Recovery Responses of Freshwater Phytoplankton Assemblage After the Disappearance of Metal Toxicity in Semi-Continuous Cultures

The present study demonstrates the recovery of phytoplankton assemblage from metal stress. Phytoplankton assemblage consisting of different freshwater algal species isolated from a tropical pond was exposed to sublethal concentrations of Cu and Zn for 25 days in semi-continuous culture (Toxicity phase). Subsequently, algal assemblage grown in the toxicity phase were transferred to the fresh culture medium without elevated levels of the test metals for 25 days in semi-continuous culture (Recovery phase). We monitored the total biovolume of each algal species during the toxicity and recovery phases. The members of Cyanophyta were most sensitive against metal toxicity, followed by the members of Bascillariophyta. However, the members of Chlorophyta showed relatively lesser sensitivity against test metals. Among chlorophytes, Scendesmus opolinensis and Cosmarium bioculatum were tolerant to both the test metals. Metal-stressed algal species showed recovery after transferring to the basal medium depending on the concentration of metals during the toxicity phase. The members of Cyanophyta were unable to recover from metal stress. However, members of Chlorophyta showed faster recovery than others from Zn stress, and the members of Bascillariophyta showed quicker recovery from Cu stress. The differential abilities of various algal species to recover from metal stress perhaps depend on their ability to counterbalance metal toxicity. Further, research is warranted to characterize the differential ability of various algal groups to recover from metal stress. The present findings would help understand the efficiency of different algal groups to restore their position within the freshwater algal community after the disappearance for metal stress.

Temporal Evolution of the Gravitaxis of Euglena gracilis from a Single Cell

Gravitaxis is one of the most important issues in the growth of microalgae in the water column; it determines how easily cells receive sunlight with a comfortable intensity that is below the damaging threshold. We quantitatively investigated and analyzed the gravitaxis and cell multiplication of Euglena gracilis using vertically placed microchambers containing a single cell. A temporal change in gravitaxis and cell multiplication was observed after transferring the cells to fresh culture medium for 9 days. We performed 29 individual experiments with 2.5 × 2.5 × 0.1 mm square microchambers and found that the cells showed positive, negative, and moderate gravitaxis in 8, 7, and 14 cases, respectively, after transferring to fresh culture medium. A common trend was observed for the temporal change in gravitaxis for the eight initially positive gravitaxis cases. The cells with initially positive gravitaxis showed a higher rate of cell multiplication than those with initially negative gravitaxis. We also discussed the gravitaxis mechanism of E. gracilis from the observed trend of gravitaxis change and swimming traces. In addition, bioconvection in a larger and thicker chamber was investigated at a millimeter scale and visualized.

Recovery Responses of Freshwater Phytoplankton Assemblage After the Disappearance of Metal Toxicity in Semi-Continuous Cultures

The present study demonstrates the recovery of phytoplankton assemblage from metal stress. Phytoplankton assemblage consisting of different freshwater algal species isolated from a tropical pond was exposed to sublethal concentrations of Cu and Zn for 25 days in semi-continuous culture (Toxicity phase). Subsequently, algal assemblage grown in the toxicity phase were transferred to the fresh culture medium without elevated levels of the test metals for 25 days in semi-continuous culture (Recovery phase). We monitored the total biovolume of each algal species during the toxicity and recovery phases. The members of Cyanophyta were most sensitive against metal toxicity, followed by the members of Bascillariophyta. However, the members of Chlorophyta showed relatively lesser sensitivity against test metals. Among chlorophytes, Scendesmus opolinensis and Cosmarium bioculatum were tolerant to both the test metals. Metal-stressed algal species showed recovery after transferring to the basal medium depending on the concentration of metals during the toxicity phase. The members of Cyanophyta were unable to recover from metal stress. However, members of Chlorophyta showed faster recovery than others from Zn stress, and the members of Bascillariophyta showed quicker recovery from Cu stress. The differential abilities of various algal species to recover from metal stress perhaps depend on their ability to counterbalance metal toxicity. Further, research is warranted to characterize the differential ability of various algal groups to recover from metal stress. The present findings would help understand the efficiency of different algal groups to restore their position within the freshwater algal community after the disappearance for metal stress.

Antagonistic effect of volatile and non-volatile compounds from Streptomyces strains on cultures of several phytopathogenic fungi

