Modular synthesis of N-glycans and arrays for the hetero-ligand binding analysis of HIV antibodies.

A new class of broadly neutralizing antibodies (bNAbs) from HIV donors has been reported to target the glycans on gp120--a glycoprotein found on the surface of the virus envelope--thus renewing hope of developing carbohydrate-based HIV vaccines. However, the version of gp120 used in previous studies was not from human T cells and so the glycosylation pattern could be somewhat different to that found in the native system. Moreover, some antibodies recognized two different glycans simultaneously and this cannot be detected with the commonly used glycan microarrays on glass slides. Here, we have developed a glycan microarray on an aluminium-oxide-coated glass slide containing a diverse set of glycans, including homo- and mixed N-glycans (high-mannose, hybrid and complex types) that were prepared by modular chemo-enzymatic methods to detect the presence of hetero-glycan binding behaviours. This new approach allows rapid screening and identification of optimal glycans recognized by neutralizing antibodies, and could speed up the development of HIV-1 vaccines targeting cell surface glycans.

Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays.

In this study, we report the fabrication of aluminum oxide-coated glass (ACG) slides for the preparation of glycan microarrays. Pure aluminum (Al, 300 nm) was coated on glass slides via electron-beam vapor deposition polymerization (VDP), followed by anodization to form a thin layer (50-65 nm) of aluminum oxide (Al-oxide) on the surface. The ACG slides prepared this way provide a smooth surface for arraying sugars covalently via phosphonate formation with controlled density and spatial distance. To evaluate this array system, a mannose derivative of α-5-pentylphosphonic acid was used as a model for the optimization of covalent arraying based on the fluorescence response of the surface mannose interacting with concanavalin A (ConA) tagged with the fluorescence probe A488. The ACG slide was characterized using scanning electron microscopy, atomic force microscopy (AFM), and ellipsometry, and the sugar loading capacity, uniformity, and structural conformation were also characterized using AFM, a GenePix scanner, and a confocal microscope. This study has demonstrated that the glycan array prepared from the ACG slide is more homogeneous with better spatial control compared with the commonly used glycan array prepared from the N-hydroxysuccinimide-activated glass slide.

Locking versus nonlocking construct in an osteoporotic, segmental fibula defect model.

Early studies on the treatment of osteoporotic distal fibular fractures suggest that poor bone quality can compromise fixation and, therefore, clinical outcome. Multiple prior biomechanical studies evaluated length-stable fracture models with destructive load-to-failure protocols, which may not represent a clinically relevant failure mode. The current authors compared a lateral locked construct with 2 distinct nonlocked constructs in an osteoporotic, segmental fibula defect model. Eighteen adult cadaveric ankles were randomized to a one-third tubular plate with nonlocking screws, a precontoured fibular plate with nonlocking screws, or the same pre-contoured plate with locking screws. Each was nondestructively loaded with an external rotation of 2.5 Nm for 10,000 cycles. The primary outcome was the change in construct stiffness. Secondary outcomes included maximal rotational displacement and change in insertion torques. Average increase in stiffness of the one-third tubular plate was 0.35 Nm/degree (P<.001), which was significantly greater than that in the nonlocking and locking precontoured plates (0.15 and 0.17 Nm/degree, respectively; P=.69). All 3 constructs externally rotated to 11.7°±4.6°, 8.6°±3.6°, and 10.9°±4.7°, respectively (P=.50). Average loss of insertion torque in the 4 proximal positions was 32.7%±19%, 41.3%±20.2%, 57.8%±19%, and 64.9%±29.2% (P>.05). Average loss in the 4 distal positions for nonlocking constructs (71.6%±21%, 70.4%±26.4%, 93.1%±11%, and 83.3%±35.4%) was significantly different from that for the locking construct (15%±21.4%, 11.7%±24%, 9.2%±9.2%, and 20.8%±23.3%) (P<.05). No evidence in the current study supports the use of a locking plate for osteoporotic specimens.

