The inhibitory effect of melatonin on osteoclastogenesis of RAW 264.7 cells in low concentrations of RANKL and MCSF

RAW 264.7 cells are one of the most recommended cell lines for investigating the activity and differentiation of osteoclasts. These cells differentiate into osteoclasts in the presence of two critical components: receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony stimulating factor (MCSF). Melatonin (MEL) hormone has recently become one of the small molecules used in the field of bone regeneration and bone disease treatment, as it has the ability to inhibit the differentiation of osteoclasts directly by suppression of the NF-κB signaling pathway. The main aim of the current study is to determine sufficient RANKL/MCSF concentrations for differentiation of the cells to osteoclasts and to describe the repressive effect of MEL on the osteoclastogenesis of these cells. In this regard, it was found that 10 ng/mL of RANKL- and MCSF-containing medium is suitable for inducing osteoclastogenesis of the cells. In addition, melatonin at doses in the range of 100–1000 μM does not have a cytotoxic effect. Subsequently, results of tartrate resistant acid phosphatase (TRAP) activity, TRAP staining, and relative expressions of cathepsin K, nuclear factor of activated T cells one (NFATC1), and TRAP genes showed a suppressive effect of MEL —especially 800 μM— on RANKL-induced osteoclastogenesis of these cells.

New Litter Trap Devices Outperform Pitfall Traps for Studying Arthropod Activity

Soil fauna play a key role in nutrient cycling and decomposition, and in recent years, researchers have become more and more interested in this compartment of terrestrial ecosystems. In addition, soil fauna can act as ecosystem engineers by creating, modifying, and maintaining the habitat for other organisms. Ecologists usually utilize live catches in pitfalls traps as a standard method to study the activity of epigeic fauna in addition to relative abundance. Counts in pitfall traps can be used as estimates of relative activity to compare among experimental treatments. This requires taking independent estimates of abundance (e.g., by sifting soil litter, mark–recapture), which can then be used as covariates in linear models to compare the levels of fauna activity (trap catches) among treatments. However, many studies show that the use of pitfall traps is not the most adequate method to estimate soil fauna relative abundances, and these concerns may be extensible to estimating activity. Here, we present two new types of traps devised to study activity in litter fauna, and which we call “cul-de-sac” and “basket traps”, respectively. We experimentally show that, at least for litter dwellers, these new traps are more appropriate to estimate fauna activity than pitfall traps because: (1) pitfall traps contain 3.5× more moisture than the surrounding environment, potentially attracting animals towards them when environmental conditions are relatively dry; (2) cul-de-sac and basket traps catch ca. 4× more of both meso- and macrofauna than pitfall traps, suggesting that pitfall traps are underestimating activity; and (3) pitfall traps show a bias towards collecting 1.5× higher amounts of predators, which suggests that predation rates are higher within pitfall traps. We end with a protocol and recommendations for how to use these new traps in ecological experiments and surveys aiming at estimating soil arthropod activity.

Stable patterns of upper limb muscle activation in different conditions of human walking

<p>Background: Arm swing during human gait is associated with contractions of upper limb muscles, which have been examined rarely.</p><p>Aim: This study aims to identify basic temporal patterns of upper limb muscle activation during walking conditions involving different modes of arm swing.</p><p>Method: Twenty volunteers were examined during (a) normal forward walking, (b) walking with immobilized arms, (c) walking while carrying loads in one or in both hands. Electromyographic (EMG) data were collected for the trapezius (TRAP), anterior (AD) and posterior deltoid (PD), biceps (BIC), triceps (TRI), latissimus dorsi (LD) and lumbar erector spinae (ES) muscles.</p><p>Results: Principal components analyses identified two basic patterns of muscle activation that remained stable across gait conditions. Some rhythmical EMG signals of arm and shoulder muscles (TRAP, PD, TRI, LD) persisted during walking with immobilized arms, indicating coupled activation of leg and arm muscles. Carrying a load in one hand resulted in stronger ipsilateral EMG activity (TRAP, AD, PD, BIC, TRI) than splitting the same load between both hands.</p><p>Interpretation: Although the amount of upper limb muscle activity varies markedly between different conditions of human walking (with/without arm sing; with/without load carriage), basic temporal activation patterns remain stable, indicating a common motor control strategy.</p><em><br clear="all" /> </em>

Stable patterns of upper limb muscle activation in different conditions of human walking

Background:Â Arm swing during human gait is associated with contractions of upper limb muscles, which have been examined rarely. Aim: This study aims to identify basic temporal patterns of upper limb muscle activation during walking conditions involving different modes of arm swing. Method: Twenty volunteers were examined during (a) normal forward walking, (b) walking with immobilized arms, (c) walking while carrying loads in one or in both hands. Electromyographic (EMG) data were collected for the trapezius (TRAP), anterior (AD) and posterior deltoid (PD), biceps (BIC), triceps (TRI), latissimus dorsi (LD) and lumbar erector spinae (ES) muscles. Results: Principal components analyses identified two basic patterns of muscle activation that remained stable across gait conditions. Some rhythmical EMG signals of arm and shoulder muscles (TRAP, PD, TRI, LD) persisted during walking with immobilized arms, indicating coupled activation of leg and arm muscles. Carrying a load in one hand resulted in stronger ipsilateral EMG activity (TRAP, AD, PD, BIC, TRI) than splitting the same load between both hands. Interpretation: Although the amount of upper limb muscle activity varies markedly between different conditions of human walking (with/without arm sing; with/without load carriage), basic temporal activation patterns remain stable, indicating a common motor control strategy.