Fungi and oomycetes are important plant pathogens that constantly attacked plants, thus compromising the production of foods worldwide. Streptomyces strains might be useful to control fungal pathogens by different mechanism. The in vitro antagonistic activity of non-volatile and volatile metabolites from four Streptomyces strains was evaluated over cultures of phytopathogenic fungi and oomycetes. The non-volatile compounds from Streptomyces strains significantly reduced (44.2 to 92.1%) the growth of aerial mycelium of pathogens. The volatile compounds (VOCs) from Streptomyces strains reduced both aerial mycelium (22.5 to 96.7%) and mycelium growing inside of culture medium (0.0 - 9.4%). The pathogens maintained their capacity to grow normally in fresh culture medium without antagonists after confrontations with antagonist VOCs. The analysis of VOCs by gas chromatography coupled to mass spectrometry revealed different kinds of VOCs included alcohols, aldehydes, ketones, esters, terpenes, terpenoids, thioethers, among others. The most abundant VOCs were trans-1,10-dimethyl-trans-9-decalol (geosmin), 2-methylisoborneol, 2-methyl-2-bornene, 1,4-dimethyladamantane, and 4-penten-1-ol, trifluoroacetate. The antipathogenic activity of nine pure VOCs that had been identified in cultures of the Streptomyces strains alone was evaluated in vitro against phytopathogenic fungi and oomycetes. Trans-2-hexenal was the most effective of these VOCs, inhibiting completely the growth of tested phytopathogens. The volatile and non-volatile compounds from Streptomyces strains effectively reduced the in vitro growth of phytopathogens and they might be used as biological control. Further studies are required to demonstrate this activity on open field conditions.

Can non-asbestiform amphibole fibers trigger carcinogenesis mechanisms?

<p>Minerals defined as asbestos include only the fibrous varieties (length > 5 µm, diameter < 3 µm and length/diameter ratio > 3:1) and asbestiform (high tensile strength or flexibility) of serpentine and amphibole.</p><p>However, there are also prismatic varieties of amphiboles, which despite the same chemical composition, are not classified as asbestos. Their geometric ratio would fall within the concept of fiber, but the minerals are not asbestiform.</p><p>Starting from a fairly contradictory context, the goal of this work was to analyze the variables inherent the morphological, but above all, the clastogenic effect determined by exposure of both asbestiform and non-asbestiform amphiboles.</p><p>The asbestiform fibers (F3), and the other three samples containing non-asbestiform amphiboles (P1, P2, P3) were tested in A549 cells line. Each sample of fiber was inoculated in A549 cells at a concentration of 100 µg/ml for 48h. Experiments were assayed in triplicate and repeated twice. To evaluate the micronuclei number for each sample the fixed cells were dropped onto clean microscope slides, stained and observed by optical microscopy at 100X.</p><p>Obtained results showed a statistically significant increase (P < 0.05) of micronuclei in F3 exposed cells when compared to negative controls. Similar results were reported when A549 cells were exposed with non-asbestiform amphiboles P2. No significant increase of micronucleated cells was observed after exposure of cells line at samples P1 and P3.</p><p>Moreover, to investigate the effect long-term triggered after 24h post fiber exposure of the medium inoculated cells were replaced with fresh culture medium and the cultures were grown for 72h. Albeit a prolonged contact of the F3 and P3 fibers resulted in a statistically significant increase (P < 0.01) of the micronuclei, no increase was reported for P2.</p><p>These results indicate that the contact of non-asbestiform amphiboles in vitro, can determine a genetic disorder, a necessary step in the cancer development. However, the kinematic of processes and the bearing of results are to be further clarified. For this purpose, the same starting materials are presently tested to determinate the transformation efficiency in no-tumoral cells and will be analyzed the different pathway involved in the etiopathogeneses of diseases trigger by inhalation of fiber.</p>

BACTERIAL AND FUNGAL CONTAMINANTS OF TISSUE-CULTURED 'LAKATAN' BANANA

This study aimed to characterize the bacterial and fungal contaminants of tissue-cultured 'Lakatan' banana (Musa acuminata) during initiation stage. This was conducted at the University of Southeastern Philippines, Tagum-Mabini Campus from October 2015 to February 2016. The sterilized banana explant was placed in a bottle of solidified nutrient agar medium. Detectable bacterial and fungal contaminants were isolated and sub-cultured into the fresh medium two to four days after incubation. The bacterial isolates were cultivated by streaking into fresh culture medium and incubated at 0 0 30-32 C for three days and fungi culture disk was transferred to fresh culture medium and incubated at 30-32 C for three to five days. Both bacterial and fungal contaminants were identified and characterized and assessed for extent of contamination. Results showed that the different contaminants occurred during the initiation stage of tissue-cultured 'Lakatan' banana meriplants were composed of Rhizopus sp., unidentified fungus and Gram-negative bacterium. Generally, 35% contamination was observed on this stage.

The H2S–Nrf2–Antioxidant Proteins Axis Protects Renal Tubular Epithelial Cells of the Native Hibernator Syrian Hamster from Reoxygenation-Induced Cell Death

