Patients with human papillomavirus associated (HPV+) head and neck cancer (HNC) demonstrate significantly improved survival outcome compared to those with HPV− negative (HPV−) tumors. Published data examining this difference offers conflicting results to date. We systematically investigated the radiation sensitivity of all available validated HPV+ HNC cell lines and a series of HPV− HNC cell lines using in vitro and in vivo techniques. HPV+ HNCs exhibited greater intrinsic radiation sensitivity (average SF2 HPV− 0.59 vs. HPV+ 0.22, p<0.0001), corresponding with a prolonged G2/M cell cycle arrest and increased apoptosis following radiation exposure (percent change 0% vs. 85%, p=0.002). A genome-wide microarray was used to compare gene-expression 24 hours following radiation between HPV+ and HPV− cell lines. Multiple genes in TP53 pathway were upregulated in HPV+ cells (Z score 4.90), including a 4.6 fold increase in TP53 (p<0.0001). Using immortalized human tonsillar epithelial cells, increased radiation sensitivity was seen in cell expressing HPV-16 E6 despite the effect of E6 to degrade p53. This suggested that low levels of normally functioning p53 in HPV+ HNC cells could be activated by radiation, leading to cell death. Consistent with this, more complete knockdown of TP53 by siRNA resulted in radiation resistance. These results provide clear evidence, and a supporting mechanism, for increased radiation sensitivity in HPV+ HNC relative to HPV− HNC. This issue is under active investigation in a series of clinical trials attempting to de-escalate radiation (and chemotherapy) in selected patients with HPV+ HNC in light of their favorable overall survival outcome.

Muscles are the actuators that drive human movement. However, despite many decades of work, we still cannot readily assess the forces that muscles transmit during human movement. Direct measurements of muscle–tendon loads are invasive and modeling approaches require many assumptions. Here, we introduce a non-invasive approach to assess tendon loads by tracking vibrational behavior. We first show that the speed of shear wave propagation in tendon increases with the square root of axial stress. We then introduce a remarkably simple shear wave tensiometer that uses micron-scale taps and skin-mounted accelerometers to track tendon wave speeds in vivo. Tendon wave speeds are shown to modulate in phase with active joint torques during isometric exertions, walking, and running. The capacity to non-invasively assess muscle–tendon loading can provide new insights into the motor control and biomechanics underlying movement, and could lead to enhanced clinical treatment of musculoskeletal injuries and diseases.

The evaluation of in vivo muscle-tendon loads is fundamental to understanding the actuation of normal and pathological human walking. However, conventional techniques for measuring muscle-tendon loads in the human body are too invasive for use in gait analysis. Here, we demonstrate the use of noninvasive measures of shear wave propagation as a proxy for Achilles tendon loading during walking. Twelve healthy young adults performed isometric ankle plantarflexion on a dynamometer. Achilles tendon wave speed, tendon moment arms, tendon cross-sectional area and ankle torque were measured. We first showed that the linear relationship between tendon stress and wave speed squared can be calibrated from isometric tasks. There was no significant effect of knee angle, ankle angle or loading rate on the subject-specific calibrations. Calibrated shear wave tensiometers were used to estimate Achilles tendon loading when walking at speeds ranging from 1 to 2 m/s. Peak tendon stresses during pushoff increased from 41 to 48 MPa as walking speed was increased, and were comparable to estimates from inverse dynamics. The tensiometers also detected Achilles tendon loading of 4 to 7 MPa in late swing. Late swing tendon loading was not discernible in the inverse dynamics estimates, but did coincide with passive stretch of the gastrocnemius muscle-tendon units. This study demonstrates the capacity to use calibrated shear wave tensiometers to evaluate tendon loading in locomotor tasks. Such technology could prove beneficial for identifying the muscle actions that underlie subject-specific movement patterns.

