In Vitro Inflammatory Cell-Induced Corrosion Using a Lymphocyte and Macrophage Coculture.

BACKGROUND: Cobalt-chromium-molybdenum (CoCrMo) and titanium alloys have been used for orthopaedic implants for decades. However, recent evidence has shown that inflammatory cell-induced corrosion (ICIC) can damage these metal alloys. This study aimed to investigate the mechanisms of ICIC by coculturing macrophages with lymphocytes. We hypothesized that macrophages would be able to alter the surface oxide layer of CoCrMo and titanium alloy (Ti6Al4V) disks, with greater oxide layer damage occurring in groups with a coculture compared to a macrophage monoculture and in groups with inflammatory activators compared to nonactivated groups. METHODS: Murine macrophages were cultured on American Society for Testing and Materials F1537 CoCrMo and F136 Ti6Al4V disks for 30 days and activated with interferon gamma and lipopolysaccharide. Interferon gamma and lipopolysaccharide were added to the culture medium to simulate local inflammation. Macrophages were either cultured alone or in a coculture with T helper lymphocytes. After the 30-day experiment, scanning electron microscopy was used to examine the disk surfaces, and oxide levels were found using energy dispersive x-ray spectroscopy. RESULTS: Pitting features consistent with previous reports of ICIC were found on disks cultured with cells. Both CoCrMo and Ti6Al4V disks had significantly lower oxide levels in all groups with cells compared to control groups with no cells (P < .01). Additionally, CoCrMo disks had significantly lower oxide levels when cultured with activated macrophages and lymphocytes compared to nonactivated macrophages alone (P < .001), activated macrophages alone (P < .01), and nonactivated macrophages and lymphocytes (P < .05). No differences in the oxide levels were found among the Ti6Al4V groups. CONCLUSIONS: This study demonstrates the ability of macrophages to alter the surface chemistry of commonly used orthopaedic alloys. We found that the addition of lymphocytes and a simulated local inflammatory response may contribute to the ICIC of CoCrMo implants.

The effect of body weight on interfragmentary fracture strain in plate fixation of distal femur fractures: A finite element analysis.

INTRODUCTION: Distal femur fractures are difficult to successfully treat due to high rates of nonunion. Obesity is an independent prognostic risk factor for nonunion. Advances in finite element analyses (FEAs) have allowed researchers to better understand the performance and behavior of constructs at the bone-implant interface under a variety of conditions. The purpose of this study is to determine the impact of body weight on fracture strain in a lateral locking plate construct for supracondylar femur fractures and whether additional construct rigidity is beneficial to optimize fracture strain in high body mass patients. HYPOTHESIS: We hypothesized that increased loads would produce a higher interfragmentary strain (IFS), which could be decreased by shortening the working length of the construct. MATERIALS AND METHODS: A 3D finite element analysis was performed on two separate femur models with a comminuted supracondylar distal femur fracture fixed with a lateral distal femoral locking plate in bridging mode with Ansys software. Axial forces were varied to recreate the effect of load from normal and high body mass patients. Working length and screw density of the construct were varied for each condition. Measurements of interfragmentary strain and shear motion (SM) were compared. RESULTS: Doubling the axial load from 70kg (control) to 140kg (high body mass) increased the interfragmentary strain by an average of 76% for the three working lengths (3.38%±1.67% to 4.37%±0.88% at the baseline working length (BWL), 1.42%±1.00% to 2.87%±2.02% at the intermediate working length (IWL) and 0.62%±0.22% to 1.22%±0.42% at the short working length (SWL)). On average, decreasing the working length in the 140kg load reduced the mean IFS to within 15% of the mean IFS of the 70kg load at the longer working length (2.87%±2.02% at IWL 140kg versus 3.38%±1.67% at BWL 70kg and 1.22%±0.45% SWL 140kg versus 1.42±1.00% IWL 70kg). DISCUSSION: Increased axial load increases interfragmentary strain in an AO/OTA 33A distal femur fracture fixed with a lateral distal femoral locking plate. Decreasing the working length of the fixation construct in the high body mass model decreased interfragmentary strain. Higher loading conditions reflective of high body mass patients should be considered in studies investigating optimization of fracture strain. LEVEL OF PROOF: V; Finite Element Analysis (FEA).

