Recent clinical trials have shown that bisphosphonate drugs improve breast cancer patient survival independent of their anti-resorptive effects on the skeleton. However, since bisphosphonates bind rapidly to bone mineral, the exact mechanisms of their anti-tumour action, particularly on cells outside of bone, remain unknown. Here we used real-time intravital two-photon microscopy to show extensive leakage of fluorescent bisphosphonate from the vasculature in 4T1 mouse mammary tumours, where it initially binds to areas of small, granular microcalcifications that are engulfed by tumour-associated macrophages (TAMs), but not tumour cells. Importantly, we also observed uptake of radiolabeled bisphosphonate in the primary breast tumour of a patient and showed the resected tumour to be infiltrated with TAMs and to contain similar granular microcalcifications. These data represent the first compelling in vivo evidence that bisphosphonates can target cells in tumours outside the skeleton and that their anti-tumour activity is likely to be mediated via TAMs.
Bisphosphonate; macrophage; myeloid cell; TAM; AF647-RIS; 99mTc-MDP; zoledronic acid; ZOL; intravital two-photon microscopy
The angiogenic switch, a rate-limiting step in tumor progression, has
already occurred by the time most human tumors are detectable. However, despite
significant study of the mechanisms controlling this switch, the kinetics and
reversibility of the process have not been explored. The stability of the
angiogenic phenotype was examined using an established human liposarcoma
xenograft model. Non-angiogenic cells inoculated into immunocompromised mice
formed microscopic tumors that remained dormant for ~125 days (vs. <40 days
for angiogenic cells) whereupon the vast majority (>95%) initiated
angiogenic growth with second-order kinetics. These original, clonally-derived
angiogenic tumor cells were passaged through four in vivo
cycles. At each cycle, a new set of single-cell clones was established from the
most angiogenic clone and characterized for in vivo for
tumorigenic activity. A total of 132 single-cell clones were tested in the 2nd,
3rd and 4th in vivo passage. Strikingly, at each passage, a
portion of the single-cell clones formed microscopic, dormant tumors. Following
dormancy, like the original cell line, these revertant tumors spontaneously
switched to the angiogenic phenotype. Finally, revertant clones were
transcriptionally profiles and their angiogenic output determined. Collectively,
these data demonstrate that the angiogenic phenotype in tumors is malleable and
can spontaneously revert to the non-angiogenic phenotype in a population of
human tumor cells.
The construct and concurrent validity of the Thera-Band Perceived Exertion Scale for Resistance Exercise with elastic bands (EB) was examined. Twenty subjects performed two separate sets of 15 repetitions of both frontal and lateral raise exercise over two sessions. The criterion variables were myoelectric activity and heart rate. One set was performed with an elastic band grip width that permitted 15 maximum repetitions in the selected exercise, and another set was performed with a grip width 50% more than the 15RM grip. Following the final repetition of each set, active muscle (AM) and overall body (O) ratings of perceived exertion (RPE) were collected from the Thera-Band® resistance exercise scale and the OMNI-Resistance Exercise Scale of perceived exertion with Thera-Band® resistance bands (OMNI-RES EB). Construct validity was established by correlating the RPE from the OMNI-RES EB with the Thera-Band RPE scale using regression analysis. The results showed significant differences (p ≤ 0.05) in myoelectric activity, heart rate, and RPE scores between the low- and high-intensity sets. The intraclass correlation coefficient for active muscles and overall RPE scale scores was 0.67 and 0.58, respectively. There was a positive linear relationship between the RPE from the OMNI-RES EB and the Thera-Band scale. Validity coefficients for the RPE AM were r2 = 0.87 and ranged from r2 = 0.76 to 0.85 for the RPE O. Therefore, the Thera-Band Perceived Exertion Scale for Resistance Exercise can be used for monitoring elastic band exercise intensity. This would allow the training dosage to be better controlled within and between sessions. Moreover, the construct and concurrent validity indicates that the OMNI-RES EB measures similar properties of exertion as the Thera-Band RPE scale during elastic resistance exercise.