Objective Evaluate the effects of aging on healthy Achilles tendon and aponeurosis shear wave speed (SWS), a quantitative metric which reflects tissue elasticity. Methods Shear wave elastography was used to measure spatial variations in Achilles tendon SWS in healthy young (n=15, 25±4 years), middle-aged (n=10, 49±4 years) and older (n=10, 68±5 years) adults. SWS was separately measured in the free Achilles tendon, soleus aponeurosis and gastrocnemius aponeurosis in resting (R), stretched (dorsiflexed 15 deg from R) and slack (plantarflexed 15 deg from R) postures. Results SWS significantly increased with stretch and varied with age in all tendon regions. Slack free tendon SWS was significantly higher in older adults than young adults (p=0.025). However, stretched soleus aponeurosis SWS was significantly lower in older adults than young adults (p=0.01). Stretched gastrocnemius aponeurosis SWS was significantly lower in both middle-aged (p=0.003) and older (p=0.001) adults, relative to younger adults. Conclusions These results suggest that aging alters spatial variations in Achilles tendon elasticity, which could alter deformations within the triceps surae muscle-tendon units, thus affecting injury potential. The observed location- and posture-dependent variations highlight the importance of controlling ankle posture and imaging location when using shear wave approaches clinically to evaluate tendon disorders.

The purpose of this study was to assess the potential for ultrasound shear wave elastography (SWE) to assess tissue elasticity and ultimate stress in both intact and healing tendons. The lateral gastrocnemius (Achilles) tendons of 41 New Zealand white rabbits were surgically severed and repaired with growth factor coated sutures. SWE imaging was used to measure shear wave speed (SWS) in both the medial and lateral tendons pre-surgery, and at 2 and 4 weeks post-surgery. Rabbits were euthanized at 4 weeks, and both medial and lateral tendons underwent mechanical testing to failure. SWS significantly (p<0.001) decreased an average of 17% between the intact and post-surgical state across all tendons. SWS was significantly (p<0.001) correlated with both the tendon elastic modulus (r = 0.52) and ultimate stress (r = 0.58). Thus, ultrasound SWE is a potentially promising noninvasive technology for quantitatively assessing the mechanical integrity of pre-operative and post-operative tendons.

Lrp5 is typically described as a Wnt signaling receptor, albeit a less effective Wnt signaling receptor than the better-studied sister isoform, Lrp6. Here we show that Lrp5 is only a minor player in the response to Wnt3a-type ligands in mammary epithelial cells; instead, Lrp5 is required for glucose uptake, and glucose uptake regulates the growth rate of mammary epithelial cells in culture. Thus, a loss of Lrp5 leads to profound growth suppression, whether growth is induced by serum or by specific growth factors, and this inhibition is not due to a loss of Wnt signaling. Depletion of Lrp5 decreases glucose uptake, lactate secretion, and oxygen consumption rates; inhibition of glucose consumption phenocopies the loss of Lrp5 function. Both Lrp5 knockdown and low external glucose induce mitochondrial stress, as revealed by the accumulation of reactive oxygen species (ROS) and the activation of the ROS-sensitive checkpoint, p38␣. In contrast, loss of function of Lrp6 reduces Wnt responsiveness but has little impact on growth. This highlights the distinct functions of these two Lrp receptors and an important Wnt ligand-independent role of Lrp5 in glucose uptake in mammary epithelial cells.A ll somatic stem cells tested to date rely on Wnt signaling to maintain their pluripotentiality (1). From the point of view of regenerative medicine, this requirement has some disadvantages, since Wnt signaling can also be highly oncogenic (2). If the molecular regulation of Wnt signaling is better understood, it may be possible to tease apart the positive and negative aspects of the pathway.Our study focuses on the signals generated at the cell surface by the Lrp5 (low-density lipoprotein receptor-related protein 5) and Lrp6 Wnt signaling receptors. Cell surface presentation of Lrp species is considered to be the limiting factor for Wnt signal generation (3). Mammary epithelial cells in vitro and in vivo grow and divide in response to ectopic Wnt signals. Thus, overexpression of Wnt1 or Wnt10B in mouse mammary glands leads to ductal hyperplasia, inducing cell division in both luminal and basal cells that together comprise the mammary ducts (4). Basal stem cells accumulate as a fraction of the total population (5), and solitary adenocarcinomas arise with a median latency of 7 months, comprising both mammary epithelial cell lineages (6). Thus, as for intestinal cell populations (2), Wnt signaling is a robust growth signal for mammary epithelial cells and acts as an oncogenic stimulus with a relatively low efficiency.We previously showed that Lrp5 is required to sustain the basal stem cell activity in mammary glands, and also for breast tumor development in response to Wnt1 (5,7,8). This was a surprising result, since Lrp5 and Lrp6 are coexpressed by basal mammary epithelial cells and because Lrp6 is known to be a more effective transducer of Wnt ligand activation (9, 10). Lrp5 and Lrp6 share 73% and 64% protein sequence identity in their extracellular and intracellular domains, respectively (11).Almost all the information we know a...