The anatomy of the supraclavicular nerve during surgical approach to the clavicular shaft.

BACKGROUND: Surgery for clavicular shaft fractures is becoming more common but incisional and chest wall numbness reportedly occurs in 10% to 29% of patients. This may be the result of iatrogenic injury to the supraclavicular nerve branches. QUESTIONS/PURPOSES: We determined if there was a predictable branching pattern of the supraclavicular nerve at the anterior clavicular border and determined the distances to these nerves from clavicular landmarks. METHODS: We performed an anatomic dissection along the anterior border of the long axis of the clavicle in 37 cadavers. The branches of the supraclavicular nerve were identified at the anterosuperior clavicular border and the distances from these nerves to palpable bony landmarks were measured. RESULTS: Ninety-seven percent of specimens had a medial and a lateral branch of the supraclavicular nerve. Nearly half (49%) possessed an additional intermediate branch. No branch was found within 2.7 cm of the sternoclavicular joint or within 1.9 cm of the acromioclavicular joint. Between these two positions, there was wide variability in nerve branch location. CONCLUSIONS: There were two or three branches of the supraclavicular nerve crossing the clavicle 97% of the time and a wide variability of the location of these branches outside the safe zones. CLINICAL RELEVANCE: There are safe zones within 2.7 cm of the sternoclavicular joint and 1.9 cm of the acromioclavicular joint. Between these safe zones, the location of the nerve branches is variable and the surgeon must use meticulous dissection if he or she wishes to prevent transection.

The retropatellar portal as an alternative site for tibial nail insertion: a cadaveric study.

OBJECTIVE: To define spatial relationships between major intra-articular structures of the knee and the entry site of a tibial nail inserted using a retropatellar portal. DESIGN: Cadaveric study using 16 fresh-frozen limbs. SETTING: University-affiliated cadaver and anatomy laboratory. RESULTS: The mean distance of the nail entry site and the medial and lateral menisci were 6.6 ± 3.2 mm and 6.4 ± 4.4 mm, respectively. The distance to the medial and lateral articular surfaces were 5.6 ± 3.6 mm and 7.4 ± 4.2 mm, respectively. The mean distance to the anterior cruciate ligament footprint was 7.5 ± 3.5 mm. The lateral meniscus was never injured during the procedure. The anterior cruciate ligament was undisturbed in all specimens. The medial meniscus was injured 1 to 2 mm in 12.5% of specimens. The intermeniscal ligament was injured 1 to 2 mm in 81.2% of the specimens. CONCLUSION: The intermeniscal ligament and medial meniscus are at the most risk during intramedullary nailing of the tibia using the retropatellar technique. This may be corrected by avoiding an excessively medial start point. Damage to the intermeniscal ligament and medial meniscus occurs more commonly with the retropatellar portal, but this damage was never more than 1 to 2 mm. This risk, however, appears similar to the pattern and incidence of injury that occurred in prior studies investigating tibial nail insertion through a standard patellar tendon approach. The retropatellar technique demands clinical investigation to further define both its safety and its use.

Retropatellar technique for intramedullary nailing of proximal tibia fractures: a cadaveric assessment.

OBJECTIVE: To investigate if the radiographically correct and anatomically safe starting point and the appropriate sagittal plane vector could be obtained using a retropatellar technique for proximal tibia fractures treated with an intramedullary device. METHODS: We performed a cadaveric and radiographic study utilizing 16 limbs. We performed a retropatellar approach via longitudinal quadriceps split, passed a specialized trocar through the patellofemoral joint and onto the superior aspect of the tibia, and inserted Kirschner wires into the anatomic safe zone of the tibial plateau at 0, 10, 20, 30, 40, and 50 degrees of knee flexion utilizing biplanar fluoroscopy. We recorded knee flexion with a goniometer and the entrance vector of the Kirschner wire in relation to the anterior tibial cortex. SETTING: University-affiliated cadaver and anatomy laboratory. RESULTS: There was a progressive increase in the ability to obtain the correct anatomical start site from 1 of 16 (6.25%) at full extension to 12 of 16 (75%) at 50 degrees of knee flexion (P = 0.00098). A statistically significant decrease in the average sagittal plane entrance vector in relation to the anterior tibial cortex was found from 23.1 degrees at full extension to -0.41 degrees at 50 degrees of knee flexion (P < 0.0001). CONCLUSIONS: The retropatellar technique allows the radiographically defined correct start site to be localized, particularly at higher degrees of knee flexion. More favorable intramedullary nail insertion angles were possible with the retropatellar technique, particularly with knee flexion angles greater than 20 degrees. The retropatellar technique demands further investigations to further delineate its advantages, limitations, and possible risks to local anatomy.