Key pointsThis new resistance intensity scale is an appropriate and valid tool for assessing perceived exertion during strength training with elastic bands.This scale can be used without reducing the accuracy of the dosage prescribed during training/rehabilitation sessions and while carrying out medium and/or long-term periodization programs or therapeutic interventions.Populations with specific physical or physiological needs could have access to an easy-to-use resource that allows them to carry out their training/rehabilitation programs with greater efficacy and without any risk to health.
RPE; myoelectric activity; heart rate; resistance exercise
A wide variety of diseases have a significant genetic component, including major causes of morbidity and mortality in the western world. Many of these diseases are also angiogenesis dependent. In humans, common polymorphisms, although more subtle in effect than rare mutations that cause Mendelian disease, are expected to have greater overall effects on human disease. Thus, common polymorphisms in angiogenesis-regulating genes may affect the response to an angiogenic stimulus and thereby affect susceptibility to or progression of such diseases. Candidate gene studies have identified several associations between angiogenesis gene polymorphisms and disease. Similarly, emerging pharmacogenomic evidence indicates that several angiogenesis-regulating polymorphisms may predict response to therapy. In contrast, genome-wide association studies have identified only a few risk alleles in obvious angiogenesis genes. As in other traits, regulatory polymorphisms appear to dominate the landscape of angiogenic responsiveness. Rodent assays, including the mouse corneal micropocket assay, tumor models, and a macular degeneration model have allowed the identification and comparison of loci that directly affect the trait. Complementarity between human and animal approaches will allow increased understanding of the genetic basis for angiogenesis-dependent disease.
Most angiogenesis genetic studies in humans have taken a candidate gene approach. Polymorphisms in several angiogenesis genes (e.g., VEGF, CXCR2, and αv and β3 integrins) are clearly disease associated.
CD248 (tumor endothelial marker 1/endosialin) is found on stromal cells and is highly expressed during malignancy and inflammation. Studies have shown a reduction in inflammatory arthritis in CD248-knockout (CD248−/−) mice. The aim of the present study was to investigate the functional effect of genetic deletion of CD248 on bone mass.
Western blotting, polymerase chain reaction, and immunofluorescence were used to investigate the expression of CD248 in humans and mice. Micro-computed tomography and the 3-point bending test were used to measure bone parameters and mechanical properties of the tibiae of 10-week-old wild-type (WT) or CD248−/− mice. Human and mouse primary osteoblasts were cultured in medium containing 10 mM β-glycerophosphate and 50 μg/ml ascorbic acid to induce mineralization, and then treated with platelet-derived growth factor BB (PDGF-BB). The mineral apposition rate in vivo was calculated by identifying newly formed bone via calcein labeling.
Expression of CD248 was seen in human and mouse osteoblasts, but not osteoclasts. CD248−/− mouse tibiae had higher bone mass and superior mechanical properties (increased load required to cause fracture) compared to WT mice. Primary osteoblasts from CD248−/− mice induced increased mineralization in vitro and produced increased bone over 7 days in vivo. There was no decrease in bone mineralization and no increase in proliferation of osteoblasts in response to stimulation with PDGF-BB, which could be attributed to a defect in PDGF signal transduction in the CD248−/− mice.
There is an unmet clinical need to address rheumatoid arthritis–associated bone loss. Genetic deletion of CD248 in mice results in high bone mass due to increased osteoblast-mediated bone formation, suggesting that targeting CD248 in rheumatoid arthritis may have the effect of increasing bone mass in addition to the previously reported effect of reducing inflammation.
The purpose of this study was to analyze upper extremity and core muscle activation when performing push-ups with different suspension devices. Young fit male university students (n = 29) performed 3 push-ups each with 4 different suspension systems. Push-up speed was controlled using a metronome and testing order was randomized. Average amplitude of the electromyographic root mean square of Triceps Brachii, Upper Trapezius, Anterior Deltoid, Clavicular Pectoralis, Rectus Abdominis, Rectus Femoris, and Lumbar Erector Spinae was recorded. Electromyographic signals were normalized to the maximum voluntary isometric contraction (MVIC). Electromyographic data were analyzed with repeated-measures analysis of variance with a Bonferroni post hoc. Based upon global arithmetic mean of all muscles analyzed, the suspended push-up with a pulley system provided the greatest activity (37.76% of MVIC; p < 0.001). Individually, the suspended push-up with a pulley system also provided the greatest triceps brachii, upper trapezius, rectus femoris and erector lumbar spinae muscle activation. In contrast, more stable conditions seem more appropriate for pectoralis major and anterior deltoid muscles. Independent of the type of design, all suspension systems were especially effective training tools for reaching high levels of rectus abdominis activation.
