Flunitrazepam Excretion Patterns using the Abuscreen OnTrak and OnLine Immunoassays: Comparison with GC-MS

A study was conducted to compare the performance of the OnLine and OnTrak immunoassays for benzodiazepines with gas chromatographic-mass spectrometric (GC-MS) analysis in detecting flunitrazepam (FNP) and its metabolites in human urine. Urine was collected over a 72-h period from six individuals (four male and two female) who had taken a single oral dose of either 1 or 4 mg of FNP. The OnTrak assay was run at a 100-ng/mL cutoff of nordiazepam (NDP), and the OnLine assay was run with a standard curve from zero to 200 ng/mL of NDP with and without β-glucuronidase treatment. Each sample was analyzed by GC-MS using FNP, 7-amino-FNP, 3-hydroxy-FNP, desmethyl-FNP, 7-amino-3-hydroxy-FNP, and desmethyl-3-hydroxy-FNP as standards with β-glucuronidase treatment. The specimens from the 1-mg dose did not yield a positive result by immunoassay over the 72-h collection period. Specimens from the 4-mg dose did yield positive results in both immunoassays. The time of the first positive result ranged from 4 to 12 h, and the time to the last positive result ranged from 18 to 60 h. Treatment of the samples with β-glucuronidase increased the OnLine values between 20 and 60%, but it did not appreciably increase the detection time. GC-MS analysis showed no detectable levels of FNP, 3-hydroxy-FNP, desmethyl-FNP, 7-amino-3-hydroxy-FNP, and desmethyl-3-hydroxy-FNP. However, all samples collected past time zero showed detectable levels of 7-amino-FNP (> 2 ng/mL) with peak concentrations at 12-36 h. The peak levels of 7-amino-FNP by GC-MS paralleled the peak levels of the immunoassay response. The amount of 7-amino-FNP metabolite quantitated by GC-MS, however, accounted for only 15-20% of the total immunoassay crossreactive FNP metabolite

Force-dependent focal adhesion assembly and disassembly: A computational study

Cells interact with the extracellular matrix (ECM) via cell–ECM adhesions. These physical interactions are transduced into biochemical signals inside the cell which influence cell behaviour. Although cell–ECM interactions have been studied extensively, it is not completely understood how immature (nascent) adhesions develop into mature (focal) adhesions and how mechanical forces influence this process. Given the small size, dynamic nature and short lifetimes of nascent adhesions, studying them using conventional microscopic and experimental techniques is challenging. Computational modelling provides a valuable resource for simulating and exploring various “what if?” scenarios in silico and identifying key molecular components and mechanisms for further investigation. Here, we present a simplified mechano-chemical model based on ordinary differential equations with three major proteins involved in adhesions: integrins, talin and vinculin. Additionally, we incorporate a hypothetical signal molecule that influences adhesion (dis)assembly rates. We find that assembly and disassembly rates need to vary dynamically to limit maturation of nascent adhesions. The model predicts biphasic variation of actin retrograde velocity and maturation fraction with substrate stiffness, with maturation fractions between 18–35%, optimal stiffness of ∼1 pN/nm, and a mechanosensitive range of 1-100 pN/nm, all corresponding to key experimental findings. Sensitivity analyses show robustness of outcomes to small changes in parameter values, allowing model tuning to reflect specific cell types and signaling cascades. The model proposes that signal-dependent disassembly rate variations play an underappreciated role in maturation fraction regulation, which should be investigated further. We also provide predictions on the changes in traction force generation under increased/decreased vinculin concentrations, complementing previous vinculin overexpression/knockout experiments in different cell types. In summary, this work proposes a model framework to robustly simulate the mechanochemical processes underlying adhesion maturation and maintenance, thereby enhancing our fundamental knowledge of cell–ECM interactions

Egr3 Dependent Sympathetic Target Tissue Innervation in the Absence of Neuron Death

Nerve Growth Factor (NGF) is a target tissue derived neurotrophin required for normal sympathetic neuron survival and target tissue innervation. NGF signaling regulates gene expression in sympathetic neurons, which in turn mediates critical aspects of neuron survival, axon extension and terminal axon branching during sympathetic nervous system (SNS) development. Egr3 is a transcription factor regulated by NGF signaling in sympathetic neurons that is essential for normal SNS development. Germline Egr3-deficient mice have physiologic dysautonomia characterized by apoptotic sympathetic neuron death and abnormal innervation to many target tissues. The extent to which sympathetic innervation abnormalities in the absence of Egr3 is caused by altered innervation or by neuron death during development is unknown. Using Bax-deficient mice to abrogate apoptotic sympathetic neuron death in vivo, we show that Egr3 has an essential role in target tissue innervation in the absence of neuron death. Sympathetic target tissue innervation is abnormal in many target tissues in the absence of neuron death, and like NGF, Egr3 also appears to effect target tissue innervation heterogeneously. In some tissues, such as heart, spleen, bowel, kidney, pineal gland and the eye, Egr3 is essential for normal innervation, whereas in other tissues such as lung, stomach, pancreas and liver, Egr3 appears to have little role in innervation. Moreover, in salivary glands and heart, two tissues where Egr3 has an essential role in sympathetic innervation, NGF and NT-3 are expressed normally in the absence of Egr3 indicating that abnormal target tissue innervation is not due to deregulation of these neurotrophins in target tissues. Taken together, these results clearly demonstrate a role for Egr3 in mediating sympathetic target tissue innervation that is independent of neuron survival or neurotrophin deregulation

