Successful weight loss initiation and maintenance among adolescents with overweight and obesity: does age matter?

Treatments for adolescents with overweight/obesity demonstrate mixed success, which may be due to a lack of consideration for developmental changes during this period. Potential developmental differences in weight loss motivations, weight maintenance behaviours and the role of parents in these efforts were examined in a sample of successful adolescent weight losers. Participants enrolled in the Adolescent Weight Control Registry (n = 49) self-reported demographic information and weight history, reasons for weight loss and weight control, weight loss approach and weight maintenance strategies, and perceived parental involvement with weight loss. Associations between age at weight loss initiation and the aforementioned factors were examined using linear and generalized regressions, controlling for highest z-BMI and sex. Adolescents who were older (≥16 years) at their weight loss initiation were more likely to report losing weight on their own (37.5% vs. 75%, P = 0.01) and reported greater responsibility for their weight loss and weight loss maintenance (P < 0.001) compared to younger adolescents. Younger age at weight loss initiation was associated with greater parental involvement (P = 0.005), whereas older age was associated with greater adolescent responsibility for the decision to lose weight (P = 0.002), the weight loss approach (P = 0.007) and food choices (P < 0.001). Findings suggest the importance of considering developmental differences in responsibility for weight loss and maintenance among adolescents with overweight/obesity.

Contribution of limb momentum to power transfer in athletic wheelchair pushing.

Pushing capacity is a key parameter in athletic racing wheelchair performance. This study estimated the potential contribution of upper limb momentum to pushing. The question is relevant since it may affect the training strategy adopted by an athlete. A muscle-free Lagrangian dynamic model of the upper limb segments was developed and theoretical predictions of power transfer to the wheelchair were computed during the push phase. Results show that limb momentum capacity for pushing can be in the order of 40J per push cycle at 10m/s, but it varies with the specific pushing range chosen by the athlete. Although use of momentum could certainly help an athlete improve performance, quantifying the actual contribution of limb momentum to pushing is not trivial. A preliminary experimental investigation on an ergometer, along with a simplified model of the upper limb, suggests that momentum is not the sole contributor to power transfer to a wheelchair. Muscles substantially contribute to pushing, even at high speeds. Moreover, an optimal pushing range is challenging to find since it most likely differs if an athlete chooses a limb momentum pushing strategy versus a muscular exertion pushing strategy, or both at the same time. The study emphasizes the importance of controlling pushing range, although one should optimize it while also taking the dynamics of the recovery period into account.

Embryonic stem cell-derived osteocytes are capable of responding to mechanical oscillatory hydrostatic pressure.

Osteoblasts can be derived from embryonic stem cells (ESCs) by a 30 day differentiation process, whereupon cells spontaneously differentiate upon removal of LIF and respond to exogenously added 1,25α(OH)2 vitamin D3 with enhanced matrix mineralization. However, bone is a load-bearing tissue that has to perform under dynamic pressure changes during daily movement, a capacity that is executed by osteocytes. At present, it is unclear whether ESC-derived osteogenic cultures contain osteocytes and whether these are capable of responding to a relevant cyclic hydrostatic compression stimulus. Here, we show that ESC-osteoblastogenesis is followed by the generation of osteocytes and then mechanically load ESC-derived osteogenic cultures in a compression chamber using a cyclic loading protocol. Following mechanical loading of the cells, iNOS mRNA was upregulated 31-fold, which was consistent with a role for iNOS as an immediate early mechanoresponsive gene. Further analysis of matrix and bone-specific genes suggested a cellular response in favor of matrix remodeling. Immediate iNOS upregulation also correlated with a concomitant increase in Ctnnb1 and Tcf7l2 mRNAs along with increased nuclear TCF transcriptional activity, while the mRNA for the repressive Tcf7l1 was downregulated, providing a possible mechanistic explanation for the noted matrix remodeling. We conclude that ESC-derived osteocytes are capable of responding to relevant mechanical cues, at least such that mimic oscillatory compression stress, which not only provides new basic understanding, but also information that likely will be important for their use in cell-based regenerative therapies.

Exogenous nitric oxide enhances calcification in embryonic stem cell-derived osteogenic cultures.

