Considerations for Glycoprotein Production.

This chapter is intended to provide insights for researchers aiming to choose an appropriate expression system for the production of recombinant glycoproteins. Producing glycoproteins is complex, as glycosylation patterns are determined by the availability and abundance of specific enzymes rather than a direct genetic blueprint. Furthermore, the cell systems often employed for protein production are evolutionarily distinct, leading to significantly different glycosylation when utilized for glycoprotein production. The selection of an appropriate production system depends on the intended applications and desired characteristics of the protein. Whether the goal is to produce glycoproteins mimicking native conditions or to intentionally alter glycan structures for specific purposes, such as enhancing immunogenicity in vaccines, understanding glycosylation present in the different systems and in different growth conditions is essential. This chapter will cover Escherichia coli, baculovirus/insect cell systems, Pichia pastoris, as well as different mammalian cell culture systems including Chinese hamster ovary (CHO) cells, human endothelial kidney (HEK) cell lines, and baby hamster kidney (BHK) cells.

Tendon biomechanical properties are altered by storage duration but not freeze-thaw temperatures or cycles.

Tendon allograft and xenograft processing often involves one or more steps of freezing and thawing. As failure strength is an important graft consideration, this study aimed to evaluate effects on failure properties when varying freeze-thaw conditions. Kangaroo tendons, a potential xenograft source, were used to evaluate changes in ultimate tensile strength (UTS), failure strain and elastic modulus after exposure to different freezer-storage temperatures (-20°C vs. -80°C), storage durations (1, 3, 6, 9, or 12 months), number of freeze-thaw cycles (1, 2, 3, 4, 5, or 10), or freeze-thaw temperature ranges (including freezing in liquid nitrogen to thawing at 37°C). Tendons stored for 6 or more months had significantly increased UTS and elastic modulus compared with 1 or 3 months of storage. This increase occurred irrespective of the freezing temperature (-20°C vs. -80°C) or the number of freeze-thaw cycles (1 vs. 10). In contrast, UTS, failure strain and the elastic modulus were no different between storage temperatures, number of freeze-thaw cycles and multiple freeze-thaw cycles across a range of freeze and thaw temperatures. Common freeze-thaw protocols did not negatively affect failure properties, providing flexibility for graft testing, storage, transportation and decellularisation procedures. However, the change in properties with the overall storage duration has implications for assessing the consistent performance of grafts stored for short versus extended periods of time (<6 months vs. >6 months), and the interpretation of data obtained from tissues of varying or unknown storage durations.

Streamlining quantitative joint-wide medial femoro-tibial histopathological scoring of mouse post-traumatic knee osteoarthritis models.

OBJECTIVES: Histological scoring remains the gold-standard for quantifying post-traumatic osteoarthritis (ptOA) in animal models, allowing concurrent evaluation of numerous joint tissues. Available systems require scoring multiple sections/joint making analysis laborious and expensive. We investigated if a single section allowed equivalent quantitation of pathology in different joint tissues and disease stages, in three ptOA models. METHOD: Male 10-12-week-old C57BL/6 mice underwent surgical medial-meniscal-destabilization, anterior-cruciate-ligament (ACL) transection, non-invasive-ACL-rupture, or served as sham-surgical, non-invasive-ACL-strain, or naïve/non-operated controls. Mice (n = 12/group) were harvested 1-, 4-, 8-, and 16-week post-intervention. Serial sagittal toluidine-blue/fast-green stained sections of the medial-femoro-tibial joint (n = 7/joint, 84 µm apart) underwent blinded scoring of 40 histology-outcomes. We evaluated agreement between single-slide versus entire slide-set maximum or median scores (weighted-kappa), and sensitivity/specificity of single-slide versus median/maximum to detect OA pathology. RESULTS: A single optimal mid-sagittal section showed excellent agreement with median (weighted-kappa 0.960) and maximum (weighted-kappa 0.926) scores. Agreement for individual histology-outcomes was high with only 19/240 median and 15/240 maximum scores having a weighted-kappa ≤0.4, the majority of these (16/19 and 11/15) in control groups. Statistically-significant histology-outcome differences between ptOA models and their controls detected with the entire slide-set were reliably reproduced using a single slide (sensitivity >93.15%, specificity >93.10%). The majority of false-negatives with single-slide scoring were meniscal and subchondral bone histology-outcomes (89%) and occurred in weeks 1-4 post-injury (84%). CONCLUSION: A single mid-sagittal slide reduced the time needed to score diverse histopathological changes by 87% without compromising the sensitivity or specificity of the analysis, across a variety of ptOA models and time-points.