Flunitrazepam Excretion Patterns using the Abuscreen OnTrak and OnLine Immunoassays: Comparison with GC-MS

A study was conducted to compare the performance of the OnLine and OnTrak immunoassays for benzodiazepines with gas chromatographic-mass spectrometric (GC-MS) analysis in detecting flunitrazepam (FNP) and its metabolites in human urine. Urine was collected over a 72-h period from six individuals (four male and two female) who had taken a single oral dose of either 1 or 4 mg of FNP. The OnTrak assay was run at a 100-ng/mL cutoff of nordiazepam (NDP), and the OnLine assay was run with a standard curve from zero to 200 ng/mL of NDP with and without β-glucuronidase treatment. Each sample was analyzed by GC-MS using FNP, 7-amino-FNP, 3-hydroxy-FNP, desmethyl-FNP, 7-amino-3-hydroxy-FNP, and desmethyl-3-hydroxy-FNP as standards with β-glucuronidase treatment. The specimens from the 1-mg dose did not yield a positive result by immunoassay over the 72-h collection period. Specimens from the 4-mg dose did yield positive results in both immunoassays. The time of the first positive result ranged from 4 to 12 h, and the time to the last positive result ranged from 18 to 60 h. Treatment of the samples with β-glucuronidase increased the OnLine values between 20 and 60%, but it did not appreciably increase the detection time. GC-MS analysis showed no detectable levels of FNP, 3-hydroxy-FNP, desmethyl-FNP, 7-amino-3-hydroxy-FNP, and desmethyl-3-hydroxy-FNP. However, all samples collected past time zero showed detectable levels of 7-amino-FNP (> 2 ng/mL) with peak concentrations at 12–36 h. The peak levels of 7-amino-FNP by GC-MS paralleled the peak levels of the immunoassay response. The amount of 7-amino-FNP metabolite quantitated by GC-MS, however, accounted for only 15–20% of the total immunoassay crossreactive FNP metabolites

Comparison of bone histomorphometry in hypophosphatemia and vitamin D deficiency osteomalacia

Background:Osteomalacia (OM) can be caused by several conditions, but the two most common causes are phosphate and vitamin D deficiency (1). Although calcium deficiency rickets and OM have been reported in children, OM due to calcium deficiency has not been reported in adults. We had the unique opportunity to compare the severity of mineralization defect in the two most common causes of OM. Methods: We examined the invivo double tetracycline labelled bone histomorphometric findings in 24 patients with vitamin D deficiency (21 patients with malabsorption and 3 with nutritional vitamin D deficiency) and 7 patients with hypophosphatemia (4 patients with tumor and 3 with tenofovir induced). Since there were no differences in bone histomorphometric findings between the two groups of either variety we combined the respective groups for analyzing the difference between vitamin D deficiency and hypophosphatemic OM. As expected, patients with vitamin D deficiency had very low or undetectable serum 25-hydroxyvitamin D (5.4±3.8ng/ml) and high PTH (277±90pg/ml) levels, whereas both were normal in hypophosphatemic patients (26.5±5.4ng/ml and 46.6±13.5pg/ml; p\u3c0.001 for the difference for both) . Mean serum phosphate level, by definition, was very low in the hypophosphatemic patients, but significantly lower than in the vitamin D deficient patient (1.6 ±0.4mg/dl Vs. 2.9±0.5mg/dl; p\u3c0.001) Results: For the entire group, osteoid indices (surface, width & volume) were high, none had double tetracycline labels and all had severe mineralization defect (mineral apposition rate =0) confirming OM in each patient. However, osteoid volume, an index of the severity of OM, was significantly higher in hypophosphatemic compared to vitamin D deficient patients (33.6 Vs. 21.7% of the bone surface; p=0.049) indicating a more severe mineralization defect in hypophosphatemic OM. In contrast resorption surface, an index of PTH effect on bone, was significantly higher (4.52 Vs. 1.42% of non-osteoid surface; p=0.009) in vitamin D deficiency compared to phosphate deficiency. Finally, cortical thickness was lower in vitamin D deficiency than in phosphate deficiency, but did not reach statistical significance (0.51 Vs. 0.94mm; p=ns), most likely due to the small sample size of hypophosphatemic OM patients. Collectively, the data suggest a more severe mineralization defect in phosphate than in vitamin D deficiency OM. Conclusions: Mineralization defect is more severe in hypophosphatemic OM. Cortical thinning, a characteristic feature of excess PTH, is seen only in vitamin D deficiency, consistent with our previous findings (2,3). To the best of our knowledge this is the first such compartive study of its kind