Key PointsCompared with standard push-ups on the floor, suspended push-ups increase core muscle activation.A one-anchor system with a pulley is the best option to increase TRICEP, TRAPS, LUMB and FEM muscle activity.More stable conditions such as the standard push-up or a parallel band system provide greater increases in DELT and PEC muscle activation.A suspended push-up is an effective method to achieve high muscle activity levels in the ABS.
EMG; unstable; core; trunk; exercise
We have observed substantial differences in angiogenic responsiveness in mice and have mapped the genetic loci responsible for these differences. We have found that the albino mutation is one of the loci responsible for such differences. Using B6.A consomic strains, we determined that chromosome 7 bears a locus that inhibits VEGF-induced corneal neovascularization. F2 crosses between B6.A consomic mice and C57BL/6J parents along with AXB and BXA recombinant inbred strains demonstrated highest linkage near the tyrosinase gene. This region was named AngVq4. Congenic animals confirmed this locus, but could not demonstrate that the classical tyrosinase albino (c) mutation was causative because of the existence of additional linked loci in the congenic region. However, in 1970, a second tyrosinase albino mutation (c-2J) arose in the C57BL/6J background at Jackson Labs. Testing this strain (C57BL/6J) demonstrated that the albino mutation is sufficient to completely explain the alteration in angiogenic response that we observed in congenic animals. Thus, we conclude that the classical tyrosinase mutation is responsible for AngVq4. In contrast to the cornea, where pigmented animals exhibit increased angiogenic responsiveness, iris neovascularization was inhibited in pigmented animals. These results may partially explain increased aggressiveness in amelanotic melanoma, as well as ethnic differences in diabetic retinopathy and macular degeneration.
Tumor marker endothelial 8 (TEM8) is a receptor for the Protective Antigen (PA) component of anthrax toxin. TEM8 is upregulated on endothelial cells lining the blood vessels within tumors, compared to normal blood vessels. A number of studies have demonstrated a pivotal role for TEM8 in developmental and tumor angiogenesis. We have also shown that targeting the anthrax receptors with a mutated form of PA inhibits angiogenesis and tumor formation in vivo.
Here we describe the development and testing of a high-throughput fluorescence resonance energy transfer assay to identify molecules that strongly inhibit the interaction of PA and TEM8. The assay we describe is sensitive and robust, with a Z-prime value of 0.8. A preliminary screen of 2310 known bioactive library compounds identified ebselen and thimerosal as inhibitors of the TEM8-PA interaction. These molecules each contain a cysteine-reactive transition metal, and complimentary studies indicate that their inhibition of interaction is due to modification of a cysteine residue in the TEM8 extracellular domain. This is the first demonstration of a high-throughput screening assay that identifies inhibitors of TEM8, with potential application for anti-anthrax and anti-angiogenic diseases.
High-throughput screening; FRET; anthrax; angiogenesis; Tumor endothelial marker 8
A number of different balance assessment techniques are currently available and widely used. These include both subjective and objective assessments. The ability to provide quantitative measures of balance and posture is the benefit of objective tools, however these instruments are not generally utilized outside of research laboratory settings due to cost, complexity of operation, size, duration of assessment, and general practicality. The purpose of this pilot study was to assess the value and validity of using software developed to access the iPod and iPhone accelerometers output and translate that to the measurement of human balance.
Thirty healthy college‐aged individuals (13 male, 17 female; age = 26.1 ± 8.5 years) volunteered. Participants performed a static Athlete's Single Leg Test protocol for 10 sec, on a Biodex Balance System SD while concurrently utilizing a mobile device with balance software. Anterior/posterior stability was recorded using both devices, described as the displacement in degrees from level, and was termed the “balance score.”