Challenging the Perceptions of Human Tendon Allografts: Influence of Donor Age, Sex, Height, and Tendon on Biomechanical Properties.

BACKGROUND: The use of allograft tendons has increased for primary and revision anterior cruciate ligament reconstruction, but allograft supply is currently limited to a narrow range of tendons and donors up to the age of 65 years. Expanding the range of donors and tendons could help offset an increasing clinical demand. PURPOSE: To investigate the effects of donor age, sex, height, and specific tendon on the mechanical properties of a range of human lower leg tendons. STUDY DESIGN: Descriptive laboratory study. METHODS: Nine tendons were retrieved from 39 fresh-frozen human cadaveric lower legs (35 donors [13 female, 22 male]; age, 49-99 years; height, 57-85 inches [145-216 cm]) including: Achilles tendon, tibialis posterior and anterior, fibularis longus and brevis, flexor and extensor hallucis longus, plantaris, and flexor digitorum longus. Tendons underwent tensile loading to failure measuring cross-sectional area (CSA), maximum load, strain at failure, ultimate tensile strength, and elastic modulus. Results from 332 tendons were analyzed using mixed-effects linear regression, accounting for donor age, sex, height, and weight. RESULTS: Mechanical properties were significantly different among tendons and were substantially greater than the effects of donor characteristics. Significant effects of donor sex, age, and height were limited to specific tendons: Achilles tendon, tibialis posterior, and tibialis anterior. All other tendons were unaffected. The Achilles tendon was most influenced by donor variables: greater CSA in men (ß = 15.45 mm2; Sidák adjusted P < .0001), decreased maximum load with each year of increased age (ß = -17.20 N per year; adjusted P = .0253), and increased CSA (ß = 1.92 mm2 per inch; adjusted P < .0001) and maximum load (ß = 86.40 N per inch; adjusted P < .0001) with each inch of increased height. CONCLUSION: Mechanical properties vary significantly across different human tendons. The effects of donor age, sex, and height are relatively small, are limited to specific tendons, and affect different tendons uniquely. The findings indicate that age negatively affected only the Achilles tendon (maximum load) and challenge the exclusion of donors aged >65 years across all tendon grafts. CLINICAL RELEVANCE: The findings support including a broader range of tendons for use as allografts for anterior cruciate ligament reconstruction and reviewing the current exclusion criterion of donors aged >65 years.

A method for mapping the linear epitopes targeted by the natural antibody response to Zika virus infection using a VLP platform technology.

Zika virus (ZIKV), a mosquito-borne pathogen, is associated with neurological complications in adults and congenital abnormalities in newborns. There are no vaccines or treatments for ZIKV infection. Understanding the specificity of natural antibody responses to ZIKV could help inform vaccine efforts. Here, we used a technology called Deep Sequence-Coupled Biopanning to map the targets of the human antibody responses to ZIKV infection. A bacteriophage virus-like particle (VLP) library displaying overlapping linear peptides derived from the ZIKV polyprotein was generated. The library was panned using IgG from 23 ZIKV-infected patients from Panama and deep sequencing identified common targets of anti-ZIKV antibodies within the ZIKV envelope glycoprotein. These included epitopes within the fusion loop within domain II and four epitopes within domain III. Additionally, we showed that VLPs displaying selected epitopes elicited antibodies that bound to native ZIKV envelope protein but failed to prevent infection in a mouse challenge model.

Animal models of osteoarthritis : the benefits and costs of reducing variability.

Cite this article: Bone Joint Res 2022;11(8):514-517.

The iminosugars celgosivir, castanospermine and UV-4 inhibit SARS-CoV-2 replication.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic poses an unprecedented challenge for health care and the global economy. Repurposing drugs that have shown promise in inhibiting other viral infections could allow for more rapid dispensation of urgently needed therapeutics. The Spike protein of SARS-CoV-2 is extensively glycosylated with 22 occupied N glycan sites and is required for viral entry. In other glycosylated viral proteins, glycosylation is required for interaction with calnexin and chaperone-mediated folding in the endoplasmic reticulum, and prevention of this interaction leads to unfolded viral proteins and thus inhibits viral replication. As such, we investigated two iminosugars, celgosivir, a prodrug of castanospermine, and UV-4, or N-(9-methoxynonyl)-1-deoxynojirimycin, a deoxynojirimycin derivative. Iminosugars are known inhibitors of the α-glucosidase I and II enzymes and were effective at inhibiting authentic SARS-CoV-2 viral replication in a cell culture system. Celgosivir prevented SARS-CoV-2-induced cell death and reduced viral replication and Spike protein levels in a dose-dependent manner in culture with Vero E6 cells. Castanospermine, the active form of celgosivir, was also able to inhibit SARS-CoV-2, confirming the canonical castanospermine mechanism of action of celgosivir. The monocyclic UV-4 also prevented SARS-CoV-2-induced death and reduced viral replication after 24 h of treatment, although the reduction in viral copies was lost after 48 h. Our findings suggest that iminosugars should be urgently investigated as potential SARS-CoV-2 inhibitors.

