope first.

Anterolateral thigh flap salvage following failed deep inferior epigastric artery perforator breast reconstruction.

The deep inferior epigastric artery perforator flap is an option for women desiring autologous tissue breast reconstruction. If this reconstruction fails, other autologous tissue flaps, including the gluteal artery perforator and latissimus dorsi flaps, may be used for salvage. The anterolateral thigh (ALT) flap offers adequate tissue volume for breast reconstruction, acceptable fat quality and a long vascular pedicle. Other advantages include obviating the need for intraoperative position changes and harvesting tissue outside of the radiation field. Two cases involving ALT flaps used in the setting of deep inferior epigastric artery perforator failure are presented with favourable results. A review of the anatomy of the ALT flap is included.

Superficialization of deep arteriovenous access procedures in obese patients using suction-assisted lipectomy: A novel approach.

Successful dialysis access necessitates superficial arteriovenous fistula (AVF) placement to facilitate identification of anatomical landmarks for safe cannulation. Suction-assisted lipectomy (SAL) may be an alternative to traditional surgical AVF revision procedures for placing fistulas more superficially. Three patients with an average body mass index of 45.2 kg/m(2), with inaccessible AVFs due to obesity, underwent ultrasound-guided SAL of their upper extremities. Successful cannulation was achieved within two weeks. A clinically insignificant hematoma and arm swelling occurred in one patient. SAL provides a safe and effective alternative for salvaging deep AVFs for dialysis access in the upper extremities of obese patients.

Muscle force and power following tendon repair at altered tendon length.

BACKGROUND: While a great deal is known regarding the performance of muscle with intact tendon, little is known about muscle performance when tendon is surgically lengthened or shortened. This knowledge may allow surgeons to more accurately predict functional outcome following tendon repair when correcting a simple tendon laceration or performing a more complex vascularized neuromuscular transfer. MATERIALS AND METHODS: We studied muscle performance 12 wk following extensor tendon repairs producing altered tendon lengths. Forty male Fischer 344 rats underwent division of the proximal and distal tendons of the extensor digitorum longus muscle. Tendons were immediately repaired producing tendons with increased length, decreased length, or presurgical length (control). Observation confirmed that altered tendon length produced inverse changes in initial resting muscle tension. RESULTS: Muscle in the decreased tendon length group demonstrated a 15.2% greater muscle mass, 4.9% greater muscle length, 9.6% greater physiological cross-sectional area, 12.6% greater maximum isometric force, and 31.9% greater maximum power relative to the control tendon length group (P < 0.05). The increased tendon length group did not differ significantly from the control tendon length group for any measurement. Histologically, muscles set with a decreased tendon length demonstrated normal appearing hypertrophied fibers, without evidence of detrimental histological effects such as fibrosis, denervation, necrosis, inflammation, fiber type changes, or fiber splitting. CONCLUSION: These data support the clinical practice of setting muscles with increased passive tension when performing tendon transfer surgeries. Conversely, setting muscles with decreased tension does not necessarily result in a force or power deficit.

Epithelioid Hemangioendothelioma of the Distal Radius: A Case Report.

Epithelioid hemangioendothelioma is a rare vascular tumor with cytologic behavior between angiosarcoma and hemangioma. We present the case of a 58-year-old male with primary epithelioid hemangioendothelioma of the distal radius measuring 6.2 × 5 cm with extension into the pronator quadratus and brachioradialis muscles. We discuss our approach to performing a limb-sparing resection combined with reconstruction to preserve upper extremity function. A review of the clinical, radiographic, and pathologic features of epithelioid hemangioendothelioma is also presented.

NMDA receptor blockade and hippocampal neuronal loss impair fear conditioning and position habit reversal in C57Bl/6 mice.

The interpretation of learning and memory deficits in transgenic mice has largely involved theories of NMDA receptor and/or hippocampal function. However, there is little empirical data that describes what NMDA receptors or the hippocampus do in mice. This research assessed the effects of different doses of the NMDA receptor antagonist, MK-801, or different-sized hippocampal lesions on several behavioral parameters in adult male C57Bl/6 mice. In the first set of experiments, different doses of MK-801 (0.05-0.3mg/kg, s.c.) were assayed in fear conditioning, shock sensitivity, locomotion, anxiety, and position habit reversal tests. Contextual and cued fear conditioning, and position habit reversal were impaired in a dose-dependent manner. Locomotor activity was increased immediately after injection of the highest dose of MK-801. A second set of experiments determined the behavioral effects of a moderate and large excitotoxic hippocampal lesion. Both lesions impaired contextual conditioning, while the larger lesion interfered with cued conditioning. Reversal learning was significantly diminished by the large lesion, while the moderate lesion had a detrimental effect at a trend level (P<0.10). These results provide important reference data for studies involving genetic manipulations of NMDA receptor or hippocampal function in mice. Furthermore, they serve as a basis for a non-transgenic mouse model of the NMDA receptor or hippocampal dysfunction hypothesized to occur in human cognitive disorders.