A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO₂ tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.     

Hand-held lactate analyzer as a tool for the real-time measurement of physical fatigue before slaughter and pork quality prediction

The objectives of this study were to assess the relationship between blood lactate variation measured at the plant, and pork quality variation on a large sample size and under commercial preslaughter handling conditions. A total of 600 pigs were randomly chosen on arrival at a commercial slaughter plant and blood samples taken from the ear vein at unloading (UN), after lairage (LA), in the restrainer (RE; before stunning) and at exsanguination (EX) were analysed for lactate content using a Lactate Scout Analyzer (LSA). In order to have a large range of measures, pigs were distributed into two groups; one kept in lairage overnight (G1) and the other for 2 to 3 h (G2) before slaughter. Meat quality was assessed in the Longissimus thoracis (LT), Semimembranosus (SM) and Adductor (AD) muscles by measuring the pH 30 min postmortem (pH1) and at 24 h postmortem (pHu), the colour and the drip loss. Blood lactate levels did not differ between G1 and G2 (P>0.05). A reduced muscle lactate and glucose contents (P=0.02 and P=0.004, respectively) resulting in a lower (P<0.001) glycolytic potential (GP) was observed in the LT muscle of G1 pigs when compared with G2 loins. In the LT muscle of G1 pigs, the lower GP resulted in an increased pHu (r=−0.67; P<0.001), decreased drip loss (r=0.57; P<0.001) and darker colour (r=0.50; P<0.001) compared with G2. In both G1 and G2 pigs, the lower GP was correlated to higher pHu value in the SM and AD muscles (r=−0.73; P<0.001). The greatest correlation was observed in G2 between blood lactate levels at LA and pHu value of the SM and AD muscles (r=0.46 and r=0.44, respectively; P<0.001 for both muscles). The second greatest correlation was found between blood lactate levels at EX and pH1 value in the SM muscle in both groups (r=−0.37 and r=−0.41, respectively; P<0.001 for both groups). Based on the results of this study, it appears that blood lactate levels, as measured by the LSA, reliably reflect the physiological response of pigs to perimortem stress and may help explain the variation in pork quality.     

Effect of ubiquinol supplementation on biochemical and oxidative stress indexes after intense exercise in young athletes

Objectives: Physical exercise significantly impacts the biochemistry of the organism. Ubiquinone is a key component of the mitochondrial respiratory chain and ubiquinol, its reduced and active form, is an emerging molecule in sport nutrition. The aim of this study was to evaluate the effect of ubiquinol supplementation on biochemical and oxidative stress indexes after an intense bout of exercise.

Methods: 21 male young athletes (26?+?5 years of age) were randomized in two groups according to a double blind cross-over study, either supplemented with ubiquinol (200?mg/day) or placebo for 1 month. Blood was withdrawn before and after a single bout of intense exercise (40 min run at 85% maxHR). Physical performance, hematochemical parameters, ubiquinone/ubiquinol plasma content, intracellular reactive oxygen species (ROS) level, mitochondrial membrane depolarization, paraoxonase activity and oxidative DNA damage were analyzed.

Results: A single bout of intense exercise produced a significant increase in most hematochemical indexes, in particular CK and Mb while, on the contrary, normalized coenzyme Q₁₀ plasma content decreased significantly in all subjects. Ubiquinol supplementation prevented exercise-induced CoQ deprivation and decrease in paraoxonase activity. Moreover at a cellular level, in peripheral blood mononuclear cells, ubiquinol supplementation was associated with a significant decrease in cytosolic ROS while mitochondrial membrane potential and oxidative DNA damage remained unchanged.

Discussion: Data highlights a very rapid dynamic of CoQ depletion following intense exercise underlying an increased demand by the organism. Ubiquinol supplementation minimized exercise-induced depletion and enhanced plasma and cellular antioxidant levels but it was not able to improve physical performance indexes or markers of muscular damage.     