Long-term Effect of a Single Subcritical Knee Injury: Increasing the Risk of Anterior Cruciate Ligament Rupture and Osteoarthritis.

BACKGROUND: Rupture of the anterior cruciate ligament (ACL) is a well-known risk factor for the development of posttraumatic osteoarthritis (PTOA), but patients with the "same injury" can have vastly different trajectories for the onset and progression of disease. Minor subcritical injuries preceding the critical injury event may drive this disparity through preexisting tissue pathologies and sensory changes. PURPOSE: To investigate the role of subcritical injury on ACL rupture risk and PTOA through the evaluation of pain behaviors, joint mechanics, and tissue structural change in a mouse model of knee injury. STUDY DESIGN: Controlled laboratory study. METHODS: Ten-week-old male C57BL/6J mice were allocated to naïve control and subcritical knee injury groups. Injury was induced by a single mechanical compression to the right hindlimb, and mice were evaluated using joint histopathology, anteroposterior joint biomechanics, pain behaviors (mechanical allodynia and hindlimb weightbearing), and isolated ACL tensile testing to failure at 1, 2, 4, or 8 weeks after injury. RESULTS: Subcritical knee injury produced focal osteochondral lesions in the patellofemoral and lateral tibiofemoral compartments with no resolution for the duration of the study (8 weeks). These lesions were characterized by focal loss of proteoglycan staining, cartilage structural change, chondrocyte pathology, microcracks, and osteocyte cell loss. Injury also resulted in the rapid onset of allodynia (at 1 week), which persisted over time and reduced ACL failure load (P = .006; mean ± SD, 7.91 ± 2.01 N vs 9.37 ± 1.01 N in naïve controls at 8 weeks after injury), accompanied by evidence of ACL remodeling at the femoral enthesis. CONCLUSION: The present study in mice establishes a direct effect of a single subcritical knee injury on the development of specific joint tissue pathologies (osteochondral lesions and progressive weakening of the ACL) and allodynic sensitization. These findings demonstrate a predisposition for secondary critical injuries (eg, ACL rupture) and an increased risk of PTOA onset and progression (structurally and symptomatically). CLINICAL RELEVANCE: Subcritical knee injuries are a common occurrence and, based on this study, can cause persistent sensory and structural change. These findings have important implications for the understanding of risk factors of ACL injury and subsequent PTOA, particularly with regard to prevention and management strategies following an often underreported event.

Sex- and injury-based differences in knee biomechanics in mouse models of post-traumatic osteoarthritis.

Sex and joint injury are risk factors implicated in the onset and progression of osteoarthritis (OA). In mouse models of post-traumatic OA (ptOA), the pathogenesis of disease is notably impacted by sex (often worse in males) and injury model (e.g. meniscal versus ligament injury). Increasing ptOA progression and severity is often associated with greater relative instability of the joint but few studies have directly quantified changes in joint mechanics after injury and compared outcomes across multiple models in both male and female mice. Passive anterior-posterior knee biomechanics were evaluated in 10-week-old, male and female C57BL/6J mice. PtOA injury models included destabilisation of the medial meniscus (DMM), anterior cruciate ligament transection (ACLT) or mechanical rupture (ACLR), and combined DMM and ACLT (DMM + ACLT). Sham operated and non-operated controls (NOC) were included for baseline comparisons. The test apparatus loaded hindlimbs at 60° flexion between ± 1 N at 0.5 mm/s (build specifications available for download: https://doi.org/10.17632/z754455x3c.1). Measures of joint laxity (range of motion, neutral zone) and stiffness were calculated. Joint laxity was comparable between male and female mice while joint stiffness was greater in females (P ≤ 0.002, correcting for body-mass and injury-model). Anterior-posterior joint mechanics were minimally altered by DMM but significantly affected by loss of the ACL (P < 0.001), with equivalent changes between ACL-injury models despite different injury mechanisms and adjacent meniscal damage. These findings suggest that despite the important role of joint injury; sex- and model-specific differences in ptOA progression and severity are not primarily driven by altered anterior-posterior knee biomechanics.