Cholesterol increases protein levels of the E3 ligase MARCH6 and thereby stimulates protein degradation.

The E3 ligase membrane-associated ring-CH-type finger 6 (MARCH6) is a polytopic enzyme bound to the membranes of the endoplasmic reticulum. It controls levels of several known protein substrates, including a key enzyme in cholesterol synthesis, squalene monooxygenase. However, beyond its own autodegradation, little is known about how MARCH6 itself is regulated. Using CRISPR/Cas9 gene-editing, MARCH6 overexpression, and immunoblotting, we found here that cholesterol stabilizes MARCH6 protein endogenously and in HEK293 cells that stably express MARCH6. Conversely, MARCH6-deficient HEK293 and HeLa cells lost their ability to degrade squalene monooxygenase in a cholesterol-dependent manner. The ability of cholesterol to boost MARCH6 did not seem to involve a putative sterol-sensing domain in this E3 ligase, but was abolished when either membrane extraction by valosin-containing protein (VCP/p97) or proteasomal degradation was inhibited. Furthermore, cholesterol-mediated stabilization was absent in two MARCH6 mutants that are unable to degrade themselves, indicating that cholesterol stabilizes MARCH6 protein by preventing its autodegradation. Experiments with chemical chaperones suggested that this likely occurs through a conformational change in MARCH6 upon cholesterol addition. Moreover, cholesterol reduced the levels of at least three known MARCH6 substrates, indicating that cholesterol-mediated MARCH6 stabilization increases its activity. Our findings highlight an important new role for cholesterol in controlling levels of proteins, extending the known repertoire of cholesterol homeostasis players.

Chromatin remodeler mutations in human cancers: epigenetic implications.

Chromatin remodeler complexes exhibit the ability to alter nucleosome composition and positions, with seemingly divergent roles in the regulation of chromatin architecture and gene expression. The outcome is directed by subunit variation and interactions with accessory factors. Recent studies have revealed that subunits of chromatin remodelers display an unexpectedly high mutation rate and/or are inactivated in a number of cancers. Consequently, a repertoire of epigenetic processes are likely to be affected, including interactions with histone modifying factors, as well as the ability to precisely modulate nucleosome positions, DNA methylation patterns and potentially, higher-order genome structure. However, the true significance of chromatin remodeler genetic aberrations in promoting a cascade of epigenetic changes, particularly during initiation and progression of cancer, remains largely unknown.

The E3 ubiquitin ligase MARCH6 degrades squalene monooxygenase and affects 3-hydroxy-3-methyl-glutaryl coenzyme A reductase and the cholesterol synthesis pathway.

The mevalonate pathway is used by cells to produce sterol and nonsterol metabolites and is subject to tight metabolic regulation. We recently reported that squalene monooxygenase (SM), an enzyme controlling a rate-limiting step in cholesterol biosynthesis, is subject to cholesterol-dependent proteasomal degradation. However, the E3-ubiquitin (E3) ligase mediating this effect was not established. Using a candidate approach, we identify the E3 ligase membrane-associated RING finger 6 (MARCH6, also known as TEB4) as the ligase controlling degradation of SM. We find that MARCH6 and SM physically interact, and consistent with MARCH6 acting as an E3 ligase, its overexpression reduces SM abundance in a RING-dependent manner. Reciprocally, knockdown of MARCH6 increases the level of SM protein and prevents its cholesterol-regulated degradation. Additionally, this increases cell-associated SM activity but is unexpectedly accompanied by increased flux upstream of SM. Prompted by this observation, we found that knockdown of MARCH6 also controls the level of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) in hepatocytes and model cell lines. In conclusion, MARCH6 controls abundance of both SM and HMGCR, establishing it as a major regulator of flux through the cholesterol synthesis pathway.

