DNA adduction by the potent carcinogen aflatoxin B1 : mechanistic studies

Aflatoxin B1, a potently carcinogenic fungal metabolite, is converted to the biologically active form by chemical oxidation using dimethyldioxirane and enzymatically by cytochrome P450 mixed-function oxidases. Both processes give rise to mixtures of the exo- and endo-8,9-epoxides. Methanolysis studies reveal exclusive trans opening of both epoxides under neutral conditions in CH3OH and CH3OH/H2O mixtures; an SN2 mechanism is postulated. Under acidic conditions, the exo isomer gives mixtures of trans and cis solvolysis products, suggesting that the reaction is, at least in part, SN1; the endo isomer gives only the trans product. The exo isomer reacts with DNA by attack of the nitrogen atom at the 7 position of guanine on C8 of the epoxide to give the trans adduct; the endo epoxide fails to form an adduct at this or any other site in DNA. The exo isomer is strongly mutagenic in a base-pair reversion assay employing Salmonella typhimurium; the endo isomer is essentially nonmutagenic. Aflatoxin B1 and its derivatives intercalate in DNA. These results are consistent with a mechanism in which intercalation of the exo epoxide optimally orients the epoxide for an SN2 reaction with guanine but intercalation of the endo isomer places the epoxide in an orientation which precludes reaction. Thus, while the exo epoxide is a potent mutagen, the endo epoxide fails to react with DNA.

The 3D kinematics of the single leg flat and decline squat

Background: Pre-participation screening is commonly used to measure and assess potential intrinsic injury risk. The single leg squat is one such clinical screening measure used to assess lumbopelvic stability and associated intrinsic injury risk. With the addition of a decline board, the single leg decline squat (SLDS) has been shown to reduce ankle dorsiflexion restrictions and allowed greater sagittal plane movement of the hip and knee. On this basis, the SLDS has been employed in the Cricket Australia physiotherapy screening protocols as a measure of lumbopelvic control in the place of the more traditional single leg flat squat (SLFS). Previous research has failed to demonstrate which squatting technique allows for a more comprehensive assessment of lumbopelvic stability. Tenuous links are drawn between kinematics and hip strength measures within the literature for the SLS. Formal evaluation of subjective screening methods has also been suggested within the literature. Purpose: This study had several focal points namely 1) to compare the kinematic differences between the two single leg squatting conditions, primarily the five key kinematic variables fundamental to subjectively assess lumbopelvic stability; 2) determine the effect of ankle dorsiflexion range of motion has on squat kinematics in the two squat techniques; 3) examine the association between key kinematics and subjective physiotherapists’ assessment; and finally 4) explore the association between key kinematics and hip strength. Methods: Nineteen (n=19) subjects performed five SLDS and five SLFS on each leg while being filmed by an 8 camera motion analysis system. Four hip strength measures (internal/external rotation and abd/adduction) and ankle dorsiflexion range of motion were measured using a hand held dynamometer and a goniometer respectively on 16 of these subjects. The same 16 participants were subjectively assessed by an experienced physiotherapist for lumbopelvic stability. Paired samples t-tests were performed on the five predetermined kinematic variables to assess the differences between squat conditions. A Bonferroni correction for multiple comparisons was used which adjusted the significance value to p = 0.005 for the paired t-tests. Linear regressions were used to assess the relationship between kinematics, ankle range of motion and hip strength measures. Bivariate correlations between hip strength measures and kinematics and pelvic obliquity were employed to investigate any possible relationships. Results: 1) Significant kinematic differences between squats were observed in dominant (D) and non-dominant (ND) end of range hip external rotation (ND p = <0.001; D p = 0.004) and hip adduction kinematics (ND p = <0.001; D p = <0.001). With the mean angle, only the non-dominant leg observed significant differences in hip adduction (p = 0.001) and hip external rotation (p = <0.001); 2) Significant linear relationships were observed between clinical measures of ankle dorsiflexion and sagittal plane kinematic namely SLFS dominant ankle (p = 0.006; R2 = .429), SLFS non-dominant knee (p = 0.015; R2 = .352) and SLFS non-dominant ankle (p = 0.027; R2 = .305) kinematics. Only the dominant ankle (p = 0.020; R2 = .331) was found to have a relationship with the decline squat. 3) Strength measures had tenuous associations with the subjective assessments of lumbopelvic stability with no significant relationships being observed. 4) For the non-dominant leg, external rotation strength and abduction strength were found to be significantly correlated with hip rotation kinematics (Newtons r = 0.458 p = 0.049; Normalised for bodyweight: r = 0.469; p = 0.043) and pelvic obliquity (normalised for bodyweight: r = 0.498 p = 0.030) respectively for the SLFS only. No significant relationships were observed in the dominant leg for either squat condition. Some elements of the hip strength screening protocols had linear relationships with kinematics of the lower limb, particularly the sagittal plane movements of the knee and ankle. Strength measures had tenuous associations with the subjective assessments of lumbopelvic stability with no significant relationships being observed; Discussion: The key finding of this study illustrated that kinematic differences can occur at the hip without significant kinematic differences at the knee as a result of the introduction of a decline board. Further observations reinforce the role of limited ankle dorsiflexion range of motion on sagittal plane movement of the hip and knee and in turn multiplanar kinematics of the lower limb. The kinematic differences between conditions have clinical implications for screening protocols that employ frontal plane movement of the knee as a guide for femoral adduction and rotation. Subjects who returned stronger hip strength measurements also appeared to squat deeper as characterised by differences in sagittal plane kinematics of the knee and ankle. Despite the aforementioned findings, the relationship between hip strength and lower limb kinematics remains largely tenuous in the assessment of the lumbopelvic stability using the SLS. The association between kinematics and the subjective measures of lumbopelvic stability also remain tenuous between and within SLS screening protocols. More functional measures of hip strength are needed to further investigate these relationships. Conclusion: The type of SLS (flat or decline) should be taken into account when screening for lumbopelvic stability. Changes to lower limb kinematics, especially around the hip and pelvis, were observed with the introduction of a decline board despite no difference in frontal plane knee movements. Differences in passive ankle dorsiflexion range of motion yielded variations in knee and ankle kinematics during a self-selected single leg squatting task. Clinical implications of removing posterior ankle restraints and using the knee as a guide to illustrate changes at the hip may result in inaccurate screening of lumbopelvic stability. The relationship between sagittal plane lower limb kinematics and hip strength may illustrate that self-selected squat depth may presumably be a useful predictor of the lumbopelvic stability. Further research in this area is required.

