Melanopsin-expressing retinal ganglion cell loss and behavioral analysis in the Thy1-CFP-DBA/2J mouse model of glaucoma

In this study,the role of melanopsin-expressing retinal ganglion cells(mRGCs) in the glaucoma-induced depressive behavioral response pattern was investigated.The CFP-D2 transgenic glaucoma animal model from five age groups was used in this study.Immunohistochemical labeling,quantitative analysis of mRGC morphology,open field test(OFT),and statistical analysis were used.In comparison with C57 BL/6 mice,the age-matched CFP-D2 mice had significantly elevated intraocular pressure(IOP).We observed parallel morphological changes in the retina,including a reduction in the density of cyan fluorescent protein(CFP) expressing cells(cells mm 2 at 2 months of age,1309±26;14 months,878±30,P<0.001),mRGCs(2 months,48±3;14 months,19±4,P<0.001),Brn3b-expressing RGCs(2 months,1283±80;14 months,950±31,P<0.001),Brn-3b expressing mRGCs(5 months,50.17%±5.5%;14 months,12.61%±3.8%,P<0.001),and reduction in the dendritic field size of mRGCs(mm2 at 2 months,0.077±0.015;14 months,0.065±0.015,P<0.05).CFP-D2 mice had hyperactive locomotor activity patterns based on OFT findings of the total distance traveled,number of entries into the center,and time spent in the center of the testing apparatus.The glaucoma induced hyperactive response pattern could be associated with dysfunctional mRGCs,most likely Brn-3b-positive mRGCs in CFP-D2 mice.     

Effect of tempol and tempol plus catalase on intra-renal haemodynamics in spontaneously hypertensive stroke-prone (SHSP) and Wistar rats

Vasoconstriction within the renal medulla contributes to the development of hypertension. This study investigated the role of reactive oxygen species (ROS) in regulating renal medullary and cortical blood perfusion (MBP and CBP respectively) in both stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar rats. CBP and MBP were measured using a laser-Doppler flow meter before and after intra-renal infusion of tempol, the superoxide dismutase (SOD) mimetic or tempol plus catalase, the hydrogen peroxide-degrading enzyme. Tempol infusion significantly elevated blood perfusion within the renal medulla (MBP) in both SHRSP (by 43 ± 7%, P < 0.001) and Wistar rats (by 17 ± 2%, P < 0.05) but the magnitude of the increase was significantly greater in the SHRSP (P < 0.01). When the enzyme catalase and tempol were co-infused, MBP was again significantly increased in SHRSP (by 57 ± 6%, P < 0.001) and Wistar rats (by 33 ± 6%, P < 0.001), with a significantly greater increase in perfusion being induced in the SHRSP relative to the Wistar rats (P < 0.01). Notably, this increase was significantly greater than in those animals infused with tempol alone (P < 0.01). These results suggest that ROS plays a proportionally greater role in reducing renal vascular compliance, particularly within the renal medulla, in normotensive and hypertensive animals, with effects being greater in the hypertensive animals. This supports the hypothesis that SHRSP renal vasculature might be subjected to elevated level of oxidative stress relative to normotensive animals.     

Proinflammatory conditions promote hepatocellular carcinoma onset and progression via activation of Wnt and EGFR signaling pathways

The aim of the current study was to investigate how proinflammatory conditions affect growth and progression of hepatocellular carcinoma. Human hepatoma cell lines were treated with lipopolysaccharide (LPS) or cyclooxygenase-2 inhibitor, Celecoxib, and in vitro proliferation, apoptosis, and cell cycle progression were assessed. This was followed up with in vivo xenograft assays to monitor tumor growth and metastatic progression under different treatment conditions. While LPS induced cell proliferation, Celecoxib induced apoptosis. Flow cytometry analysis demonstrated that S-phase cell count in LPS group was higher than control group (41.9 ± 3.2 vs 30.6 ± 0.1 %, respectively), whereas G0/G1-phase cells were significantly higher in the Celecoxib group in comparison with the control group (69.6 ± 5.0 vs 50.4 ± 1.6 %, respectively) (p < 0.05). Immunoblot analyses showed induction of epidermal growth factor receptor expression and induction and nuclear accumulation of Wnt/β-catenin and p65 in LPS group. Xenograft assays showed that LPS treatment induced comparatively large, rapidly growing tumors (2,702 ± 572 mm³) that metastasized to lungs, whereas Celecoxib treatment alone (1,008 ± 296 mm³) or in combination with LPS (1,303 ± 283 mm³) suppressed tumor growth in comparison to control groups (2,072 ± 456 mm³) (n = 5; p < 0.05). Inflammation can thus promote hepatoma cell proliferation and growth, and enhance the invasion and metastatic ability of hepatocarcinoma cells through inducing tumor angiogenesis, which in turn may be related to the activation of Wnt/β-catenin and EGFR signaling pathways.     