Association of shiftwork and immune cells among police officers from the Buffalo Cardio-Metabolic Occupational Police Stress study

Shift workers suffer from a constellation of symptoms associated with disruption of circadian rhythms including sleep abnormalities, and abnormal hormone secretion (e.g. melatonin, cortisol). Recent, but limited, evidence suggests that shift workers have elevated levels of circulating white blood cells (WBCs) compared to their day working counterparts. Interestingly, recent reviews highlight the strong linkage between the immune system and circadian rhythms which includes, but is not limited to, circulating cell populations and functions. The elevated levels of these WBCs may be associated with the increased chronic disease risk observed among this group. The purpose of this analysis was to examine the cross-sectional association between long- and short-term (3, 5, 7, and 14 days) shiftwork (SW) and counts of WBCs among officers in the Buffalo Cardio-Metabolic Occupational Police Stress (BCOPS) cohort. Data collection for this analysis took place among 464 police officers working in Buffalo, New York, USA between 2004 and 2009. Precise SW histories were obtained using electronic payroll records. Officers were assigned a shift type based on the shift (i.e. day, evening, night) that they spent a majority (i.e. ≥50%) of their time from 1994 to the data collection date for long-term SW. The same process was applied to SW over 3, 5, 7, and 14 days prior to data collection. A fasted blood sample collected in the morning of a non-work day was used for characterization of WBCs (total), neutrophils, monocytes, lymphocytes, eosinophils, and basophils. Potential confounding factors included demographic characteristics (e.g. age, sex, race), occupational characteristics (e.g. rank), health behaviors (e.g. smoking, alcohol consumption, diet), anthropometrics, and other biomarkers (e.g. lipids, hemoglobin A1C, leptin). Generalized linear models were used to estimate least square means of the immune cells according to SW categorization for long- and short-term SW histories. Compared to the day shift group, those working long-term night shifts had greater absolute numbers of total WBCs, neutrophils, lymphocytes, and monocytes (all p < 0.05). Those working mainly on the night shift over 7-days had elevated counts of WBCs, lymphocytes, and monocytes (p < 0.05) compared to those mainly working day shifts. Results based on 3-, 5-, and 14-day SW were similar to the 7-day results. This study corroborates other studies with similar findings. However, this analysis provided insights into the effect of both long- and short-term SW on the number of circulating WBCs. SW may lead to disruption of circadian-influenced components of the immune system, which in term, may result in various chronic diseases. These findings, plus previous findings, may provide evidence that SW may lead to immune system dysregulation. Future research is needed to understand whether increases in immune cells among shift workers may be associated with the increased disease risk among this group.     

The lungs of Polypterus senegalus and Erpetoichthys calabaricus: Insights into the structure and functional distribution of the pulmonary epithelial cells

The present article is a comparative, structural study of the lung of Polypterus senegalus and Erpetoichthys calabaricus , two species representative of the two genera that constitute the Polypteriformes. The lung of the two species is an asymmetric, bi‐lobed organ that arises from a slit‐like opening in the ventral side of the pharynx. The wall is organized into layers, being thicker in P. senegalus . The inner epithelium contains ciliated and non‐ciliated bands. The latter constitute the respiratory surface and are wider in E. calabaricus . The air‐blood barrier is thin and uniform in P. senegalus and thicker and irregular in E. calabaricus . In the two species, the ciliated areas contain ciliated cells, mucous cells and cells with lamellar bodies. Additionally, P. senegalus contains polymorphous granular cells (PGCs) and neuroendocrine cells (NECs) while E. calabaricus lacks PGCs but shows granular leukocytes and a different type of NEC. Interestingly, ciliated cells and secretory cells show a dual morphology in E. calabaricus indicating the presence of cellular subtypes and suggesting more complex secretory activity. Also in E. calabaricus , cilia show a novel doublet‐membrane interaction that may control the displacement of the microtubule doublets. The subepithelium is a connective layer that appears thicker in P. senegalus and contains, in the two species, fibroblasts and granulocytes. The outer layer contains bundles of richly innervated striated muscle. This layer is likely involved in the control of lung motion. In the two species, smooth muscle cells constitute a limiting layer between the subepithelium and the striated muscle compartment. The role of this layer is unclear.     