Material properties of trans-iliac bone biopsies, as assessed by nanoindentation (NI), in patients with and without atypical femur fractures (AFF) on long-term bisphosphonate (BP) therapy for low bone density: Preliminary results of a prospective nested case-controlled study

Objective & Background: AFFs are now a well-established complication of long-term BP therapy, but its pathogenesis still remains unclear. Although severe suppression of bone turnover (SSBT), as we initial proposed as a major contributor, many patients with SSBT as defined may not develop AFF. Accordingly, we studied the material properties of iliac bone biopsy specimens by NI in 20 women (10 with and 10 without AFF matched for age and race). Methods: Nanoindentation data were analyzed using a mixed model ANOVA (SAS 9.4) for 20 subjects, 10 subjects with atypical femur fractures, and 10 without atypical fractures. The model was fit to using either elastic modulus, contact hardness, or resistance to plastic deformation as a function of bone type (cortical - 1, or trabecular -2), femur fracture (no fracture - 0, or fracture - 1) and all first order interactions between bone type and fracture. Post hoc analysis of the least squared means were analyzed and statistically significant differences we reported if p≤0.05. Results: Cortical bone with atypical femur fractures has a higher elastic modulus (is stiffer) than cortical bone without atypical fractures (ECB=17.49±0.16 GPa, ECB=16.56±0.15 GPa p=0.0005). This result suggests that bone with an atypical fracture has a higher level of mineralization, which implies that it has not been recently remodeled and therefore more likely to accumulate microdamage that would contribute to failure. Furthermore, for bone with an atypical femur fracture, the trabecular bone had a significantly lower elastic modulus than that of cortical bone (ETB=16.19±0.17 GPa, ECB=17.49±0.16 GPa p=0.0001). The trabecular bone with a fracture had the lowest mean value elastic modulus that we tested. This result suggests that the trabecular bone in the fracture case may not provide sufficient internal structural rigidity to accommodate applied loads, thus contributing to the overall failure of the bone. For bone with atypical fractures, cortical bone resistance to plastic deformation was significantly less than trabecular bone (HCB=1.97 ± 0.028 GPa, HTB =2.19±0.030 GPa p\u3c0.0001). This result further suggests that the integrity of the cortical bone has been compromised by microdamage, which may contribute to the atypical fracture of the bone. Conclusions: For bone that exhibits atypical femur fractures, the primary damage at the microstructural level appears to be associated with microdamage accumulation within the cortical tissue as evidenced by changes in the elastic modulus and decreased resistance to plastic deformation. In general, the trabecular bone does not appear to be as strongly affected by microdamage

Bone Structure and Turnover Status in Postmenopausal Women with Atypical Femur Fracture After Prolonged Bisphosphonate Therapy

Atypical femur fracture (AFF), a serious complication of long-term bisphosphonate therapy, is usually preceded by an incomplete fracture appearing on the lateral femur. AFF is most likely the result of severely suppressed bone turnover (SSBT). However, the differences in bone structure and turnover between patients with incomplete and complete AFF remain unknown. We examined trans-iliac bone biopsies from 12 white postmenopausal women with AFF (incomplete = 5; complete = 7) on BP therapy of \u3e5 years and 43 healthy white premenopausal women. Histomorphometric measurements were performed separately in cancellous, intracortical and endosteal envelopes. Of the 43 histomorphometric measurements on 3 difference bone surfaces (cancellous, intracortical and endosteal), only 2 bone resorption variables (Oc.S/BS and Oc.S/NOS) on the endosteal surface were significantly lower in patients with complete AFF than those with incomplete AFF. Compared to healthy premenopausal women, the trabecular bone volume, thickness and number were all significantly lower in patients with AFF. The dynamic bone formation variables in patients with AFF were significantly reduced on all bone surfaces. The likelihood of a biopsy with no tetracycline labeling was significantly higher in AFF patients than in healthy premenopausal women. Based on these results, we conclude that there are no significant differences in bone turnover between patients with incomplete and complete AFF, suggesting that the suppression of bone turnover had already existed in the femur with incomplete AFF. Compared to healthy premenopausal women, bone turnover is similarly suppressed in patients with either type of AFF

Schematic showing the flowchart for simulation and force quantification.

Schematic showing the flowchart for simulation and force quantification.</p