Effects of streptozotocin-induced long-term diabetes on parietal cell function and morphology in rats

Gastric pathology is a common complication in diabetes mellitus. The aim of the study was to evaluate the functions and morphological changes of the parietal cells of the rat stomach after streptozotocin-induced diabetes. Diabetes mellitus was induced in Wistar rats by a single intraperitoneal injection of streptozotocin (60 mg/kg body weight). The rats were weighed weekly and sacrificed after 6 months. The glandular portion of the stomach was removed and processed for H⁺-K⁺-ATPase immunohistochemistry and light and electron microscopy studies. Acid secretion was measured in vivo. After 6 months of diabetes, the mean weight of the rats was significantly lower (P < 0.001) compared to control. The mean weight of the stomach to body weight percentage increased significantly (P < 0.001) compared to control. The blood glucose level in diabetic rats was significantly higher (P < 0.001) than in normal control. Diabetic rats showed significant (P < 0.001) decrease in basal and stimulated acid secretion when compared to control. Electron micrographs of the parietal cells of glandular stomach of diabetic rats revealed significant (P < 0.0002) reduction in the number of mitochondria and a small though not significant increase in the number of canaliculi in the parietal cells compared with normal. Immunohistochemistry showed reduced H⁺-K⁺-ATPase (P < 0.00001) compared to control. Long-term diabetes induces morphological as well as functional changes in gastric parietal cells. The decrease in the number of mitochondria accompanied by reduced in H⁺-K⁺-ATPase in parietal cells may explain the reduced acid secretion observed in diabetics.     

Cysteine protects freshly isolated cardiomyocytes against oxidative stress by stimulating glutathione peroxidase

Cysteine has been implicated in myocardial protection, although this is controversial and constrained by limited knowledge about the effects of cysteine at the cellular level. This study tested the hypothesis that a physiologically relevant dose of l-cysteine could be safely loaded into isolated cardiomyocytes leading to improved protection against oxidative stress. Freshly isolated adult rat ventricular cardiomyocytes were incubated for 2 h at 37°C with (cysteine incubated) or without (control) 0.5 mM cysteine prior to washing and suspension in fresh cysteine-free media. Cysteine incubated cells had higher intracellular cysteine levels compared to controls (9.6 ± 0.78 vs. 6.5 ± 0.65 nmol/mg protein, P < 0.02, n = 6 ± SE). Cell homeostasis indicators were similar in the two groups. Cysteine incubated cells had significantly higher glutathione peroxidase (GPx) activity (1.11 ± 0.23 vs. 0.54 ± 0.1 U/mg protein, P < 0.05, n = 5 ± SE) and significantly greater expression of GPx-1 (5.01 ± 0.48 vs. 3.01 ± 0.25 OD units/mm², P < 0.05, n = 4 ± SE) compared to controls. Upon exposure to H₂O₂, cysteine incubated cells generated fewer reactive oxygen species and took longer to show contractile changes and undergo hypercontracture. However, when cells were exposed to H₂O₂ in the presence of 0.05 mM of the GPx inhibitor mercaptosuccinic acid, this increased the control cells’ susceptibility to H₂O₂ and completely abolished the cysteine mediated protection. These results suggest a new role for cysteine in myocardial protection involving stimulation of glutathione peroxidase.     