While the involvement of nitric oxide in bone formation, homeostasis and healing has been extensively characterized, its role in directing pluripotent stem cells to the osteogenic lineage has not been described. Yet, the identification of chemical inducers that improve differentiation output to a particular lineage is highly valuable to the development of such cells for the cell-based treatment of osteo-degenerative diseases. This study aimed at investigating the instructive role of nitric oxide (NO) and its synthesizing enzymes on embryonic stem cell (ESC) osteogenic differentiation. Our findings showed that NO levels may support osteogenesis, but that the effect of nitric oxide on osteoblast differentiation may be specific to a particular time phase during the development of osteoblasts in vitro. Endogenously, nitric oxide was specifically secreted by osteogenic cultures during the calcification period. Simultaneously, messenger RNAs for both the endothelial and inducible nitric oxide synthase isoforms (eNOS and iNOS) were upregulated during this late phase development. However, the specific eNOS inhibitor L-N(5)-(1-Iminoethyl)ornithine dihydrochloride attenuated calcification more so than the specific iNOS inhibitor diphenyleneiodonium. Exogenous stage-specific supplementation of culture medium with the NO donor S-nitroso-N-acetyl-penicillamine increased the percentage of cells differentiating into osteoblasts and enhanced calcification. Our results point to a primary role for eNOS as a pro-osteogenic trigger in ESC differentiation and expand on the variety of supplements that may be used to direct ESC fate to the osteogenic lineage, which will be important in the development of cell-based therapies for osteo-degenerative diseases.

Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis.

To study spermatogonial stem cells the heterogeneous testicular cell population first needs to be enriched for undifferentiated spermatogonia, which contain the stem cell population. When working with non-rodent models, this step requires working with large numbers of cells. Available cell separation methods rely on differential properties of testicular cell types such as expression of specific cell surface proteins, size, density, or differential adhesion to substrates to separate germ cells from somatic cells. The objective of this study was to develop an approach that allowed germ cell enrichment while providing efficiency of handling large cell numbers. Here, we report the use of stirred suspension bioreactors (SSB) to exploit the adhesion properties of Sertoli cells to enrich cells obtained from pre-pubertal porcine testes for undifferentiated spermatogonia. We also compared the bioreactor approach with an established differential plating method and the combination of both: SSB followed by differential plating. After 66 h of culture, germ cell enrichment in SSBs provided 7.3 ± 1.0-fold (n = 9), differential plating 9.8 ± 2.4-fold (n = 6) and combination of both methods resulted in 9.1 ± 0.3-fold enrichment of germ cells from the initial germ cell population (n = 3). To document functionality of cells recovered from the bioreactor, we demonstrated that cells retained their functional ability to reassemble seminiferous tubules de novo after grafting to mouse hosts and to support spermatogenesis. These results demonstrate that the SSB allows enrichment of germ cells in a controlled and scalable environment providing an efficient method when handling large cell numbers while reducing variability owing to handling.

Embryonic stem cell therapy improves bone quality in a model of impaired fracture healing in the mouse; tracked temporally using in vivo micro-CT.

In the current study, we used an estrogen-deficient mouse model of osteoporosis to test the efficacy of a cell-generated bone tissue construct for bone augmentation of an impaired healing fracture. A reduction in new bone formation at the defect site was observed in ovariectomized fractures compared to the control group using repeated measures in vivo micro-computed tomography (µCT) imaging over 4 weeks. A significant increase in the bone mineral density (BMD), trabecular bone volume ratio, and trabecular number, thickness and connectivity were associated with fracture repair in the control group, whereas the fractured bones of the ovariectomized mice exhibited a loss in all of these parameters (p<0.001). In a separate group, ovariectomized fractures were treated with murine embryonic stem (ES) cell-derived osteoblasts loaded in a three-dimensional collagen I gel and recovery of the bone at the defect site was observed. A significant increase in the trabecular bone volume ratio (p<0.001) and trabecular number (p<0.01) was observed by 4 weeks in the fractures treated with cell-loaded collagen matrix compared to those treated with collagen I alone. The stem cell-derived osteoblasts were identified at the fracture site at 4 weeks post-implantation through in situ hybridization histochemistry. Although this cell tracking method was effective, the formation of an ectopic cellular nodule adjacent to the knee joints of two mice suggested that alternative in vivo cell tracking methods should be employed in order to definitively assess migration of the implanted cells. To our knowledge, this study is the first of its kind to examine the efficacy of stem cell therapy for fracture repair in an osteoporosis-related fracture model in vivo. The findings presented provide novel insight into the use of stem cell therapies for bone injuries.

Hermitian splines for modeling biological soft tissue systems that exhibit nonlinear force-elongation curves.