Steps toward the validation of the Trendelenburg Test : the effect of experimentally reduced hip abductor muscle function on frontal plane mechanics

Objective: To investigate the validity of the Trendelenburg test (TT) using an ultrasound-guided nerve block (UNB) of the superior gluteal nerve and determine whether the reduction in hip abductor muscle (HABD) strength would result in the theorized mechanical compensatory strategies measured during the TT. Design: Quasi-experimental. Setting: Hospital. Participants: Convenience sample of 9 healthy men. Only participants with no current or previous injury to the lumbar spine, pelvis, or lower extremities, and no previous surgeries were included. Interventions: Ultrasound-guided nerve block. Main Outcome Measures: Hip abductor muscle strength (percent body weight [%BW]), contralateral pelvic drop (cPD), change in contralateral pelvic drop (Delta cPD), ipsilateral hip adduction, and ipsilateral trunk sway (TRUNK) measured in degrees. Results: The median age and weight of the participants were 31 years (interquartile range [IQR], 22-32 years) and 73 kg (IQR, 67-81 kg), respectively. An average 52% reduction of HABD strength (z = 2.36, P = 0.02) resulted after the UNB. No differences were found in cPD or Delta cPD (z = 0.01, P = 0.99, z = 20.67, P = 0.49, respectively). Individual changes in biomechanics showed no consistency between participants and nonsystematic changes across the group. One participant demonstrated the mechanical compensations described by Trendelenburg. Conclusions: The TT should not be used as a screening measure for HABD strength in populations demonstrating strength greater than 30% BW but should be reserved for use with populations with marked HABD weakness. Clinical Relevance: This study presents data regarding a critical level of HABD strength required to support the pelvis during the TT.

Steps towards the validation of the Trendelenburg test : The effect of experimentally reduced hip abductor muscle function on frontal plane mechanics