Ambroxol and Ciprofloxacin Show Activity Against SARS-CoV2 in Vero E6 Cells at Clinically-Relevant Concentrations.

We studied the activity of a range of weakly basic and moderately lipophilic drugs against SARS CoV2 in Vero E6 cells, using Vero E6 survival, qPCR of viral genome and plaque forming assays. No clear relationship between their weakly basic and hydrophobic nature upon their activity was observed. However, the approved drugs ambroxol and ciprofloxacin showed potent activity at concentrations that are clinically relevant and within their known safety profiles, and so may provide potentially useful agents for preclinical and clinical studies in COVID-19.

The use of mice lacking type I or both type I and type II interferon responses in research on hemorrhagic fever viruses. Part 1: Potential effects on adaptive immunity and response to vaccination.

For more than 20 years, researchers have used laboratory mice lacking type I or both type I and type II interferon (IFN) responses to study viruses that cause hemorrhagic fever (HF) in humans. Whereas immunocompetent mice do not become ill when infected with Ebola, Lassa, dengue and other HF viruses, IFN-deficient mice typically develop severe or fatal disease when inoculated with these pathogens. The ease of employment of these "mouse models" has led to their extensive use in biocontainment laboratories to assess the efficacy of novel vaccines, often without consideration of whether adaptive immune responses in IFN-deficient mice accurately mirror those in humans. Failure to consider these questions may lead to inappropriate expectations of the predictive value of mouse experiments. In two invited articles, we investigate this question. This paper examines how the lack of type I or both type I and type II IFN signaling may affect the development of adaptive immune responses in mice and the outcome of vaccine studies. A second article reviews the published literature on the use of IFN-deficient mice for the assessment of novel vaccines against HF viruses.

A Biomechanical Comparison of Different Suture Materials Used for Arthroscopic Shoulder Procedures.

PURPOSE: To evaluate the viscoelastic properties of 4 commercially available cord-like sutures and 2 commercially available suture tapes when subjected to physiological loads, as well as to compare them with each other and to identify the clinically most desirable combination of suture material properties. METHODS: Six suture materials (Ethibond, FiberWire, FiberTape, Orthocord, Ultrabraid, and Ultratape) underwent creep testing (n = 7, 60 N, 10 minutes) to determine specimen stiffness, initial elongation at 60 N of load, static creep (during 10 minutes of loading), and relaxed elongation (material recovery 3 minutes after removal of load). Furthermore, cyclic testing (n = 7, 10-45 N, 0.5 Hz, 500 cycles) was carried out to determine dynamic creep, peak-to-peak displacement, and relaxed elongation. Mechanical testing was conducted on a material testing machine in 37°C phosphate-buffered saline solution. RESULTS: FiberTape showed the greatest stiffness (23.9 ± 3.2 N/mm, P < .001), the smallest amounts of static (0.38 ± 0.10 mm, P < .001) and dynamic (0.16 ± 0.09 mm, P = .003) creep, and the smallest peak-to-peak displacement (0.20 ± 0.02 mm, P < .001). FiberTape and FiberWire showed the smallest initial elongation (1.17 ± 0.17 mm and 1.63 ± 0.25 mm, respectively; P < .001). Ultrabraid showed the greatest relaxed elongation, both statically (4.73 ± 0.73 mm, P < .001) and dynamically (4.18 ± 0.83 mm, P = .002). CONCLUSIONS: FiberTape consistently displayed less creep, greater stiffness, and less extensibility than the other suture types. Ultrabraid showed the largest amount of relaxed elongation on both static and dynamic testing. CLINICAL RELEVANCE: When considering high stiffness in combination with low initial extension and low static creep to be ideal parameters to achieve optimal initial construct stability and considering low dynamic creep in combination with low peak-to-peak displacement to be ideal conditions for the repetitive loading of the construct during the healing process, tapes seem to be superior to cord-like sutures for performing rotator cuff repair.

Functionally distinct tendons have different biomechanical, biochemical and histological responses to in vitro unloading.