Size at maturity of Mediterranean marine fishes

In this review we collected data on the length at maturity (L_m) and maximum reported total length (L_max) of 565 Mediterranean marine fish stocks, representing 150 species, 68 families, 24 orders and 3 classes. Overall, L_m ranged from 2 cm, for the males of the toothcarp Aphanius fasciatus, to 350 cm, for the females of the bluntnose sixgill shark Hexanchus griseus. L_m was positively linearly related with L_max for Actinopterygii (logL_m = ?0.123 + 0.92 × logL_max; r ² = 0.87, n = 471, P < 0.001) and Elasmobranchii (logL_m = ?0.008 + 0.922 × logL_max; r ² = 0.90, n = 92, P < 0.001) with the two slopes being significantly different (ANCOVA: F = 2,904, P < 0.001). The reproductive load (L_m/L_max) ranged between 0.23 (sand steenbras Lithognathus mormyrus) and 0.94 (angular roughshark Oxynotus centrina and thornback ray Raja clavata). The mean L_m/L_max was significantly (ANOVA, F = 34.14, P < 0.001) lower for Actinopterygii (mean = 0.59, SD = 0.122, n = 471) compared to Elasmobranchii (mean = 0.70, SD = 0.132, n = 92) and Holocephali (mean = 0.77, SD = 0.077, n = 2). The L_m/L_max was significantly (ANOVA, F = 43.80, P < 0.001) higher for species providing some form of parental care, i.e. guarders, bearers, nesters (mean L_m/L_max ± SD = 0.68 ± 0.141, n = 111) compared to non-guarders (mean L_m/L_max ± SD = 0.59 ± 0.123, n = 454). The mean L_m/L_max displayed a remarkable constancy with longitude (northern and southern Mediterranean coastline: ANOVA, F = 0.01, P = 0.93), latitude (western, central and eastern regions: ANOVA, F = 1.25, P = 0.29) and habitat (ANOVA, F = 0.85, P = 0.51).     

Decreased Serum Selenium Levels are Correlated with Diminished Coronary Flow Reserve Among Hemodialysis Patients

Cardiovascular diseases are the main reason of high mortality among hemodialysis patients. Decreased serum selenium levels may have a role in accelerated atherosclerosis in this patient group. The hypothesis of this study was to show a correlation between decreased serum selenium levels and coronary flow reserve as an indicator of endothelial dysfunction and atherosclerosis in HD patients. Seventy-one chronic hemodialysis patients and age 65 and sex-matched healthy controls were included in the study. Plasma selenium levels were measured by spectrophotometry, and coronary flow reserve was assessed by transthoracic Doppler echocardiography. Serum selenium levels (34.16?±?6.15 ng/ml vs. 52.4?±?5.51 ng/ml, P?<?0.001) and coronary flow reserve values (1.73?±?0.11 vs. 2.32?±?0.28, P?<?0.001) were significantly lower in hemodialysis patients compared with controls, respectively. There was a significant positive correlation between coronary flow reserve and serum levels of selenium (r?=?0.676, P?<?0.001). A linear regression analysis showed that serum levels of selenium were independently and positively correlated with coronary flow reserve (regression coefficient?=?0.650, P?<?0.05). This study was the first to show a positive and independent correlation between decreased selenium levels and diminished coronary flow reserve as an indicator of endothelial dysfunction and atherosclerosis in hemodialysis patients. Our data suggest that decreased serum selenium levels may facilitate the development of endothelial dysfunction and disruption of coronary flow reserve which occur before the development of overt atherosclerosis.     

Reproductive biology of the invasive Asian freshwater leech Barbronia weberi (Blanchard, 1897)

Barbronia weberi were collected from Hydrilla verticillata purchased from aquarium suppliers in Melbourne, Australia and additional B. weberi were obtained from aquatic plants in Canberra, ACT, Australia. Adult leeches fed Tubifex sp. ad libitum successfully reproduced under laboratory conditions (21 ± 5?°C ). Reproductively mature B. weberi produced cocoons every week for up to three months at which time the adults began to senesce. Cocoons contained one to five (barx=2.41±0.78 SD, N=58) eggs with juveniles leaving the cocoon within 27 ± 3.4 SD days (N=13) of cocoon production. Juvenile B. weberi reached reproductive maturity within four months (N=7) of hatching and had a maximum growth rate of 1.10 mm² d^?1 once they left the cocoon at around 30 days. Individual egg volume (r=0.63, scaling exponent = 1.21) and cocoon volume (r=0.65, scaling exponent = 1.24) showed a significant (P<0.001) and nearly isometric relationship when scaled with maternal body size. Because this species can grow rapidly and produce a large number of eggs over a short period of time and can piggyback with plant species and travel through the aquarium trade, there is potential for B. weberi to rapidly invade new localities.     