Glycan array on aluminum oxide-coated glass slides through phosphonate chemistry.

A new type of glycan array covalently or noncovalently attached to aluminum oxide-coated glass (ACG) slides has been developed for studies of enzymatic reactions and protein binding. To prepare the noncovalent array, glycans with a polyfluorinated hydrocarbon (-C(8)F(17)) tail are spotted robotically onto the ACG slide surface containing a layer of polyfluorinated hydrocarbon terminated with phosphonate. After incubation and washing, the noncovalent array can be characterized by MS-TOF via ionization/desorption at a low laser energy without addition of matrix. A representative cellotetraose array was developed to study the activity and specificity of different cellulases and to differentiate the exo- and endoglucanase activities. To prepare the covalent array, glycans with a phosphonic acid tail were synthesized and spotted robotically onto the ACG slide surface. After incubation, the slides can be used directly for quantitative protein binding analysis. Compared to the preparation of glycan arrays on glass slides and other surfaces, this method of arraying using phosphonic acid reacting with ACG is more direct, convenient, and effective and represents a new platform for the high-throughput analysis of protein-glycan interactions.

Neurovascular risks of anteroinferior clavicular plating.

Anteroinferior plating has been described for internal fixation of clavicular fractures, citing improved bicortical fixation, less hardware prominence, and safer drill trajectories compared with other plate configurations. This anatomic study defined structures at risk during anteroinferior clavicular plating. Four paired cadaveric specimens (8 clavicles) from ages 75 to 93 years were systematically dissected. Using the screw paths associated with an anteroinferior plate (anteroinferior to superoposterior), the distance from the posterior clavicle cortex to the nearest vital structure was measured at 5 different positions along the clavicle. In the medial half of the clavicle, the subclavian artery is in closest proximity to the clavicle, measuring, on average, 22.95 mm and 15.10 mm at point A and B. At the lateral three-fifths point of the clavicle (point C), the brachial plexus is 12.76 mm from the posterior clavicle and is more at risk than the subclavian artery. Lateral to the three-fifth point of the clavicle, there were no vital structures that could be injured by overdrilling. Our results suggest that more care should be observed with placement of screws in the medial half of the clavicle where subclavian artery damage is more likely.

Cartilage Oligomeric Matrix Protein (COMP): A Biomarker of Arthritis.

Arthritis is a chronic disease with a significant impact on the population. It damages the cartilage, synovium, and bone of the joints causing pain, impairment, and disability in patients. Current methods for diagnosis of and monitoring the disease are only able to detect clinical manifestations of arthritis late in the process. However, with the recent onset of successful treatments for rheumatoid arthritis and osteoarthritis, it becomes important to identify prognostic factors that can predict the evolution of arthritis. This is especially critical in the early phases of disease so that these treatments can be started as soon as possible to slow down progression of the disease. A valuable approach to monitor arthritis would be by measuring biological markers of cartilage degradation and repair to reflect variations in joint remodeling. One such potential biological marker of arthritis is cartilage oligomeric matrix protein (COMP). In various studies, COMP has shown promise as a diagnostic and prognostic indicator and as a marker of the disease severity and the effect of treatment. This review highlights the progress in the utilization of COMP as a biomarker of arthritis.

