Effects of Piper hispidinervum on spermatogenesis and histochemistry of ovarioles of Spodoptera frugiperda

The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), not only damages crops, but controlling its population also requires synthetic insecticides, which leads to selection of resistant populations and environmental contamination. Essential oils are an alternative for controlling this insect. There are few studies of the effects of these oils on the insect's reproductive system. We evaluated the effects of the long pepper, Piper hispidinervum, essential oil on the gonads of the armyworm and tested its possible influence on the fertility of this insect. Dosages of 30 and 50 mg/ml were tested in 3^rd instar caterpillars using the leaf immersion method. Testes and ovarioles were collected, fixed with 10% formalin and embedded in Historesin. The sections were stained with toluidine blue and Mallory trichrome to detect connective tissue, periodic acid-Schiff to detect neutral carbohydrates, and bromophenol blue to detect proteins. We found that the long pepper essential oil affected negatively the spermatogenesis and altered the histochemistry of the ovarioles of S. frugiperda. The effects of long pepper oil suggest that it is a promising tool for controlling the armyworm pest.     

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50–60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.     

Measuring cell cycle progression kinetics with metabolic labeling and flow cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments. Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.     

Relationship of biomechanical factors to baseball pitching velocity: within pitcher variation

To reach the level of elite, most baseball pitchers need to consistently produce high ball velocity but avoid high joint loads at the shoulder and elbow that may lead to injury. This study examined the relationship between fastball velocity and variations in throwing mechanics within 19 baseball pitchers who were analyzed via 3-D high-speed motion analysis. Inclusion in the study required each one to demonstrate a variation in velocity of at least 1.8 m/s (range 1.8-3.5 m/s) during 6 to 10 fastball pitch trials. Three mixed model analyses were performed to assess the independent effects of 7 kinetic, 11 temporal, and 12 kinematic parameters on pitched ball velocity. Results indicated that elbow flexion torque, shoulder proximal force, and elbow proximal force were the only three kinetic parameters significantly associated with increased ball velocity. Two temporal parameters (increased time to max shoulder horizontal adduction and decreased time to max shoulder internal rotation) and three kinematic parameters (decreased shoulder horizontal adduction at foot contact, decreased shoulder abduction during acceleration, and increased trunk tilt forward at release) were significantly related to increased ball velocity. These results point to variations in an individual's throwing mechanics that relate to pitched ball velocity, and also suggest that pitchers should focus on consistent mechanics to produce consistently high fastball velocities. In addition, pitchers should strengthen shoulder and elbow musculature that resist distraction as well as improve trunk strength and flexibility to maximize pitching velocity and help prevent injury.     

Adenoviral oncoprotein E1B55K mediates colocalization of SSBP2 and PML in response to stress

Transient expression of adenoviral oncoprotein E1B55K in normal cells induces aggresome formation and sequestration of critical host proteins in aggresomes. Our previous studies reported that Sequence Specific Binding Protein 2 (SSBP2), a candidate tumor suppressor is recruited to aggresomes in adenovirally transformed human embryonal kidney 293 (HEK293) cells. To understand the extent and significance of the E1B55K-SSBP2 interactions in these cells, we have examined SSBP2 localization under conditions of stress in HEK293 cells. SSBP2 localizes to PML- Nuclear Bodies (PML-NBs) in response to inhibition of nuclear export, treatment with etoposide, hydroxyurea or gamma irradiation only in HEK293 cells. Furthermore, the PML-NBs grow in size and number in response to radiation over a 24 hour period in HEK293 cells analogous to previous findings for other cell types. Nonetheless, we conclude that E1B55K subverts SSBP2 function in HEK293 cells. These findings demonstrate the limitations in using HEK293 cells to study DNA damage response and other cellular processes since SSBP2 and similar regulatory proteins are aberrantly localized due to constitutive E1B55K expression.     

Low sclerostin levels: a predictive marker of persistent inflammation in ankylosing spondylitis during anti-tumor necrosis factor therapy?