The Effect of Enriched Milk with Selenium and Vitamin E on Growth Rate, Hematology, Some Blood Biochemical Factors, and Immunoglobulins of Newborn Goat Kids

Thirty male and female (n?=?15 for each one) Markhoz newborn goat kids (aged 7?±?3 days) were distributed in a randomized block design in a 2?×?2?+?1 factorial arrangement: two levels of sodium selenite as a source of selenium (0.2 or 0.3 ppm Se), two levels of α-tocopherol acetate as a source of vitamin E (150 or 200 IU Vit E), and one control treatment with six repetitions per treatment (each replicate included three male and three female kids). Animals were fed daily by Se-Vit E-enriched milk (Se-Vit E treatments) or non-enriched milk (control treatment). Growth rate, hematology, and serum biological parameters were measured. The levels of serum albumin (P?<?0.01), serum globulin (P?<?0.05), total serum protein levels (P?<?0.01), erythrocyte counts (RBC) (P?<?0.001), hemoglobin (P?<?0.001), hematocrit (P?<?0.001), leukocyte counts (WBC) (P?<?0.001), IgA (P?<?0.05), IgG (P?<?0.01), and IgM (P?<?0.01) significantly differed among treatments, while no significant differences were observed for calcium, lymphocyte, neutrophil average daily gain and body weight among treatments. Kids feeding by enriched milk with 0.3 ppm Se and 200 IU Vit E had significantly higher serum total protein, globulin, RBC, IgA, IgG, and IgM compared to control and those fed by enriched milk to 0.2 ppm Se and 200 IU Vit E had significantly higher WBC counts.     

Is high prevalence of vitamin D deficiency a correlate for attention deficit hyperactivity disorder?

The aim of the study was to determine the association between vitamin D and attention deficit hyperactivity disorder (ADHD), and difference in the level of vitamin D in ADHD children and control. This a case–control study carried out in school health and primary health care clinics. A total of 1,331 children and adolescents who were diagnosed with ADHD based on clinical criteria and standardized questionnaires were enrolled in this study and were matched with 1,331 controls, aged 5–18 years old. Data on body mass index (BMI), clinical biochemistry variables including serum 25-hydroxyvitamin D were collected. The study found significant association between ADHD and vitamin D deficiency after adjusting for BMI and sex (adj. OR 1.54; 95 % CI 1.32–1.81; P < 0.001). Majority of the ADHD children were in the age group 5–10 years (40.7 %), followed by 11–13 years (38.4 %). The proportion of BMI <85th percentile was significantly over represented in ADHD group as compared to healthy control (87.8 vs. 83 %; P < 0.001, respectively), while on the other hand, BMI >95th percentile was over represented in the control than ADHD group (7.6 vs. 4.6 %; P < 0.001, respectively). Mean values of vitamin D (ng/mL) were significantly lower in ADHD children (16.6 ± 7.8) than in healthy children (23.5 ± 9.0) (P < 0.001). There was significant correlation between vitamin D deficiency and age (r = ?0.191, P = 0.001); calcium (r = 0.272, P = 0.001); phosphorous (r = 0.284, P = 0.001); magnesium (r = 0.292, P = 0.001); and BMI (r = 0.498, P = 0.001) in ADHD children. The vitamin D deficiency was higher in ADHD children compared to healthy children.     

A role for membrane transport in modulation of intramuscular free glutamine turnover in streptozotocin diabetic rats