A practical comparison of ligation-independent cloning techniques.

The precise assembly of specific DNA sequences is a critical technique in molecular biology. Traditional cloning techniques use restriction enzymes and ligation of DNA in vitro, which can be hampered by a lack of appropriate restriction-sites and inefficient enzymatic steps. A number of ligation-independent cloning techniques have been developed, including polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC), and overlap extension cloning (OEC). These strategies rely on the generation of complementary overhangs by DNA polymerase, without requiring specific restriction sites or ligation, and achieve high efficiencies in a fraction of the time at low cost. Here, we outline and optimise these techniques and identify important factors to guide cloning project design, including avoiding PCR artefacts such as primer-dimers and vector plasmid background. Experiments made use of a common reporter vector and a set of modular primers to clone DNA fragments of increasing size. Overall, PIPE achieved cloning efficiencies of ∼95% with few manipulations, whereas SLIC provided a much higher number of transformants, but required additional steps. Our data suggest that for small inserts (<1.5 kb), OEC is a good option, requiring only two new primers, but performs poorly for larger inserts. These ligation-independent cloning approaches constitute an essential part of the researcher's molecular-tool kit.

A key regulator of cholesterol homoeostasis, SREBP-2, can be targeted in prostate cancer cells with natural products.

There is growing evidence showing that prostate cancer cells have perturbed cholesterol homoeostasis, accumulating cholesterol to promote cell growth. Consequently, cholesterol-lowering drugs such as statins are being evaluated in prostate cancer treatment. Furthermore, natural products such as betulin (from birch tree bark) and tocotrienol (a minor form of vitamin E) have been shown to lower cholesterol levels. Using these drugs and oxysterols, we have determined which aspects of cholesterol homoeostasis should be targeted in prostate cancer, e.g. cellular cholesterol levels are increased by the transcription factor SREBP-2 (sterol-regulatory-element-binding protein isoform 2), whereas LXR (liver X receptor) promotes cholesterol efflux. Whereas betulin exerted non-specific effects on cell viability, tocotrienols produced a strong direct correlation between SREBP-2 activity and cell viability. Mechanistically, tocotrienols lowered SREBP-2 activity by degrading mature SREBP-2 independently of the proteasome. In contrast, no correlation was seen between LXR activity and cell viability, implying that SREBP-2 is a better target than LXR for prostate cancer treatment. Lastly, androgen-dependent and -independent LNCaP cells were both sensitive to tocotrienols. Overall, this suggests that tocotrienols and other drugs targeting the SREBP-2 pathway are a potential therapeutic option for prostate cancer.

A randomized trial to assess the effect of a research informational pamphlet on telephone survey completion rates among older Latinos.

PURPOSE OF THE STUDY: To assess the effects of receiving a research informational pamphlet produced by the federal Office for Human Research Protections on telephone survey participation rates of older Latinos, and illustrate the feasibility of nesting recruitment studies within other funded studies when stand alone funding for recruitment studies is limited. DESIGN AND METHODS: Latino patients aged ≥50 with ≥1 visit during the preceding year (N=1314) were sampled from three community clinics and a multi-specialty medical group. Patients were randomly assigned to receive or not receive a pamphlet that contained information on research participation in the initial mailing for the telephone survey study. Survey participation rates were compared between the pamphlet and no pamphlet groups. RESULTS: In a multivariate model, women (OR=1.4; 95% CI 1.1, 1.8), and those with public insurance (vs. no insurance; OR=1.7; 95% CI 1.1, 2.5) were more likely to participate, while those age 65+ (vs. age 50-54; OR=0.6; 95% CI 0.4, 0.8) were less likely to participate; there was no significant difference by pamphlet group (OR=0.8; 95% CI 0.7, 1.1). Nesting of the randomized trial of the recruitment pamphlet within the funded study required minimal additional resources. IMPLICATIONS: Recruitment methods that are more intensive than a pamphlet may be needed to enhance survey participation rates among older Latinos. Nesting recruitment trials within funded studies is a promising and efficient approach for testing recruitment strategies.