Introduction

Sclerostin levels have been reported to be low in ankylosing spondylitis (AS), but there is no data regarding the possible role of this Wnt inhibitor during anti-tumor necrosis factor (TNF) therapy. The present study longitudinally evaluated sclerostin levels, inflammatory markers and bone mineral density (BMD) in AS patients under anti-TNF therapy.

Methods

Thirty active AS patients were assessed at baseline, 6 and 12 months after anti-TNF therapy regarding clinical parameters, inflammatory markers, BMD and baseline radiographic damage (mSASSS). Thirty age- and sex-matched healthy individuals comprised the control group. Patients'' sclerostin levels, sclerostin binding low-density lipoprotein receptor-related protein 6 (LRP6) and BMD were evaluated at the same time points and compared to controls.

Results

At baseline, AS patients had lower sclerostin levels (60.5 ± 32.7 vs. 96.7 ± 52.9 pmol/L, P = 0.002) and comparable sclerostin binding to LRP6 (P = 0.387) than controls. Improvement of Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Bath Ankylosing Spondylitis Metrology Index (BASMI), Ankylosing Spondylitis quality of life (ASQoL) was observed at baseline vs. 6 vs. 12 months (P < 0.01). Concomitantly, a gradual increase in spine BMD (P < 0.001) and a positive correlation between baseline mSASSS and spine BMD was found (r = 0.468, P < 0.01). Inflammatory parameters reduction was observed comparing baseline vs. 6 vs. 12 months (P <0.01). Sclerostin levels progressively increased [baseline (60.5 ± 32.7) vs. 6 months (67.1 ± 31.9) vs. 12 months (72.7 ± 32.3) pmol/L, P <0.001]. At 12 months, the sclerostin levels remained significantly lower in patients compared to controls (72.7 ± 32.3 vs. 96.70 ± 52.85 pmol/L, P = 0.038). Moreover, sclerostin serum levels at 12 months were lower in the 10 patients with high C reactive protein (CRP) (≥ 5 mg/l) compared to the other 20 patients with normal CRP (P = 0.004). Of note, these 10 patients with persistent inflammation also had lower sclerostin serum levels at baseline compared to the other patients (P = 0.023). Univariate logistic regression analysis demonstrated that AS patients with lower sclerostin serum levels had an increased risk to have high CRP at 12 months (odds ratio = 7.43, 95% CI 1.23 to 45.01, P = 0.020) than those with higher sclerostin values.

Conclusions

Persistent low sclerostin levels may underlie continuous inflammation in AS patients under anti-TNF therapy.     

Kinematic and kinetic comparison of baseball pitching among various levels of development

Proper biomechanics help baseball pitchers minimize their risk of injury and maximize performance. However previous studies involved adult pitchers only. In this study, 23 youth, 33 high school, 115 college, and 60 professional baseball pitchers were analyzed. Sixteen kinematic (11 position and five velocity), eight kinetic, and six temporal parameters were calculated and compared among the four levels of competition. Only one of the 11 kinematic position parameters showed significant differences among the four levels, while all five velocity parameters showed significant differences. All eight kinetic parameters increased significantly with competition level. None of the six temporal parameters showed significant differences. Since 16 of the 17 position and temporal parameters showed no significant differences, this study supports the philosophy that a child should be taught ‘proper’ pitching mechanics for use throughout a career. Kinetic differences observed suggest greater injury risk at higher competition levels. Since adult pitchers did not demonstrate different position or temporal patterns than younger pitchers, increases in joint forces and torques were most likely due to increased strength and muscle mass in the higher level athlete. The greater shoulder and elbow angular velocities produced by high-level pitchers were most likely due to the greater torques they generated during the arm cocking and acceleration phases. The combination of more arm angular velocity and a longer arm resulted in greater linear ball velocity for the higher level pitcher. Thus, it appears that the natural progression for successful pitching is to learn proper mechanics as early as possible, and build strength as the body matures.