We wished to examine the effects of diabetes on muscle glutamine kinetics. Accordingly, female Wistar rats (200 g) were made diabetic by a single injection of streptozotocin (85 mg/kg) and studied 4 days later; control rats received saline. In diabetic rats, glutamine concentration of gastrocnemius muscle was 33% less than in control rats: 2.60 ± 0.06 μmol/g vs. 3.84 ± 0.13 μmol/g (P < 0.001). In gastrocnemius muscle, glutamine synthetase activity (V_max) was unaltered by diabetes (approx. 235 nmol/min per g) but glutaminase V_max increased from 146 ± 29 to 401 ± 94 nmol/min per g; substrate K_m values of neither enzyme were affected by diabetes. Net glutamine efflux (AZ concentration difference × blood flow) from hindlimbs of diabetic rats in vivo was greater than control values (?30.0 ± 3.2 vs. ?1.9 ± 2.6 nmol/min per g (P < 0.001) and hindlimb NH₃ uptake was concomitantly greater (about 27 nmol/min per g). The glutamine transport capacity (V_max) of the Na-dependent System N^m in perfused hindlimb muscle was 29% lower in diabetic rats than in controls (820 ± 50 vs. 1160 ± 80 nmol/min per g (P < 0.01)), but transporter K_m was the same in both groups (9.2 ± 0.5 nM). The difference between inward and net glutamine fluxes indicated that glutamine efflux in perfused hindlimbs was stimulated in diabetes at physiological perfusate glutamine (0.5 mM); ammonia (1 mM in perfusate) had little effect on net glutamine flux in control and diabetic muscles. In Intramuscular Na⁺ was 26% greater in diabetic (13.2 μmol/g) than control muscle, but muscle K⁺ (100 μmol/g) was similar. The accelerated rate of glutamine release from skeletal muscle and the lower muscle free glutamine concentration observed in diabetes may result from a combination of; (i), a diminished Na⁺ electrochemical gradient (i.e., the net driving force for glutamine accrual in muscle falls); (ii), a faster turnover of glutamine in muscle and (iii), an increased V_max/K_m for sarcolemmal glutamine efflux.     

Circulating microparticles in patients with coronary heart disease and its correlation with interleukin-6 and C-reactive protein

Microparticles (MPs) are vesicles released from activated or apoptotic cells. MP derive from various cells, most notably platelets, but also leucocytes, lymphocytes, erythrocytes, and endothelial cells. The aim of this study was to investigate endothelial MP (EMP), platelet MP (PMP), lymphocyte MP and monocyte MP and TF-positive MPs (TF⁺ MPs) in patients with coronary heart disease (CHD), and to evaluate the correlation of these MPs with Interleukin-6 (IL-6) and C-reactive protein (CRP). Different cell-derived MPs and TF⁺ MPs were analyzed by flow cytometry in 40 patients with myocardial infarction (MI), 30 unstable angina (UA), 20 stable angina (SA) and 20 healthy individuals, and IL-6 and CRP were determined by ELISA and special protein analyzer, respectively. Compared with SA and control, EMP and PMP was significantly elevated in MI and UA (P < 0.001), and TF⁺ MPs was significantly elevated in MI and UA (P < 0.001). EMP and PMP correlated with IL-6 (r = 0.822, P < 0.001 and r = 0.567, P < 0.001; respectively) or CRP level (r = 0.597, P < 0.001 and r = 0.66, P < 0.001; respectively). Different cell-derived MPs in CHD may indicate the different pathophysiological changes in vessels, and MPs may both participate in the development of thrombosis and enhance the vascular inflammation.     

Oxidative Stress-Related Genes in Type 2 Diabetes: Association Analysis and Their Clinical Impact

Worldwide prevalence of diabetes mellitus motivates a number of association studies to be conducted throughout the world. Eleven polymorphisms from nine candidate genes in oxidative stress pathway have been analyzed in eastern Indian type 2 diabetic patients (n = 145) and healthy controls (n = 100). Different biochemical parameters were also analyzed for their association with the disease. Significant associations were observed for rs2070424 A>G SOD1 (OR 3.91, 95% CI 2.265–8.142, P < 0.001), rs854573 A>G PON1 (OR 3.415, 95% CI 2.116–5.512, P < 0.001), rs6954345 G>C PON2 (OR 3.208, 95% CI 2.071–4.969, P < 0.001), RAGE rs1800624 ?374 T>A (OR 3.58, 95% CI 2.218–5.766, P < 0.001), and NOS3 ?786 T>C (OR 3.75, 95% CI 2.225–6.666, P < 0.001). Haplotype containing two risk alleles of PON1 and PON2 genes was significantly associated with disease (OR 8.34, 95% CI 1.554–44.804, P < 0.002). Our results suggest that carriers of major and efficient alleles of oxidative stress genes are more likely to survive the comorbid complications and single copy of risk allele is sufficient for developing the disease.     