Numerical simulation of soft tissue mechanical properties is a critical step in developing valuable biomechanical models of live organisms. A cubic Hermitian spline optimization routine is proposed in this paper to model nonlinear experimental force-elongation curves of soft tissues, in particular when modeled as lumped elements. Boundary conditions are introduced to account for the positive definiteness and the particular curvature of the experimental curve to be fitted. The constrained least-square routine minimizes user intervention and optimizes fitting of the experimental data across the whole fitting range. The routine provides coefficients of a Hermitian spline or corresponding knots that are compatible with a number of constraints that are suitable for modeling soft tissue tensile curves. These coefficients or knots may become inputs to user-defined component properties of various modeling software. Splines are particularly advantageous over the well-known exponential model to account for the traction curve flatness at low elongations and to allow for more flexibility in the fitting process. This is desirable as soft tissue models begin to include more complex physical phenomena.

Attenuated reovirus displays oncolysis with reduced host toxicity.

BACKGROUND: Although the naturally occurring reovirus causes only mild symptoms in humans, it shows considerable potential as an oncolytic agent because of its innate ability to target cancer cells. In immunocompromised hosts, however, wild-type reovirus can target healthy tissues, including heart, liver, pancreas and neural structures. METHODS: We characterized an attenuated form of reovirus (AV) derived from a persistently infected cell line through sequence analysis, as well as western blot and in vitro transcription and translation techniques. To examine its pathogenesis and oncolytic potential, AV reovirus was tested on healthy embryonic stem cells, various non-transformed and transformed cell lines, and in severe combined immunodeficiency (SCID) mice with tumour xenografts. RESULTS: Sequence analysis of AV reovirus revealed a premature STOP codon in its sigma 1 attachment protein. Western blot and in vitro translation confirmed the presence of a truncated σ1. In comparison to wild-type reovirus, AV reovirus did not kill healthy stem cells or induce black tail formation in SCID mice. However, it did retain its ability to target cancer cells and reduce tumour size. CONCLUSION: Despite containing a truncated attachment protein, AV reovirus still preferentially targets cancer cells, and compared with wild-type reovirus it shows reduced toxicity when administered to immunodeficient hosts, suggesting the potential use of AV reovirus in combination cancer therapy.

Loss of discordant cells during micro-mass differentiation of embryonic stem cells into the chondrocyte lineage.

Embryonic stem cells (ESCs) have attracted particular interest in regenerative medicine because of their unlimited self-renewal and multipotentiality for differentiation. Spontaneous differentiated ESCs display heterogeneous multipotent cell populations and generate teratomas in vivo, with process by which ESCs differentiate into specific lineages remaining unclear. In this study, we focused on the in vitro chondrocyte differentiation of ESCs through micro-mass without using an embryoid body (EB) step and observed the unique characteristics of cartilage formation coupled with endochondral ossification in vivo. This approach resulted in an aggressive loss of discordant cells by apoptosis, which was accompanied by significant changes in gene expression during the course of ESC differentiation into chondrocytes. Unlike EB formation where discordant cells remain trapped within aggregates, micro-mass permits cells to die, leave the group and/or form a new group in response to changes in gene expression. Our observations suggest that the cell death that accompanies ESC micro-mass differentiation helps purify a terminally differentiated cell population and selects for targeted end points within a suitable microenvironment.

Properties of murine embryonic stem cells maintained on human foreskin fibroblasts without LIF.

In embryonic stem (ES) cells, leukemia inhibitory factor (LIF)/STAT3, wnt and nodal/activin signaling are mainly active to control pluripotency during expansion. To maintain pluripotency, ES cells are typically cultured on feeder cells of varying origins. Murine ES cells are commonly cultured on murine embryonic fibroblasts (MEFs), which senesce early and must be frequently prepared. This process is laborious and leads to batch variation presenting a challenge for high-throughput ES cell expansion. Although some cell lines can be sustained by exogenous LIF, this method is costly. We present here a novel and inexpensive culture method for expanding murine ES cells on human foreskin fibroblast (HFF) feeders. After 20 passages on HFFs without LIF, ES cell lines showed normal expression levels of pluripotency markers, maintained a normal karyotype and retained the ability to contribute to the germline. As HFFs do not senesce for at least 62 passages, they present a vast supply of feeders.

RED: the analysis, management and dissemination of expressed sequence tags.

The Rancourt EST Database (RED) is a web-based system for the analysis, management, and dissemination of expressed sequence tags (ESTs). RED represents a flexible template DNA sequence database that can be easily manipulated to suit the needs of other laboratories undertaking mid-size sequencing projects.

Bacteriophage gene targeting vectors generated by transplacement.