Glycan arrays on aluminum-coated glass slides.

We have developed a novel method of immobilizing glycans onto aluminum-coated glass (ACG) slides for potential use in disease diagnosis and drug discovery. The quality of these sugar chips can be assessed by mass spectrometry and fluorescence measurements with high sensitivity. The unique properties of ACG slides include: 1) the metal oxide layer on the surface can be activated for grafting organic compounds such as modified oligosaccharides; 2) the surface remains electrically conductive, and the grafted oligosaccharides can be simultaneously characterized by mass spectrometry and carbohydrate-binding assay; and 3) the slides are more sensitive than transparent glass slides in binding analysis. To demonstrate this, we synthesized a model compound of mannose with a built-in photocleavable linker bound to the ACG slide surface. The molecular weight of the grafted mannose was identified by mass spectrometry, and the slide was subjected to biotinylated ConA binding followed by Cy3-tagged streptavidin detection. This method was further extended to the preparation of glycan arrays containing lactose and the cancer antigen Globo H.

Desorption ionization of biomolecules on metals.

Direct desorption ionization of various types of biomolecules on metal substrates without the need of matrices was observed by a time-of-flight mass spectrometer. It provides a new convenient method for detection of small biomolecules without the confusion of ion peaks from matrix compounds. Simple commercial Al foil can be used as the substrate to obtain mass spectra of biomolecules without the need of an etching process to produce a porous surface such as with direct ionization on silicon (DIOS). The desorption and ionization mechanism is also discussed.

Nonunions and the potential of stem cells in fracture-healing.

Recent progress in human embryonic and adult stem cell research is a cause for much enthusiasm in bone and joint surgery. Stem cells have therapeutic potential in the realm of orthopaedic surgery because of their capacity to self-renew and differentiate into various types of mature cells and tissues, including bone. Because nonunions remain a clinically important problem, there is interest in the use of cell-based strategies to augment fracture repair. Such strategies are being investigated with variations in the model systems, sources of stem cells, and methods for the application and enhancement of osseous healing, including genetic modifications and tissue-engineering. This review highlights the recent progress in the utilization of stem cells and cell-based gene therapy in promoting fracture-healing and its potential utility in the clinical setting.

Percutaneous management of Morel-Lavallee lesions.

BACKGROUND: Previous recommendations for treatment of Morel-Lavallee soft-tissue degloving lesions have included open débridement with packing or delayed closure. The purpose of this study was to review the use of percutaneous drainage for the initial management of these lesions. METHODS: Nineteen patients with a Morel-Lavallee lesion were managed with percutaneous drainage and débridement of the lesion within three days after the injury. Drainage was usually completed through two 2-cm incisions: one over the distal aspect of the lesion and one over the most superior and posterior extent of the lesion. A plastic brush was used to débride the injured fatty tissue, which was washed from the wound with pulsed lavage. A medium Hemovac drain was placed within the lesion and was removed when drainage was <30 mL over twenty-four hours. RESULTS: Fifteen of the nineteen patients had surgery for an associated pelvic or acetabular fracture. Seven of the nine patients in whom a pelvic fracture was treated surgically had percutaneous fixation of the posterior part of the pelvic ring as well as treatment of the Morel-Lavallee lesion during the same operative setting. Fixation of the remaining two pelvic fractures and the six acetabular fractures was deferred until at least twenty-four hours after the drain was removed. Three of sixteen cultures of specimens taken from the wounds were positive. None of the patients with percutaneous fixation of the pelvis had wound complications. One wound required surgical exploration because of persistent drainage, but the culture was negative and the wound healed with no sequelae. No patient required débridement of skin and, at a minimum of six months, no deep infection had occurred. CONCLUSIONS: Early percutaneous drainage with débridement, irrigation, and suction drainage for the treatment of Morel-Lavallee lesions appears to be safe and effective. Percutaneous procedures for pelvic fixation were well tolerated by the small number of patients in this series, and open procedures appeared to be safe when performed in a delayed fashion.