Effect of intramuscular baculovirus encoding mutant hypoxia-inducible factor 1-alpha on neovasculogenesis and ischemic muscle protection in rabbits with peripheral arterial disease

Background aimsPeripheral arterial disease (PAD) is a progressive, disabling ailment for which no effective treatment exists. Gene therapy-mediated neovascularization has emerged as a potentially useful strategy. We tested the angiogenic and arteriogenic efficacy and safety of a baculovirus (BV) encoding mutant, oxygen-resistant hypoxia-inducible factor 1-alpha (mHIF-1α), in rabbits with PAD.MethodsAfter assessing the transfection efficiency of the BV.mHIF-1α vector and its tubulogenesis potential in vitro, we randomized rabbits with experimental PAD to receive 1 × 10⁹ copies of BV.mHIF-1α or BV.null (n = 6 per group) 7 days after surgery. Two weeks post-treatment, collateralization (digital angiography) and capillary and arteriolar densities (immunohistochemistry) were measured in the posterior limbs. Ischemic damage was evaluated in adductor and gastrocnemius muscle samples. Tracking of viral DNA in injected zones and remote tissues at different time points was performed in additional rabbits using a BV encoding GFP.ResultsAngiographically visible collaterals were more numerous in BV.mHIF-1α-treated rabbits (8.12 ± 0.42 vs 6.13 ± 1.15 collaterals/cm², P < 0.05). The same occurred with arteriolar (27.9 ± 7.0 vs 15.3 ± 4.0 arterioles/mm²) and capillary (341.8 ± 109.9 vs 208.8 ± 87.7 capillaries/mm², P < 0.05) densities. BV.mHIF-1α-treated rabbits displayed less ischemic muscle damage than BV.null-treated animals. Viral DNA and GFP mRNA were detectable only at 3 and 7 days after injection in hind limbs. Neither the virus nor GFP mRNA was detected in remote tissues.ConclusionsIn rabbits with PAD, BV.mHIF-1α induced neovascularization and reduced ischemic damage, exhibiting a good safety profile at 14 days post-treatment. Complementary studies to evaluate its potential usefulness in the clinic are needed.     

The Degree of Resistance of Erythrocyte Membrane Cytoskeletal Proteins to Supra-Physiologic Concentrations of Calcium: An In Vitro Study

Calcium is a key regulator of cell dynamics. Dysregulation of its cytosolic concentration is implicated in the pathophysiology of several diseases. This study aimed to assess the effects of calcium on the network of membrane cytoskeletal proteins. Erythrocyte membranes were obtained from eight healthy donors and incubated with 250 µM and 1.25 mM calcium solutions. Membrane cytoskeletal proteins were quantified using SDS-PAGE at baseline and after 3 and 5 days of incubation. Supra-physiologic concentrations of calcium (1.25 mM) induced a significant proteolysis in membrane cytoskeletal proteins, compared with magnesium (p < 0.001). Actin exhibited the highest sensitivity to calcium-induced proteolysis (6.8 ± 0.3 vs. 5.3 ± 0.6, p < 0.001), while spectrin (39.9 ± 1.0 vs. 40.3 ± 2.0, p = 0.393) and band-6 (6.3 ± 0.3 vs. 6.8 ± 0.8, p = 0.191) were more resistant to proteolysis after incubation with calcium in the range of endoplasmic reticulum concentrations (250 µM). Aggregation of membrane cytoskeletal proteins was determined after centrifugation and was significantly higher after incubation with calcium ions compared with control, EDTA and magnesium solutions (p < 0.001). In a supra-physiologic range of 1.25–10 mM of calcium ions, there was a nearly perfect linear relationship between calcium concentration and aggregation of erythrocyte membrane cytoskeletal proteins (R ² = 0.971, p < 0.001). Our observation suggests a strong interaction between calcium ions and membrane cytoskeletal network. Cumulative effects of disrupted calcium homeostasis on cytoskeletal proteins need to be further investigated at extended periods of time in disease states.     