A rate-determining step in gene targeting is the generation of the targeting vector. We have developed bacteriophage gene targeting vectorology, which shortens the timeline of targeting vector construction. Using retro-recombination screening, we can rapidly isolate targeting vectors from an embryonic stem cell genomic library via integrative and excisive recombination. We have demonstrated that recombination can be used to introduce specific point mutations or unique restriction sites into gene targeting vectors via transplacement. Using the choline/ethanolamine kinase alpha and beta genes as models, we demonstrate that transplacement can also be used to introduce specifically a neo resistance cassette into a gene targeting phage. In our experience, the lambdaTK gene targeting system offers considerable flexibility and efficiency in TV construction, which makes generating multiple vectors in one week's time possible.

Dynamics of pushing.

A standing individual can use several strategies for modulating pushing force magnitude. Using a static model, researchers have shown that the efficacy of those strategies varies considerably. In the present article, the authors propose a human motor control dynamic model for analyzing transients that occur when an individual is asked to modulate force magnitude. According to the model, the impedances of both the upper and the lower limbs influence the time course of force variations and foot placement has a profound effect on pushing force dynamics. With a feet-together posture, the center of pressure has a limited range of motion and changes in force may be preceded by initial changes in the opposite direction; that is, to decrease force, an individual must first increase force. When the feet are placed apart, individuals can move the center of pressure over a much larger range, thereby modulating pushing force magnitude, without reversing behavior, over a larger range of force magnitudes. Therefore, the best way to control pushing force at the hand may be by using the foot.

Increased risk for falling associated with obesity: mathematical modeling of postural control.

Recent epidemiological studies report that obesity is positively related to fracture incidence. In the present experiment, a model of postural control was used to examine the impact of an abnormal distribution of body fat in the abdominal area upon postural stability. Obese and lightweight humanoids were destabilized by imposing a small initial angular speed from a neutral standing position. To avoid a loss of stability yielding a stepping reaction or a fall, an ankle torque is necessary to counteract the perturbation. Three torque parameters--ankle torque onset, time to peak torque, and muscular ankle torque--were entered in a program to simulate the intrinsic variability of the human postural control system. A loss of stability was detected when the center of pressure exceeded stability margins. The most striking observation is the nonlinear increase of torque needed to stabilize the humanoid when the motor response was characterized by delayed temporal parameters. The effect was more pronounced when an anterior position of the center of mass was included in the simulations. This suggests that, when submitted to daily postural stresses and perturbations, obese persons (particularly those with an abnormal distribution of body fat in the abdominal area) may be at higher risk of falling than lightweight individuals.

Regulation of the strypsin-related proteinase ISP2 by progesterone in endometrial gland epithelium during implantation in mice.

Hormones prepare the uterus for the arrival and subsequent invasion of the embryo during pregnancy. Extracellular matrix-degrading proteinases and their inhibitors are involved in this integration process. Recent genetic evidence indicates that there is redundancy within the implantation proteinase cascade, indicating that additional proteinases may be involved. Recently, we described a novel implantation serine proteinase (ISP1) gene that encodes the embryo-derived enzyme strypsin, which is necessary for blastocyst hatching in vitro and the initiation of invasion. The evidence presented in the present study indicates that a second proteinase secreted from the uterus also participates in lysis of the zona pellucida. A second implantation serine proteinase gene (ISP2) was isolated, which encodes a related secreted tryptase expressed specifically within uterine endometrial glands. In pseudopregnancy, ISP2 gene expression is dependent on progesterone priming and is inhibited by the antiprogestin RU486. On the basis of similarities between ISP2 gene expression and that of a progesterone-regulated luminal proteinase associated with lysis of the zona pellucida, it is possible that the strypsin-related protein, ISP2, may encode a zona lysin proteinase.

Stability in force-production tasks.

Exerting a force on a mechanical system can induce mechanical instability. To overcome that instability, humans may take advantage of their upper limb mechanical impedance (e.g., hand stiffness). The authors investigated what stiffness is required to maintain static stability and how humans can achieve that stiffness in the context of the task of pushing on a pivoting stick. Results showed that the stiffness required is in the range of measured human upper limb stiffness. To avoid an ill-posed problem, one can better express the requirements for stability as a simple geometrical criterion related to the curvature of the potential energy field at the hand. A planar model of the upper limb revealed that individuals can use both hand rotational and translational stiffness to stabilize a stick. Although hand rotational stiffness does not participate in producing the axial force on the stick, it can significantly contribute to achieving a limb stiffness appropriate for maintaining static stability. Hand rotational stiffness can be important for the design of hand tools, because humans can increase it only by augmenting grip force, a biomechanical factor associated with cumulative trauma injuries of the upper extremities.