There were no significant differences between the two reported balance scores (p = 0.818. Mean balance score on the balance platform was 1.41 ± 0.90, as compared to 1.38 ± 0.72 using the mobile device.
There is a need for a valid, convenient, and cost‐effective tool to objectively measure balance. Results of this study are promising, as balance score derived from the Smartphone accelerometers were consistent with balance scores obtained from a previously validated balance system. However, further investigation is necessary as this version of the mobile software only assessed balance in the anterior/posterior direction. Additionally, further testing is necessary on a healthy populations and as well as those with impairment of the motor control system.
Level of Evidence:
Level 2b (Observational study of validity)1
Accelerometer; Single Leg Balance Test; Smartphone Application; Stability assessment
CMG2 is a transmembrane extracellular matrix binding protein that is also an anthrax toxin receptor. We have shown that high affinity CMG2 binders can inhibit angiogenesis and tumor growth. We recently described a high throughput FRET assay to identify CMG2 inhibitors. We now report the serendipitous discovery that PGG (1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose) is a CMG2 inhibitor with anti-angiogenic activity. PGG is a gallotannin produced by a variety of medicinal plants that exhibits a wide variety of anti-tumor and other activities. We find that PGG inhibits CMG2 with a submicromolar IC50 and it also inhibits the migration of human dermal microvascular endothelial cells at similar concentrations in vitro. Finally, oral or intraperitoneal administration of PGG inhibits angiogenesis in the mouse corneal micropocket assay in vivo. Together, these results suggest that a portion of the in vivo anti-tumor activity of PGG may be the result of antiangiogenic activity mediated by inhibition of CMG2.
pentagalloyl glucose; 5GG; angiogenesis; corneal neovascularization; cancer; polyphenol
Huanglongbing (HLB) or citrus greening is a devastating disease of citrus. The gram-negative bacterium Candidatus Liberibacter asiaticus (Las) belonging to the α-proteobacteria is responsible for HLB in North America as well as in Asia. Currently, there is no cure for this disease. Early detection and quarantine of Las-infected trees are important management strategies used to prevent HLB from invading HLB-free citrus producing regions. Quantitative real-time PCR (qRT-PCR) based molecular diagnostic assays have been routinely used in the detection and diagnosis of Las. The oligonucleotide primer pairs based on conserved genes or regions, which include 16S rDNA and the β-operon, have been widely employed in the detection of Las by qRT-PCR. The availability of whole genome sequence of Las now allows the design of primers beyond the conserved regions for the detection of Las explicitly.
We took a complimentary approach by systematically screening the genes in a genome-wide fashion, to identify the unique signatures that are only present in Las by an exhaustive sequence based similarity search against the nucleotide sequence database. Our search resulted in 34 probable unique signatures. Furthermore, by designing the primer pair specific to the identified signatures, we showed that most of our primer sets are able to detect Las from the infected plant and psyllid materials collected from the USA and China by qRT-PCR. Overall, 18 primer pairs of the 34 are found to be highly specific to Las with no cross reactivity to the closely related species Ca. L. americanus (Lam) and Ca. L. africanus (Laf).
We have designed qRT-PCR primers based on Las specific genes. Among them, 18 are suitable for the detection of Las from Las-infected plant and psyllid samples. The repertoire of primers that we have developed and characterized in this study enhanced the qRT-PCR based molecular diagnosis of HLB.
Detection system; Diagnostic; Candidatus Liberibacter asiaticus; Greening; Huanglongbing; Bacteria; Psyllid; Citrus
Studies have established that pigmentation can provide strong, protective effects against certain human diseases. For example, angiogenesis-dependent diseases such as wet age-related macular degeneration and infantile hemangioma are more common in light-skinned individuals of mixed European descent than in African-Americans. Here we found that melanocytes from light-skinned humans and albino mice secrete high levels of fibromodulin (FMOD), which we determined to be a potent angiogenic factor. FMOD treatment stimulated angiogenesis in numerous in vivo systems, including laser-induced choroidal neovascularization, growth factor–induced corneal neovascularization, wound healing, and Matrigel plug assays. Additionally, FMOD enhanced vascular sprouting during normal retinal development. Deletion of Fmod in albino mice resulted in a marked reduction in the amount of neovascularization induced by retinal vein occlusion, corneal growth factor pellets, and Matrigel plugs. Our data implicate the melanocyte-secreted factor FMOD as a key regulator of angiogenesis and suggest an underlying mechanism for epidemiological differences between light-skinned individuals of mixed European descent and African-Americans. Furthermore, inhibition of FMOD in humans has potential as a therapeutic strategy for treating angiogenesis-dependent diseases.