Tendons with different in vivo functions are known to have different baseline biomechanics, biochemistry and ultrastructure, and these can be affected by changes in loading. However it is not know whether different tendon types respond in the same, or different ways, to changes in loading. This study performed in vitro un-loading (stress deprivation) in culture on ovine medial extensor tendons (MET, a positional tendon), and superficial and deep digital flexor tendons (SDFTs and DDFTs, with energy-storing and intermediate functions respectively), for 21 days (n = 14 each). Tensile strength and elastic modulus were then measured, followed by biochemical assays for sulphated glycosaminoglycan (sGAG) and hydroxyproline content. Histological inspection for cell morphology, cell density and collagen alignment was also performed. The positional tendon (MET) had a significant reduction (∼50%) in modulus and strength (P < 0.001) after in vitro stress-deprivation, however there were no significant effects on the energy-storing tendons (SDFT and DDFT). In contrast, sGAG was not affected in the MET, but was reduced in the SDFT and DDFT (P < 0.001). All tendons lost compactness and collagen organisation, and had reduced cell density, but these were more rapid in the MET than the SDFT and DDFT. These results suggest that different tendon types respond to identical stimuli in different ways, thus; (i) the results from an experiment in one tendon type may not be as applicable to other tendon types as previously thought, (ii) positional tendons may be particularly vulnerable to clinical stress-deprivation, and (iii) graft tendon source may affect the biological response to loading in ligament and tendon reconstruction.

Generation and Selection of a Panel of Pan-Filovirus Single-Chain Antibodies using Cell-Free Ribosome Display.

Filoviruses, which include ebolaviruses and marburgvirus, can cause outbreaks of highly lethal hemorrhagic fever. This disease causes significant morbidity and mortality in humans and non-human primates, with human fatality rates reaching 90% during some outbreaks. Currently, there is lack of licensed vaccines or antivirals for these viruses. Since early symptoms of filovirus infection mimic more common diseases, there is a strong unmet public health and biodefense need for broad-spectrum filovirus rapid diagnostics. We have generated a panel of mouse single-chain Fv-antibodies (scFvs) to filovirus glycoproteins (GPs) using cell-free ribosome display and determined their cross-reactivity profiles to all known filovirus species. Two scFvs (4-2 and 22-1) were able to detect all known Ebolavirus and Marburgvirus species. This is the first report on ribosome display scFvs that can detect a broad set of filovirus GPs, which demonstrates the potential for use in diagnostics.

The Effect of Extensor Tendon Adhesions on Finger Motion.

PURPOSE: To quantify the amount and pattern of finger range of motion loss at the metacarpophalangeal (MCP), proximal interphalangeal (PIP), and distal interphalangeal (DIP) joints with a simulated extensor tendon adhesion at the level of the proximal phalanx or metacarpal. METHODS: In 10 cadaveric specimens, traction sutures were placed in the forearm extensor digitorum communis and flexor digitorum profundus tendons of the middle and ring fingers. Active motion was simulated by suspending weights from the traction sutures via pulleys. The angles of the MCP, PIP, and DIP joints were measured at the position of maximum flexion and extension. Extensor tendon adhesions were simulated alternately at the proximal phalanx and metacarpal levels of the middle and ring fingers, using suture anchors. Repeat measurements were taken using the same amount of force. RESULTS: There was an average total loss of flexion of 38° and of extension of 6° with a proximal phalanx adhesion, with a greater contribution of flexion loss at the PIP joint. The loss of flexion was 17° and of extension was 50° with a metacarpal adhesion, with a loss of extension mostly at the MCP joint. CONCLUSIONS: The results of this study identified clear patterns of motion loss that are associated with isolated simulated adhesions in different locations along the extensor mechanism. The greatest motion loss occurred at the joint immediately distal to the simulated adhesion. CLINICAL RELEVANCE: Although extrapolation of these findings to clinical relevance remains unclear, the ability to predict the level of adhesion by the pattern of motion restriction may allow for a targeted tenolysis procedure. This would reduce the amount of soft tissue dissection required, which in turn, could be expected to reduce the degree of repeat adhesion formation.

Engineering approaches to understanding mechanisms of spinal column injury leading to spinal cord injury.

BACKGROUND: The mechanical interactions occurring between the spinal column and spinal cord during an injury event are complex and variable, and likely have implications for the clinical presentation and prognosis of the individual. METHODS: The engineering approaches that have been developed to better understand spinal column and cord interactions during an injury event are discussed. These include injury models utilising human and animal cadaveric specimens, in vivo anaesthetised animals, finite element models, inanimate physical systems and combinations thereof. FINDINGS: The paper describes the development of these modelling approaches, discusses the advantages and disadvantages of the various models, and the major outcomes that have had implications for spinal cord injury research and clinical practice. INTERPRETATION: The contribution of these four engineering approaches to understanding the interaction between the biomechanics and biology of spinal cord injury is substantial; they have improved our understanding of the factors contributing to the spinal column disruption, the degree of spinal cord deformation or motion, and the resultant neurological deficit and imaging features. Models of the injury event are challenging to produce, but technological advances are likely to improve these models and, consequently, our understanding of the mechanical context in which the biological injury occurs.