Quantitative Analysis of Telomerase in Feline Mammary Tissues

The purpose of this study was to validate immunohistochemistry (IHC) as an alternative to telomerase repeat amplification protocol (TRAP) analysis to detect telomerase activity. TRAP–enzyme-linked immunosorbent assay (ELISA) reactivity was compared with telomerase reverse transcription (TERT) IHC staining in 22 feline mammary tissues (6 normal mammary glands, 2 dysplastic mammary glands, 1 fibroadenoma, and 13 malignant neoplasms [6 solid mammary carcinomas, 2 squamous-cell carcinomas, 4 tubulopapillary mammary carcinomas, and 1 mammary carcinosarcoma]). TERT IHC staining revealed enzymatic expression in nuclear, nucleolar, cytoplasmic, and combined nuclear and nucleolar staining patterns that were separately quantified by image analysis and expressed as the absolute number (average) of positive cells or percentage of positive cells with respect to overall cellularity. With TERT IHC staining, the absolute number and percentage of cells with positive nuclei and nucleoli within the same cell were the variables with the greatest discrimination between benign and malignant mammary lesions (analysis of variance [ANOVA], average P < 0.0001; percentage P < 0.001). For TRAP-ELISA–positive versus TRAP-ELISA–negative tissues, a positive test result provided greater differentiation between malignant versus benign mammary lesions (ANOVA, average P = 0.00038; percentage P = 0.0022). The same IHC pattern of expression showed a proportional and significant (average P = 0.004; percentage P = 0.002) but low (average R = 0.60; percentage R = 0.63) correlation with TRAP-ELISA by the Pearson test. The correlation coefficients obtained show that IHC and TRAP cannot be considered interchangeable because the 2 methods are more complementary than exclusive.

High Dose Simvastatin Has No Beneficial Effect on Markers of Bone Turn-Over in Multiple Myeloma.

Background: Bone degradation in multiple myeloma (MM) is a result of increased bone degradation by osteoclasts that is not compensated for by bone forming osteoblasts. Ideally new drugs used for treatment of MM should target not only the myeloma cells but also the imbalance between bone resorption and bone formation. Statins have been shown to inhibit myeloma cell proliferation and induce apoptosis in vitro. Furthermore statins have been shown to stimulate osteoblasts and inhibit osteoclasts both in vitro and in animal models. Statins are normally used at doses around 20–80 mg/day, but in order to reach serum concentrations that can match the in vitro experiments MM patients were treated with 15 mg/kg/day of Simvastatin (HD-Sim) divided in two daily doses in this study. This high dose has previously been found to be safe for MM patients (Haematologica 2006, 91,542–545) Patients and methods: Six patients with advanced MM have been included in this pilot study, 4 males and 2 females with an average age of 68 years and an average duration of disease of 43 months. The patients were treated with 2 cycles of HD-Sim for seven days followed by a break of 21 days in a 4-weeks cycle. Two of the patients were treated with bisphosphonates during the study, and 4 had previously been treated with bisphosphonates. Endpoints are change in concentrations of markers of osteoclast activity (TRAP) or bone resorption (CTX, NTX, ICTP) or markers of bone formation (Osteocalcin and PINP). Cholesterol, OPG and DDK-1 were also measured. Results: Two patients completed the protocol with two cycles of HD-Sim at full dose, 2 patients were reduced to 7.5 mg/kg/day simvastatin in cycle 2 due to nausea and diarrhea and 2 patients left the protocol after 3 weeks (deaths not related to high dose simvastatin). All patients experienced gastrointestinal toxicity grade 1–2. Myalgia and other muscular symptoms grade 1–2 were reported by 5 patients but were not associated with an increase in creatin kinase. TRAP and NTX activity in serum increased for all 6 patients during the seven days of treatment with HD-Sim indicating that bone resorption may have been stimulated rather than inhibited. The other markers of bone resorption and the bone formation markers showed no change. All patients responded with a significantly reduced level of cholesterol in serum. None of the patients showed any reduction in free monoclonal light chains or monoclonal proteins in serum during treatment with HD-Sim and 2 of the 4 patients completing the protocol showed progression of diseases. Conclusion: This pilot study of HD-Sim in advanced MM has been terminated due to lack of response and evidence from two markers of osteoclast activity (TRAP) and bone resorption (NTX) that HD-Sim may be harmful rather than beneficial in MM.