Introduction: The Trendelenburg Test (TT) is used to assess the functional strength of the hip abductor muscles (HABD), their ability to control frontal plane motion of the pelvis, and the ability of the lumbopelvic complex to transfer load into single leg stance. Rationale: Although a standard method to perform the test has been described for use within clinical populations, no study has directly investigated Trendelenburg’s hypotheses. Purpose: To investigate the validity of the TT using an ultrasound guided nerve block (UNB) of the superior gluteal nerve and determine whether the reduction in HABD strength would result in the theorized mechanical compensatory strategies measured during the TT. Methods: Quasi-experimental design using a convenience sample of nine healthy males. Only subjects with no current or previous injury to the lumbar spine, pelvis, or lower extremities, and no previous surgeries were included. Force dynamometry was used to evaluation HABD strength (%BW). 2D mechanics were used to evaluate contralateral pelvic drop (cMPD), change in contralateral pelvic drop (∆cMPD), ipsilateral hip adduction (iHADD) and ipsilateral trunk sway (TRUNK) measured in degrees (°). All measures were collected prior to and following a UNB on the superior gluteal nerve performed by an interventional radiologist. Results: Subjects’ age was median 31yrs (IQR:22-32yrs); and weight was median 73kg (IQR:67-81kg). An average 52% reduction of HABD strength (z=2.36,p=0.02) resulted following the UNB. No differences were found in cMPD or ∆cMPD (z=0.01,p= 0.99, z=-0.67,p=0.49). Individual changes in biomechanics show no consistency between subjects and non-systematic changes across the group. One subject demonstrated the mechanical compensations described by Trendelenburg. Discussion: The TT should not be used as screening measure for HABD strength in populations demonstrating strength greater than 30%BW but reserved for use with populations with marked HABD weakness. Importance: This study presents data regarding a critical level of HABD strength required to support the pelvis during the TT.

Elucidation of double bond position in unsaturated lipids by ozone electrospray ionization mass spectrometry

The position(s) of carbon-carbon double bonds within lipids can dramatically affect their structure and reactivity and thus has a direct bearing on biological function. Commonly employed mass spectrometric approaches to the characterization of complex lipids, however, fail to localize sites of unsaturation within the molecular structure and thus cannot distinguish naturally occurring regioisomers. In a recent communication \[Thomas, M. C.; Mitchell, T. W.; Blanksby, S. J. J. Am. Chem. Soc. 2006, 128, 58-59], we have presented a new technique for the elucidation of double bond position in glycerophospholipids using ozone-induced fragmentation within the source of a conventional electrospray ionization mass spectrometer. Here we report the on-line analysis, using ozone electrospray mass spectrometry (OzESI-MS), of a broad range of common unsaturated lipids including acidic and neutral glycerophospholipids, sphingomyelins, and triacylglycerols. All lipids analyzed are found to form a pair of chemically induced fragment ions diagnostic of the position of each double bond(s) regardless of the polarity, the number of charges, or the adduction (e.g., \[M - H](-), \[M - 2H](2-), \[M + H](+), \[M + Na](+), \[M + NH4](+)). The ability of OzESI-MS to distinguish lipids that differ only in the position of the double bonds is demonstrated using the glycerophosphocholine standards, GPCho(9Z-18:1/9Z-18:1) and GPCho(6Z-18:1/6Z-18:1). While these regioisomers cannot be differentiated by their conventional tandem mass spectra, the OzESI-MS spectra reveal abundant fragment ions of distinctive mass-to-charge ratio (m/z). The approach is found to be sufficiently robust to be used in conjunction with the m/z 184 precursor ion scans commonly employed for the identification of phosphocholine-containing lipids in shotgun lipidomic analyses. This tandem OzESI-MS approach was used, in conjunction with conventional tandem mass spectral analysis, for the structural characterization of an unknown sphingolipid in a crude lipid extract obtained from a human lens. The OzESI-MS data confirm the presence of two regioisomers, namely, SM(d18:0/15Z-24:1) and SM(d18:0/17Z-24:1), and suggest the possible presence of a third isomer, SM(d18:0/19Z-24:1), in lower abundance. The data presented herein demonstrate that OzESI-MS is a broadly applicable, on-line approach for structure determination and, when used in conjunction with established tandem mass spectrometric methods, can provide near complete structural characterization of a range of important lipid classes. As such, OzESI-MS may provide important new insight into the molecular diversity of naturally occurring lipids.

Characterization of acyl chain position in unsaturated phosphatidylcholines using differential mobility-mass spectrometry

Glycerophospholipids (GPs) that differ in the relative position of the two fatty acyl chains on the glycerol backbone (i.e., sn-positional isomers) can have distinct physicochemical properties. The unambiguous assignment of acyl chain position to an individual GP represents a significant analytical challenge. Here we describe a workflow where phosphatidylcholines (PCs) are subjected to ESI for characterization by a combination of differential mobility spectrometry and MS (DMS-MS). When infused as a mixture, ions formed from silver adduction of each phospholipid isomer {e.g., [PC (16:0/18:1) + Ag]+ and [PC (18:1/16:0) + Ag]+} are transmitted through the DMS device at discrete compensation voltages. Varying their relative amounts allows facile and unambiguous assignment of the sn-positions of the fatty acyl chains for each isomer. Integration of the well-resolved ion populations provides a rapid method (< 3 min) for relative quantification of these lipid isomers. The DMS-MS results show excellent agreement with established, but time-consuming, enzymatic approaches and also provide superior accuracy to methods that rely on MS alone. The advantages of this DMS-MS method in identification and quantification of GP isomer populations is demonstrated by direct analysis of complex biological extracts without any prior fractionation.