The human vagina is colonized by a variety of indigenous microflora; in healthy individuals the predominant bacterial genus is Lactobacillus while those with bacterial vaginosis (BV) carry a variety of anaerobic representatives of the phylum Actinobacteria. In this study, we evaluated the antimicrobial activity of benzoyl peroxide (BPO) encapsulated in a hydrogel against Gardnerella vaginalis, one of the causative agents of BV, as well as indicating its safety for healthy human lactobacilli. Herein, it is shown that in well diffusion assays G. vaginalis is inhibited at 0.01% hydrogel-encapsulated BPO and that the tested Lactobacillus spp. can tolerate concentrations of BPO up to 2.5%. In direct contact assays (cells grown in a liquid culture containing hydrogel with 1% BPO or BPO particles), we demonstrated that hydrogels loaded with 1% BPO caused 6-log reduction of G. vaginalis. Conversely, three of the tested Lactobacillus spp. were not inhibited while L. acidophilus growth was slightly delayed. The rheological properties of the hydrogel formulation were probed using oscillation frequency sweep, oscillation shear stress sweep, and shear rate sweep. This shows the gel to be suitable for vaginal application and that the encapsulation of BPO did not alter rheological properties.
Older women may have chronic or age-related conditions that increase the risk of falls or that limit their ability to remain active. It is unclear if a water-based exercise program provides a safe and effective alternative to land-based exercise. The purpose of this study was to evaluate the impact of a water-based exercise program method on land-based functional activities of daily living (ADL) for women 60 years and older. This study used a quasi- experimental, nonequivalent control group design. Sixty-six women (60-89 yr of age) self- selected to a water exercise (WEX) group (n = 48) or control (C) group (n = 18). The training consisted of a 16-week (45 min·day-1, 3 d·wk-1) supervised WEX program that included 10 min of warm-up and warm down/stretching and 35 min training using the S.W.E.A.T.™ method in shallow water 1.0-1.2 m, with water temperature approximately 28-29°C. Participants were required to attendat least 94% of the sessions. Assessments for participants included ADL functional field tests. In comparison to the C group, WEX participantsimproved (p < 0.05) flexibility (8%), sit- to-stand (31%), walking speed (16%) and stride length (10%), agility (20%), stair climb (22%), arm curl (39%), and static (42-48%) balance, but not dynamic balance. Results indicate that the S.W.E.A.T.™ method applied to this water exercise program provides a well-rounded, safe, and effective exercise program where older women can improve functional ADL and static balance.
Key PointsOlder women with a variety of health conditions participated in 16 weeks of exercise (92% adherence) with no injuries.The S.W.E.A.T.™ method applied to this water-based program was found to significantly improve several aspects of physical function, including postural balance.This shallow water program provided a well-rounded, safe and effective activity for women to improve functional ADL on land.
Functional ADL; water/aquatic exercise; older women
Targeting and inhibiting CMG2 (Capillary Morphogenesis Gene protein 2) represents a new strategy for therapeutic agents for cancer and retinal diseases due to CMG2’s role in blood vessel growth (angiogenesis). A high throughput FRET (Förster Resonance Energy Transfer) assay was developed for the identification of CMG2 inhibitors as anti-angiogenetic agents. Bioassay-guided separation led to the isolation and identification of two new compounds (1 and 2) from CR252M, an endophytic fungus Coccomyces proteae collected from a Costa Rican rainforest, and one known compound (3) from CR1207B (Aurapex penicillata). Secondary in vitro assays indicated anti-angiogenic activity. Compound 3 inhibited the endothelial cell migration at 52 µM, but did not show any endothelial cell antiproliferative effect at 156 µM. The structure of the two new compounds, A (1) and B (2), were elucidated on the basis of extensive spectroscopic analysis, including 1D and 2D NMR experiments.