Effects of extracellular calcium on viability and osteogenic differentiation of bone marrow stromal cells in vitro

Bone marrow stromal cells (BMSCs) have been extensively used for tissue engineering. However, the effect of Ca²⁺ on the viability and osteogenic differentiation of BMSCs has yet to be evaluated. To determine the dose-dependent effect of Ca²⁺ on viability and osteogenesis of BMSCs in vitro, BMSCs were cultured in calcium-free DMEM medium supplemented with various concentrations of Ca²⁺ (0, 1, 2, 3, 4, and 5 mM) from calcium citrate. Cell viability was analyzed by MTT assay and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) assay, Von Kossa staining, and real-time PCR. Ca²⁺ stimulated BMSCs viability in a dose-dependent manner. At slightly higher concentrations (4 and 5 mM) in the culture, Ca²⁺ significantly inhibited the activity of ALP on days 7 and 14 (P < 0.01 or P < 0.05), significantly suppressed collagen synthesis (P < 0.01 or P < 0.05), and significantly elevated calcium deposition (P < 0.01) and mRNA levels of osteocalcin (P < 0.01 or P < 0.05) and osteopontin (P < 0.01 or P < 0.05). Therefore, elevated concentrations of extracellular calcium may promote cell viability and late-stage osteogenic differentiation, but may suppress early-stage osteogenic differentiation in BMSCs.     

Comparing soil carbon sequestration in coastal freshwater wetlands with various geomorphic features and plant communities in Veracruz,Mexico

Background and aims

Wetlands are important carbon sinks across the planet. However, soil carbon sequestration in tropical freshwater wetlands has been studied less than its counterpart in temperate wetlands. We compared carbon stocks and carbon sequestration in freshwater wetlands with various geomorphic features (estuarine, perilacustrine and depressional) and various plant communities (marshes and swamps) on the tropical coastal plain of the Gulf of Mexico in the state of Veracruz, Mexico. These swamps are dominated by Ficus insipida, Pachira aquatic and Annona glabra and the marshes by Typha domingensis, Thalia geniculata, Cyperus giganteus, and Pontederia sagittata.

Methods

The soil carbon concentration and bulk density were measured every 2 cm along 80 cm soil profiles in five swamps and five marshes. Short-term sediment accretion rates were measured during a year using horizontal makers in three of the five swamps and marshes, the carbon sequestration was calculated using the accretion rates, and the bulk density and the percentage of organic carbon in the surficial layer was measured.

Results

The average carbon concentration ranged from 50 to 150 gC kg^?1 in the marshes and 50 to 225 gC kg^?1 in the swamps. When the wetlands were grouped according to their geomorphic features, no significant differences in the carbon stock (P?=?0.095) were found (estuarine (25.50?±?2.26 kgC m^?2), perilacustrine (28.33?±?2.74 kgC m^?2) and depressional wetlands (34.93?±?4.56 kgC m^?2)). However, the carbon stock was significantly higher (P?=?0.030) in the swamps (34.96?±?1.3 kgC m^?2) than in the marshes (25.85?±?1.19 kgC m^?2). The average sediment accretion rates were 1.55?±?0.09 cm yr^?1 in the swamps and 0.84?±?0.02 cm yr^?1 in the marshes with significant differences (P?=?0.040). The rate of carbon sequestration was higher (P?=?0.001) in swamp soils (0.92?±?0.12 kgC m^?2 yr^?1) than marsh soils (0.31?±?0.08 kgC m^?2 yr^?1). Differences in the rates of carbon sequestration associated with geomorphic features were found between the swamp ecosystems (P?<?0.05); i.e., higher values were found in the swamps than in the marshes in perilacustrine and estuarine wetlands (P?<?0.05). However, no significant differences (P?=?0.324) in carbon sequestration rates were found between the marsh and swamp areas of the depressional site.

Conclusions

Swamp soils are more important contributors to the carbon stock and sequestration than are marsh soils, resulting in a reduction in global warming, which suggests that the plant community is an important factor that needs to be considered in global carbon budgets and projects of restoration and conservation of wetlands.     