Biomechanics of transfemoral amputees fitted with osseointegrated fixation: Loading data for evidence-based practice

This presentation will provide an overview of the load applied on the residuum of transfemoral amputees fitted with an osseointegrated fixation during (A) rehabilitation, including static and dynamic load bearing exercises (e.g., rowing, adduction, abduction, squat, cycling, walking with aids), and (B) activities of daily living including standardized activities (e.g., level walking in straight line and around a circle, ascending and descending slopes and stairs) and activities in real world environments.

Experimentally reduced hip-abductor muscle strength and frontal-plane biomechanics during walking

Researchers have postulated that reduced hip-abductor muscle strength may have a role in the progression of knee osteoarthritis by increasing the external knee-adduction moment. However, the relationship between hip-abductor strength and frontal-plane biomechanics remains unclear. To experimentally reduce hip-abduction strength and observe the subsequent changes in frontal-plane biomechanics. Descriptive laboratory study. Research laboratory. Eight healthy, recreationally active men (age = 27 ± 6 years, height = 1.75 ± 0.11 m, mass = 76.1 ± 10.0 kg). All participants underwent a superior gluteal nerve block injection to reduce the force output of the hip-abductor muscle group. Maximal isometric hip-abduction strength and gait biomechanical data were collected before and after the injections. Gait biomechanical variables collected during walking consisted of knee- and hip-adduction moments and impulses and the peak angles of contralateral pelvic drop, hip adduction, and ipsilateral trunk lean. Hip-abduction strength was reduced after the injection (P = .001) and remained lower than baseline values at the completion of the postinjection gait data collection (P = .02). No alterations in hip- or knee-adduction moments (hip: P = .11; knee: P = .52) or impulses (hip: P = .16; knee: P = .41) were found after the nerve block. Similarly, no changes in angular kinematics were observed for contralateral pelvic drop (P = .53), ipsilateral trunk lean (P = .78), or hip adduction (P = .48). A short-term reduction in hip-abductor strength was not associated with alterations in the frontal-plane gait biomechanics of young, healthy men. Further research is needed to determine whether a similar relationship is true in older adults with knee osteoarthritis.

Errors in the knee joint forces and moments during gait depending on the foot and knee prosthetic components

Background Previously studies showed that inverse dynamics based on motion analysis and force-plate is inaccurate compared to direct measurements for individuals with transfemoral amputation (TFA). Indeed, direct measurements can appropriately take into account the absorption at the prosthetic foot and the resistance at the prosthetic knee. [1-3] However, these studies involved only a passive prosthetic knee. Aim The objective of the present study was to investigate if different types of prosthetic feet and knees can exhibit different levels of error in the knee joint forces and moments. Method Three trials of walking at self-selected speed were analysed for 9 TFAs (7 males and 2 females, 47±9 years old, 1.76±0.1 m 79±17 kg) with a motion analysis system (Qualisys, Goteborg, Sweden), force plates (Kitsler, Winterthur, Switzerland) and a multi-axial transducer (JR3, Woodland, USA) mounted above the prosthetic knee [1-17]. TFAs were all fitted with an osseointegrated implant system. The prostheses included different type of foot (N=5) and knee (N=3) components. The root mean square errors (RMSE) between direct measurements and the knee joint forces and moments estimated by inverse dynamics were computed for stance and swing phases of gait and expressed as a percentage of the measured amplitudes. A one-way Kruskal-Wallis ANOVA was performed (Statgraphics, Levallois-Perret, France) to analyse the effects of the prosthetic components on the RMSEs. Cross-effects and post-hoc tests were not analysed in this study. Results A significant effect (*) was found for the type of prosthetic foot on anterior-posterior force during swing (p=0.016), lateral-medial force during stance (p=0.009), adduction-abduction moment during stance (p=0.038), internal-external rotation moment during stance (p=0.014) and during swing (p=0.006), and flexion-extension moment during stance (p = 0.035). A significant effect (#) was found for the type of prosthetic knee on anterior-posterior force during swing (p=0.018) and adduction-abduction moment during stance (p=0.035). Discussion & Conclusion The RMSEs were larger during swing than during stance. It is because the errors on accelerations (as derived from motion analysis) become substantial with respect to the external loads. Thus, inverse dynamics during swing should be analysed with caution because the mean RMSEs are close to 50%. Conversely, there were fewer effects of the prosthetic components on RMSE during swing than during stance and, accordingly, fewer effects due to knees than feet. Thus, inverse dynamics during stance should be used with caution for comparison of different prosthetic components.