Fungus; Coccomyces proteae; Aurapex penicillata; CMG2; Phenolic
The purpose of this study was to compare the effects of Nordic walking with conventional walking and band-based resistance exercise on functional fitness, static balance and dynamic balance in older adults. Volunteers (n = 65) were divided into four groups: Nordic walking (NW), conventional walking (CW), resistance (RES), and control. Each group performed activity 50-70 min·day−1 (warm-up 10-15 min, main exercise 30-40, and cool down 10-15 min), 3 days·week−1 (NW and CW) or 2 day·week−1 (RES) for 12 wks. Upper-body strength improved (p < 0. 05) in the RES (22.3%) and the NW (11.6%) groups compared to the CW and control groups. Cardio- respiratory fitness improved more in the NW (10.9%) and CW (10.6%) groups compared to the RES and control groups. Upper- and lower-body flexibility also improved in all exercise groups compared to the control group. There were no improvements in balance measures in any group. While all modes of exercise improved various components of fitness, Nordic walking provided the best well-rounded benefits by improving upper-body strength, cardiovascular endurance, and flexibility. Therefore, Nordic walking is recommended as an effective and efficient mode of concurrent exercise to improve overall functional fitness in older adults.
Key PointsNordic walking, conventional walking, and resistance training are beneficial for older adults.Nordic walking and conventional walking both improve cardio-respiratory fitness while resistance training does not.Nordic walking provides additional benefits in upper-body muscular strength compared to conventional walking.Nordic walking is an effective and efficient mode of exercise to improve overall fitness in older adults.
Walking; resistance exercise; concurrent exercise; aging; functional fitness
As the older adult population increases in size, the number of older adults participating in sport activities will also likely increase proportionally with a concomitant increase in musculoskeletal injuries. Age-associated functional declines in muscle strength and the sensory systems, in addition to several other issues, contribute to reductions in balance that may increase fall risk There are a variety of ways to evaluate balance and fall-risk, and each older adult should be regularly screened in order to evaluate any changes in the ability to maintain postural stability. Balance training is a useful intervention in rehabilitation of postural stability impairments as well as in training programs for performance enhancement. One scientifically-based approach is Sensorimotor Training (SMT) which can be characterized as a progressive balance training program using labile surfaces to provide adequate and safe challenges to the older athlete's balance. SMT addresses both static and dynamic components of balance as well as the multitude of systems that control balance in order to train effective strategies and elicit automatic postural responses in order to enhance postural stability. The authors believe that SMT should become part of the regular training regimen for the aging athlete. For the sport and orthopedic healthcare professional, an understanding of the physiologic changes that occur with age, the means by which balance can be assessed, and how SMT programs can be developed and implemented is crucial in addressing the growing number of older athletes that they will see.
Level of Evidence:
Aging; balance assessment; balance training; postural stability
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The article entitled “Monosodium glutamate (MSG) intake is associated with the prevalence of metabolic syndrome in a rural Thai population”, concluded that higher amounts of individual’s MSG consumption are associated with the risk of having the metabolic syndrome and being overweight independent of other major determinants. However, this epidemiological study is the only study indicating such a relationship between MSG intake and the prevalence of metabolic syndrome and there is no direct supporting evidence for a causal relationship between MSG intake and prevalence of metabolic syndrome. This study does not indicate that MSG causes metabolic syndrome. Furthermore, there are several questionable points concerning study methods. Further carefully designed studies taking into account all glutamate sources are necessary to demonstrate the relationship between overweight, metabolic syndrome, MSG intake and umami sensitivity.
Monosodium glutamate; Intake; Overweight; Metabolic syndrome
Determine the reliability of two different modified (MOD1 and MOD2) testing methods compared to a standard method (ST) for testing trunk flexion and extension endurance.
Twenty‐eight healthy individuals (age 26.4 ± 3.2 years, height 1.75 ± m, weight 71.8 ± 10.3 kg, body mass index 23.6 ± 3.4 m/kg2).