Fertility preservation and preimplantation genetic assessment for women with breast cancer

Breast cancer is the most common cancer diagnosed among reproductive aged women, and its treatment can compromise future fertility. Options for fertility preservation include oocyte or embryo cryopreservation after ovarian stimulation (OS), which are the most established choices and are applicable for adult women with cancer. Ovarian tissue freezing may also be appropriate, as it offers potentially the least delay. The recognisation of the role of BRCA1 and BRCA2 mutations in some women has led to the involvement of preimplantation genetic diagnosis (PGD), recently renamed preimplantation genetic testing for monogenic disorder (PGT-M), whereby embryos are created by IVF and cell(s) are removed and genetically analyzed for specific disease-related mutations. PGT-M offers a valid option for women wishing to avoid transmission of the predisposition for hereditary breast cancer to their offspring. The aim of this paper is to provide an overview of the factors that influence fertility preservation in newly diagnosed breast cancer patients, and to illustrate the option of PGT-M to enable conception of an unaffected child.     

Antibodies specific for gp40 inhibit cell-cell adhesion by cross-linking the protein on the surface of Dictyostelium purpureum

We have previously suggested a role for gp40 in cell-cell adhesion in Dictyostelium purpureum from the fact that antibodies specific for this protein inhibited adhesion in an in vitro assay [Springer: Dev Biol 133:447–455, 1989]. To further confirm this role mutants lacking the protein were isolated and characterized. To our surprise, the mutants had normal adhesive properties both in vitro and in situ. These results lead us to the conclusion that gp40 is not necessary for the cell-cell adhesions observed and may not be a molecule which directly participates in these adhesions. When studied further, we found that adhesion-inhibitory antibodies were only effective as divalent IgG. Monovalent Fab fragments of the same antibodies could not inhibit adhesion. The inhibitory antibodies also caused the cells to remain rounded and incapable of attaching to plastic surfaces. We conclude that when divalent antibodies specific for gp40 cross-link this protein on the cell surface a cytoskeletal change prevents them from attaching to substratum as well as to other cells, thereby inhibiting cell-cell adhesion. We suggest that an alternative mechanism for inhibition of cell-cell adhesion by divalent antibodies exists and should be considered before proposing a direct role for a protein in adhesion.     

On the relation between gene flow theory and genetic gain

In conventional gene flow theory the rate of genetic gain is calculated as the summed products of genetic selection differential and asymptotic proportion of genes deriving from sex-age groups. Recent studies have shown that asymptotic proportions of genes predicted from conventional gene flow theory may deviate considerably from true proportions. However, the rate of genetic gain predicted from conventional gene flow theory was accurate. The current note shows that the connection between asymptotic proportions of genes and rate of genetic gain that is embodied in conventional gene flow theory is invalid, even though genetic gain may be predicted correctly from it.     

Ecological and genetic data indicate recovery of the endangered coral Acropora palmata in Los Roques, Southern Caribbean

The rapid decline of Acropora cervicornis and Acropora palmata has often been linked with coral reef deterioration in the Caribbean; yet, it remains controversial whether these species are currently recovering or still declining. In this study, the status of ten populations of A. palmata in Los Roques National Park (LRNP), Venezuela is presented. Six of these populations showed signs of recovery. Ten 80 m² belt-transects were surveyed at each of the ten reef sites. Within belt-transects, each colony was measured (maximum diameter and height) and its status (healthy, diseased or injured) was recorded. Populations in recovery were defined by a dominance of small to medium-sized colonies in densities >1 colony per 10 m², together with 75% undamaged colonies, a low prevalence of diseases (<10%), and a low density of predators (0.25 snails per colony). Based on allozyme analysis of seven polymorphic loci in four populations (N = 30), a moderate to high-genetic connectivity among these populations (F _ST = 0.048) was found with a predominance of sexual over asexual reproduction (N* : N = 1; N _go : N = 0.93–1). Both ecological and molecular data support a good prognosis for the recovery of this species in Los Roques.