Three-dimensional architecture of the whole human soleus muscle in vivo.

BACKGROUND: Most data on the architecture of the human soleus muscle have been obtained from cadaveric dissection or two-dimensional ultrasound imaging. We present the first comprehensive, quantitative study on the three-dimensional anatomy of the human soleus muscle in vivo using diffusion tensor imaging (DTI) techniques. METHODS: We report three-dimensional fascicle lengths, pennation angles, fascicle curvatures, physiological cross-sectional areas and volumes in four compartments of the soleus at ankle joint angles of 69 ± 12° (plantarflexion, short muscle length; average ± SD across subjects) and 108 ± 7° (dorsiflexion, long muscle length) of six healthy young adults. Microdissection and three-dimensional digitisation on two cadaveric muscles corroborated the compartmentalised structure of the soleus, and confirmed the validity of DTI-based muscle fascicle reconstructions. RESULTS: The posterior compartments of the soleus comprised 80 ± 5% of the total muscle volume (356 ± 58 cm3). At the short muscle length, the average fascicle length, pennation angle and curvature was 37 ± 8 mm, 31 ± 3° and 17 ± 4 /m, respectively. We did not find differences in fascicle lengths between compartments. However, pennation angles were on average 12° larger (p < 0.01) in the posterior compartments than in the anterior compartments. For every centimetre that the muscle-tendon unit lengthened, fascicle lengths increased by 3.7 ± 0.8 mm, pennation angles decreased by -3.2 ± 0.9° and curvatures decreased by -2.7 ± 0.8 /m. Fascicles in the posterior compartments rotated almost twice as much as in the anterior compartments during passive lengthening. DISCUSSION: The homogeneity in fascicle lengths and inhomogeneity in pennation angles of the soleus may indicate a functionally different role for the anterior and posterior compartments. The data and techniques presented here demonstrate how DTI can be used to obtain detailed, quantitative measurements of the anatomy of complex skeletal muscles in living humans.

Effects of Removal and Reinsertion of Headless Compression Screws.

PURPOSE: This study investigates the loss of compression when 3 commonly used headless compression screws are backed out (reversed), and assesses the ability to re-establish compression with screws of greater diameter. METHODS: Two investigators tested 3 screw designs (Acutrak 2, Synthes HCS, Medartis SpeedTip CCS) in 2 diameters and lengths. Each design had 10 test cycles in a polyurethane foam bone model with compression recorded using a washer load cell. A 28-mm screw of the narrower diameter was inserted until 2 mm recessed and then reversed 30°, 60°, 90°, 180°, 270°, 360°, and 720°. After this the screw was removed completely and a 24-mm screw of greater diameter inserted until recessed 2 mm with the compressive force again recorded. RESULTS: All screws showed an immediate, statistically significant loss of compression at 30° of reversing. The Acutrak 2 Micro screw demonstrated not only the greatest mean compressive force, but also the fastest compressive loss. Insertion of the shorter screw of greater diameter was associated with re-establishment of compression to levels comparable with the original screw. CONCLUSIONS: This study reaffirms the importance of establishing the correct screw length before insertion due to the immediate loss of compression with reversal of these devices. CLINICAL RELEVANCE: If a headless compression screw penetrates the far joint surface, the screw should be completely removed and replaced with a shorter screw of greater diameter.

Production and Purification of Filovirus Glycoproteins in Insect and Mammalian Cell Lines.

Filoviruses are highly virulent pathogens capable of causing severe disease. The glycoproteins of filoviruses are the only virally expressed proteins on the virion surface and are required for receptor binding. As such, they are the main candidate vaccine antigen. Despite their virulence, most filoviruses are not comprehensively characterized, and relatively few commercially produced reagents are available for their study. Here, we describe two methods for production and purification of filovirus glycoproteins in insect and mammalian cell lines. Considerations of expression vector choice, modifications to sequence, troubleshooting of purification method, and glycosylation differences are all important for successful expression of filovirus glycoproteins in cell lines. Given the scarcity of commercially available filovirus glycoproteins, we hope our experiences with possible difficulties in purification of the proteins will facilitate other researchers to produce and purify filovirus glycoproteins rapidly.