Thermoregulatory plasticity in free-ranging vervet monkeys,Chlorocebus pygerythrus

We used implanted miniature data loggers to obtain the first measurements of body temperature from a free-ranging anthropoid primate. Vervet monkeys (Chlorocebus pygerythrus) living in a highly seasonal, semi-arid environment maintained a lower mean 24-h body temperature in winter (34.6 ± 0.5 °C) than in summer (36.2 ± 0.1 °C), and demonstrated increased heterothermy (as indexed by the 24-h amplitude of their body temperature rhythm) in response to proximal environmental stressors. The mean 24-h amplitude of the body temperature rhythm in summer (2.5 ± 0.1 °C) was lower than that in winter (3.2 ± 0.4 °C), with the highest amplitude for an individual monkey (5.6 °C) recorded in winter. The higher amplitude of the body temperature rhythm in winter was a consequence primarily of lower 24-h minimum body temperatures during the nocturnal phase, when monkeys were inactive. These low minimum body temperatures were associated with low black globe temperature (GLMM, β = 0.046, P < 0.001), short photoperiod (β = 0.010, P < 0.001) and low rainfall over the previous 2 months, which we used as a proxy for food availability (β = 0.001, P < 0.001). Despite the lower average winter minimum body temperatures, there was no change in the lower modal body temperature between winter and summer. Therefore, unlike the regulated physiological adjustments proposed for torpor or hibernation, these minimum winter body temperatures did not appear to reflect a regulated reduction in body temperature. The thermoregulatory plasticity nevertheless may have fitness benefits for vervet monkeys.     

Preliminary findings on the influence of FTO rs9939609 and MC4R rs17782313 polymorphisms on resting energy expenditure,leptin and thyrotropin levels in obese non-morbid premenopausal women

Given that leptin, ghrelin and thyrotropin play a major role in the regulation of resting energy expenditure (REE) and that the FTO rs9939609 and the MC4R rs17782313 polymorphisms have been proposed to affect energy homeostasis, we hypothesized that both polymorphisms are associated with REE and that these relationships can be mediated by leptin, ghrelin and thyrotropin in obesity. Therefore, the present study aimed to examine the relationships between FTO rs9939609 and the MC4R rs17782313 with REE, leptin, ghrelin and thyrotropin levels in obese women. The study comprised 77 obese (body mass index 34.0?±?2.8 kg/m²) women (age 36.7?±?7 years). We measured body composition by dual-energy X-ray absorptiometry and REE by indirect calorimetry. We analysed fasting leptin, ghrelin and thyrotropin levels and the ratio of leptin to fat mass was calculated. Genotype distributions of the polymorphisms did not deviate from Hardy–Weinberg expectations (P values >0.2). Women carrying the A allele of the FTO rs9939609 had lower REE (1,580?±?22 vs. 1,739?±?35 kcal/day, P?<?0.001) and higher leptin to fat mass ratio (1.33?±?0.05 vs. 1.13?±?0.08 ng/ml kg, P?<?0.05) and thyrotropin levels (1.93?±?0.10 vs. 1.53?±?0.16 μU/ml, P?<?0.05) regardless of age and body mass index. We found no significant influence of the MC4R rs17782313 on energy metabolism or biochemical variables. Our findings confirm that the A allele of the FTO rs9939609 is associated with lower REE and increased plasma leptin levels. We also found an association between the FTO rs9939609 and thyrotropin, suggesting the possible influence of FTO in the hypothalamic–pituitary–thyroid axis as a potential mechanism of the increased adiposity.     

Proliferation and apoptosis in subcutaneous adipose tissue of lactating cows with different genetic merit for milk yield

The aim of this study was to investigate the adipocyte size and fate in subcutaneous fat (scAT) of cows diverging for genetic merit at mid lactation stage, when anabolic activity increases and animals are in a state of positive energy balance. Twenty mid lactation cows (180 ± 20 days in milk) grouped according to the Estimated Breeding Values (EBV) for milk yield in plus (EBVp) and minus (EBVm) variants were selected. Average of adipocytes area, proliferation and apoptotic labelling index as well as DLK-1 expression, a marker of pre-adipocytes, were immunohistochemically evaluated in scAT biopsies. In EBVp cows, the BCS was lower (P < 0.01) whereas milk yield, protein, fat yield (P < 0.001) and plasma free fatty acid concentration (P < 0.05) were higher. The scAT of EBVp cows showed a significantly (P < 0.001) higher frequency between 500 and 3000 μm² classes in comparison to EBVm cows, that showed a significantly (P < 0.01) higher apoptotic labeling index. The immunohistochemical reaction showed DLK-1 positivity in scAT of EBVp cows. Taking together, the data indicate a link between milk yield genetic merit of cows, scAT morphology and function, suggesting greater dynamics and metabolic flexibility in EBVp cows.