Trunk endurance time was measured in seconds for flexion and extension under the three different stabilization conditions. The MOD1 testing procedure utilized a female clinician (70.3 kg) and MOD2 utilized a male clinician (90.7 kg) to provide stabilization as opposed to the ST method of belt stabilization.
No significant differences occurred between flexion and extension times. Intraclass correlations (ICCs3,1) for the different testing conditions ranged from .79 to .95 (p <.000) and are found in Table 3. Concurrent validity using the ST flexion times as the gold standard coefficients were .95 for MOD1 and .90 for MOD2. For ST extension, coefficients were .91 and .80, for MOD1 and MOD2 respectively (p <.01).
These methods proved to be a reliable substitute for previously accepted ST testing methods in normal college‐aged individuals. These modified testing procedures can be implemented in athletic training rooms and weight rooms lacking appropriate tables for the ST testing.
Level of Evidence:
Core; stabilization; trunk endurance
The adenosine diphosphate (ADP) receptor P2RY12 (purinergic receptor P2Y, G protein coupled, 12) plays a critical role in platelet aggregation, and P2RY12 inhibitors are used clinically to prevent cardiac and cerebral thrombotic events. Extracellular ADP has also been shown to increase osteoclast (OC) activity, but the role of P2RY12 in OC biology is unknown. Here, we examined the role of mouse P2RY12 in OC function. Mice lacking P2ry12 had decreased OC activity and were partially protected from age-associated bone loss. P2ry12–/– OCs exhibited intact differentiation markers, but diminished resorptive function. Extracellular ADP enhanced OC adhesion and resorptive activity of WT, but not P2ry12–/–, OCs. In platelets, ADP stimulation of P2RY12 resulted in GTPase Ras-related protein (RAP1) activation and subsequent αIIbβ3 integrin activation. Likewise, we found that ADP stimulation induced RAP1 activation in WT and integrin β3 gene knockout (Itgb3–/–) OCs, but its effects were substantially blunted in P2ry12–/– OCs. In vivo, P2ry12–/– mice were partially protected from pathologic bone loss associated with serum transfer arthritis, tumor growth in bone, and ovariectomy-induced osteoporosis: all conditions associated with increased extracellular ADP. Finally, mice treated with the clinical inhibitor of P2RY12, clopidogrel, were protected from pathologic osteolysis. These results demonstrate that P2RY12 is the primary ADP receptor in OCs and suggest that P2RY12 inhibition is a potential therapeutic target for pathologic bone loss.
Anti-angiogenic therapies are effective for the treatment of cancer, a variety of ocular diseases, and have potential benefits in cardiovascular disease, arthritis, and psoriasis. We have previously shown that anthrax protective antigen (PA), a non-pathogenic component of anthrax toxin, is an inhibitor of angiogenesis, apparently as a result of interaction with the cell surface receptors capillary morphogenesis gene 2 (CMG2) protein and tumor endothelial marker 8 (TEM8). Hence, molecules that bind the anthrax toxin receptors may be effective to slow or halt pathological vascular growth. Here we describe development and testing of an effective homogeneous steady-state fluorescence resonance energy transfer (FRET) high throughput screening assay designed to identify molecules that inhibit binding of PA to CMG2. Molecules identified in the screen can serve as potential lead compounds for the development of anti-angiogenic and anti-anthrax therapies. The assay to screen for inhibitors of this protein–protein interaction is sensitive and robust, with observed Z' values as high as 0.92. Preliminary screens conducted with a library of known bioactive compounds identified tannic acid and cisplatin as inhibitors of the PA-CMG2 interaction. We have confirmed that tannic acid both binds CMG2 and has anti-endothelial properties. In contrast, cisplatin appears to inhibit PA-CMG2 interaction by binding both PA and CMG2, and observed cisplatin anti-angiogenic effects are not mediated by interaction with CMG2. This work represents the first reported high throughput screening assay targeting CMG2 to identify possible inhibitors of both angiogenesis and anthrax intoxication.