Prior studies have observed an age-related decline in net ankle power and work at faster walking speeds. However, the underlying changes in muscle-tendon behavior are not well-understood, and are challenging to infer from joint level analyses. This study used shear wave tensiometry to investigate the modulation of force and work done by the triceps surae across walking speeds. Fourteen healthy young (7F/7M, 26 ± 5 years) and older (7F/7M, 67 ± 5 years) adults were tested. Subjects walked on an instrumented treadmill at four walking speeds (0.75, 1.00, 1.25, and 1.50 m/s) while lower extremity kinematics and Achilles tendon shear wave speeds were collected. Subject-specific calibrations were used to compute Achilles tendon force from wave speed. Excursions of the soleus and gastrocnemius muscle-tendon units were computed from the kinematic data and subject-specific measures of the Achilles tendon moment arm. Work loop plots were then used to assess effective muscle-tendon stiffness during lengthening, and positive, negative, and net work production during stance. Two-way mixed ANOVAs were used to evaluate the effects of age group and walking speed on each outcome measure. Tendon loading during muscle-tendon lengthening (effective stiffness) did not differ between age groups, but did vary with speed. The soleus became effectively stiffer with increasing speed while the gastrocnemius became effectively more compliant. There was a marked age-related deficit in net soleus (−66% on average) and gastrocnemius (−36%) work across all walking speeds. We did not observe an age-speed interaction effect on net work production. These results suggest the age-related deficit in triceps surae output in walking is pervasive across speed, and hence seemingly not linked to absolute mechanical demands of the task.

The purpose of this study was to investigate age-related differences in Achilles tendon loading during gait. Fourteen young (7F/7M, 26 ± 5 years) and older (7F/7M, 67 ± 5 years) adults without current neurological or orthopaedic impairment participated. Shear wave tensiometry was used to measure tendon stress by tracking Achilles tendon wave speed. The wave speed-stress relationship was calibrated using simultaneously collected tensiometer and force plate measures during a standing sway task. Tendon stress was computed from the force plate measures using subjectspecific ultrasound measures of tendon moment arm and cross-sectional area. All subjects exhibited a highly linear relationship between wave speed squared and tendon stress (mean R 2 >0.9), with no significant age-group differences in tensiometer calibration parameters. Tendon wave speed was monitored during treadmill walking at four speeds (0.75, 1.00, 1.25, and 1.50 m/s) and used to compute the stress experienced by the tendon. Relative to young adults, older adults exhibited 22% lower peak tendon wave speeds. Peak tendon stress during push-off in older adults (24.8 MPa) was 32% less than that in the young adults (36.7 MPa) (p = 0.01). There was a moderate increase (+11%) in peak tendon stress across both groups when increasing speed from 0.75 to 1.50 m/s (main effect of speed, p = 0.01). Peak tendon loading during late swing did not differ between age groups (mean 3.8 MPa in young and 4.2 MPa in older adults). These age-related alterations in tendon tissue loading may affect the mechanobiological stimuli underlying tissue remodeling and thereby alter the propensity for tendon injury and disease.