A novel murine tryptase involved in blastocyst hatching and outgrowth.

Before implantation the blastocyst is maintained within a proteinaceous coat, the zona pellucida, which prevents polyspermy and ectopic pregnancy. An extracellular trypsin-like activity, which is necessary for hatching from the zona pellucida in vitro, is localized to the abembryonic pole of the blastocyst. Upon hatching, the extracellular matrix-degrading proteinases urokinase plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9) are thought to promote blastocyst invasion. However, gene disruption experiments have demonstrated that uPA and MMP-9 are dispensable and, thus, that other key enzymes are involved in implantation. In this study, a novel implantation serine proteinase (ISP1) gene, which is distantly related to haematopoietic tryptases and represents a novel branch of the S1 proteinase family, was cloned. ISP1 is expressed throughout morulae and blastocysts during hatching and outgrowth. Abrogation of ISP1 mRNA accumulation using antisense oligodeoxynucleotides disrupts blastocyst hatching and outgrowth in vitro. The results of this study indicate that the ISP1 gene probably encodes the long sought after 'hatching enzyme' that is localized to the abembryonic pole during hatching in vitro. ISP1 is the earliest embryo-specific proteinase to be expressed in implantation and may play a critical role in connecting embryo hatching to the establishment of implantation competence at the abembryonic pole of the blastocyst.

Germ cell specific promoter drives ectopic transgene expression during embryogenesis.

In this study, we used the male germ cell-specific phosphoglycerate kinase 2 (Pgk2) promoter to generate Pgk2Cre transgenic mice to allow investigation of genes critically involved in meiosis. The Pgk2 promoter had been used previously to target transgene expression to spermatocytes and spermatids in several laboratories including ours. In several Cre targeting experiments using other promoters, ectopic Cre expression had been observed, but the timing and extent of this expression was not analyzed. We demonstrate that in adult mice the Pgk2Cre transgene is expressed specifically in spermatocytes and spermatids, as expected. However, in offspring from matings of Pgk2Cre mice and an H19loxP indicator strain, we discovered that recombination events had occurred in several, but not all, tissues to varying extents. The lacZ-loxP transgenic indicator strain was next used to uncover ectopic Cre expression even in single cells, which indicated that the Pgk2Cre transgene is expressed between days 11 and 15 during embryogenesis in several tissues and organs. Using an RT PCR assay we were unable to detect endogenous Pgk2 mRNA during embryogenesis or in adult tissues other than testis. In conclusion, the Pgk2 promoter is a valid choice for targeting gene expression to meiotic male germ cells, since transient ectopic expression is unlikely to have a discernable effect in most studies, but it may be inappropriate for utilization with Cre recombinase.

Estimation and application of EMG amplitude during dynamic contractions.

The sections above have described an EMG amplitude estimator and an initial application of this estimator to the EMG-torque problem. The amplitude estimator consists of six stages. In the first stage, motion artifact and power-line interference are attenuated. Motion artifact is typically removed with a highpass filter. Elimination of power-line noise is more difficult. Commercial systems tend to use notch filters, accepting the concomitant loss of "true" signal power in exchange for simplicity and robustness. Adaptive methods may be preferable, however, to preserve more "true" signal power. In stage two, the signal is whitened. One fixed whitening technique and two adaptive whitening methods were described. For low-amplitude levels, the adaptive whitening technique that includes adaptive noise cancellation may be necessary. In stage three, multiple EMG channels (all overlying the same muscle) are combined. For most applications, simple gain normalization is all that is required. Stage four rectifies the signal and then applies the power law required to demodulate the signal. In stage six, the inverse of the power law is applied to relinearize the signal. Direct comparison of MAV (first power) to RMS (second power) processing demonstrates little difference between the two. Therefore, unless there is reason to believe that the EMG density departs strongly from that found in the existing studies, RMS and MAV processing are essentially identical. In stage five, the demodulated samples are averaged across all channels and then smoothed (time averaged) to reduce the variance of the amplitude estimate, but at the expense of increasing the bias. For best performance, the window length that best trades off variance and bias error is selected. The advanced EMG processing was next applied to dynamic EMG-torque estimation about the elbow joint. Results showed that improved EMG amplitude estimates led to improved EMG-torque estimates. An initial comparison of different system-identification techniques and model orders was reported. It is expected that these advanced processing and identification algorithms will also improve performance in other EMG applications, including myoelectrically controlled prostheses, biofeedback, and ergonomic assessment.