To determine the effects of a supervised strength training program on body composition and physical capacity of older women using three different devices: weight machines, elastic bands, and aquatic devices that increase drag forces (ADIDF). Four groups were formed: control group, weight machine group (WMG), elastic band group (EBG) and a group that used ADIDF (ADIDFG). Body composition and physical capacity were assessed before and after the intervention period. The ADIDFG showed improvements in fat mass (FM), fat-free mass of the left arm (FFM-LA) and right arm (FFM-RA), knee push-up test (KPT), squat test (ST) and crunch test (CT) (p <0.05). Individuals in the EBG and WMG also improved their FM, fat free mass (FFM), FFM-LA, FFM-RA, KPU, ST and CT. ADIDF training improves body composition and physical capacity of postmenopausal women as does performing land-based training programs.
aquatic training; weight machines; elastic bands; fitness
Angiogenesis is the process by which new blood vessels are formed from existing vessels. Mammalian populations, including humans and mice, harbor genetic variations that alter angiogenesis. Angiogenesis-regulating gene variants can result in increased susceptibility to multiple angiogenesis-dependent diseases in humans. Our efforts to dissect the complexity of the genetic diversity that regulates angiogenesis have used laboratory animals due to the availability of genome sequence for many species and the ability to perform high volume controlled breeding. Using the murine corneal micropocket assay, we have observed more than ten-fold difference in angiogenic responsiveness among various mouse strains. This degree of difference is observed with either bFGF or VEGF induced corneal neovascularization. Ongoing mapping studies have identified multiple loci that affect angiogenic responsiveness in several mouse models. In this study, we used F2 intercrosses between C57BL/6J and the 129 substrains 129P1/ReJ and 129P3/J, as well as the SJL/J strain, where we have identified new QTLs that affect angiogenic responsiveness. In the case of AngFq5, on chromosome 7, congenic animals were used to confirm the existence of this locus and subcongenic animals, combined with a haplotype-based mapping approach that identified the pink-eyed dilution mutation as a candidate polymorphism to explain AngFq5. The ability of mutations in the pink-eyed dilution gene to affect angiogenic response was demonstrated using the p-J allele at the same locus. Using this allele, we demonstrate that pink-eyed dilution mutations in Oca2 can affect both bFGF and VEGF-induced corneal angiogenesis.
Progress in the synthesis of novel fluorescent conjugates of N-heterocyclic bisphosphonate drugs and related analogues, together with some recent applications of these compounds as imaging probes, are briefly discussed.
Fluorescent; bisphosphonate; imaging; conjugates
Originally identified as axonal guidance cues, semaphorins are expressed throughout many different tissues and regulate numerous non-neuronal processes. We demonstrate that most class III semaphorins are expressed in mouse osteoblasts and are differentially regulated by cell growth and differentiation: Sema3d expression is increased and Sema3e expression decreased during proliferation in culture, while expression of Sema3a is unaffected by cell density but increases in cultures of mineralizing osteoblasts. Expression of Sema3a, -3e, and -3d is also differentially regulated by osteogenic stimuli; inhibition of GSK3β decreased expression of Sema3a and -3e, while 1,25-(OH)2D3 increased expression of Sema3e. Parathyroid hormone had no effect on expression of Sema3a, -3b, or -3d. Osteoblasts, macrophages, and osteoclasts express the Sema3e receptor PlexinD1, suggesting an autocrine and paracrine role for Sema3e. No effects of recombinant Sema3e on osteoblast proliferation, differentiation, or mineralization were observed; but Sema3e did inhibit the migration of osteoblasts in a wound-healing assay. The formation of multinucleated, tartrate-resistant acid phosphatase–positive osteoclasts was decreased by 81% in cultures of mouse bone marrow macrophages incubated with 200 ng/mL Sema3e. Correspondingly, decreased expression of osteoclast markers (Itgb3, Acp5, Cd51, Nfatc1, CalcR, and Ctsk) was observed by qPCR in macrophage cultures differentiated in the presence of Sema3e. Our results demonstrate that class III semaphorins are expressed by osteoblasts and differentially regulated by differentiation, mineralization, and osteogenic stimuli. Sema3e is a novel inhibitor of osteoclast formation in vitro and may play a role in maintaining local bone homeostasis, potentially acting as a coupling factor between osteoclasts and osteoblasts.
Macrophage; Plexin; Mineralization; Vitamin D3; Migration