During hibernation, repeated cycles of ischemia-reperfusion (I-R) leave vital organs without injury. Studying this phenomenon may reveal pathways applicable to improving outcomes in I-R injury-induced human diseases. We evaluated whether the H2S–nuclear factor erythroid 2-like 2 (Nrf2)–antioxidant proteins axis protects renal proximal tubular epithelial cells (RPTECs) of the native hibernator, the Syrian hamster, from reperfusion-induced cell death. To imitate I-R, the hamsters’, and control mice’s RPTECs were subjected to warm anoxia, washed, and then subjected to reoxygenation in fresh culture medium. Whenever required, the H2S-producing enzymes inhibitor aminooxyacetate or the lipid peroxidation inhibitor α-tocopherol were used. A handmade H2S detection methylene blue assay, a reactive oxygen species (ROS) detection kit, a LDH release cytotoxicity assay kit, and western blotting were used. Reoxygenation upregulated the H2S-producing enzymes cystathionine beta-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase in the hamster, but not in mouse RPTECs. As a result, H2S production increased only in the hamster RPTECs under reoxygenation conditions. Nrf2 expression followed the alterations of H2S production leading to an enhanced level of the antioxidant enzymes superoxide dismutase 3 and glutathione reductase, and anti-ferroptotic proteins ferritin H and cystine-glutamate antiporter. The upregulated antioxidant enzymes and anti-ferroptotic proteins controlled ROS production and rescued hamster RPTECs from reoxygenation-induced, lipid peroxidation-mediated cell death. In conclusion, in RPTECs of the native hibernator Syrian hamster, reoxygenation activates the H2S–Nrf2–antioxidant proteins axis, which rescues cells from reoxygenation-induced cell death. Further studies may reveal that the therapeutic activation of this axis in non-hibernating species, including humans, may be beneficial in I-R injury-induced diseases.

Adhesion of Sickle RBCs to Heme-Activated Endothelial Cells Correlates with Patient Clinical Phenotypes

Severe hemolysis and associated high levels of hemolytic biomarkers, including LDH and heme, are among major constituents of the pathophysiology of sickle cell disease (SCD). Elevated extracellular heme due to hemolysis overwhelms endogenous detoxification mechanisms and leads to oxidative stress, triggering systemic endothelium activation, vascular dysfunction, and end organ damage. To understand the role of red blood cells (RBC) in this process, we assessed sickle RBC adhesion to heme-activated endothelial cells utilizing an endothelialized microfluidic platform in a clinically diverse patient population. Human umbilical vein endothelial cells (HUVECs) were seeded into fibronectin (FN) functionalized microfluidic channels and incubated for 4 hours in a 37°C and 5% CO2 environment. Next, the confluent monolayers were loaded and incubated for 45 minutes with fresh culture medium or at one of two concentrations of heme solutions: (1) 20 μM that corresponds to physiological heme levels in SCD patients, and (2) 40 μM. SCD blood samples, collected from 8 patients (7 HbSS and 1 HbS/β0 thal; 3 males and 5 females), were centrifuged to remove the plasma and washed with PBS thrice prior to flow experiments. RBCs were re-suspended in culture medium at a hematocrit of 25%. A total volume of 15 µl RBC suspension was perfused into the microchannels followed by rinsing with fresh culture medium at 1 dyne/cm2, which corresponds to the typical shear stress value observed in post-capillary venules. The fully closed and hermetically sealed microfluidic system design ensured the stability of gas composition in the culture medium during the experiments. Endothelialized microchannels supported sickle RBC adhesion to non-treated, 20 µM heme-activated, and 40 µM heme-activated HUVECs (Fig. 1A, B, C). Adhesion results suggested that activation of HUVECs by heme mediated RBC adhesion in a concentration-dependent manner, with a significant difference observed at 40 µM (Fig. 1D, paired t-test, p<0.05). Notably, a heterogeneous heme-mediated adhesion profile was seen among patients. Sickle RBC adhesion to 20 μM heme-activated HUVECs was significantly increased in patients with higher LDH levels (Fig. 1E, p=0.024, ANOVA), higher absolute reticulocyte counts (Fig. 1F, p=0.002, ANOVA), and lower total hemoglobin (Fig. 1G, p=0.016, ANOVA), which are indicative of elevated hemolysis. All patients in the high adhesion group (>200 adherent RBCs) had elevations in serum LDH levels and in absolute reticulocyte counts. Moreover, patients with a recent transfusion had higher RBC adhesion to 40 µM heme-activated HUVECs compared to patients with no transfusion in the last 3 months (Fig. 1H, p<0.05, ANOVA). Here, we report a direct link between heme-driven endothelial activation and RBC adhesion in a patient-specific manner. In patients with a more severe clinical phenotype, as reflected in LDH, total hemoglobin, and absolute reticulocyte or recent blood transfusions, we found greater RBC adhesion to heme-activated HUVECs. These findings suggest that RBCs from those patients most likely to experience hemolysis in vivo may also be those RBCs most likely to adhere to heme-activated endothelium. Acknowledgments: This work was supported by grants #2013126 and #2015191 from the Doris Duke Charitable Foundation, National Heart Lung and Blood Institute R01HL133574, and National Science Foundation CAREER Award 1552782. Figure 1: Sickle RBC adhesion to heme-activated HUVECs and clinical associations. Sickle RBCs adherent to immobilized HUVECs in (A) non-activated, (B) 20 µM heme-activated, and (C) 40 µM heme-activated microchannels. The microscope images illustrate a small portion of the full microchannel surface. (D) RBC adhesion to HUVECs increased depending on the heme concentration, with a significant difference at the 40 µM level (paired t test, p<0.05). Patients with higher LDH (E) as well as absolute reticulocyte counts (F), and lower total hemoglobin (G) showed significantly greater RBC adhesion to 20 µM heme-activated HUVECs (ANOVA). (H) Furthermore, patients with a recent transfusion history displayed elevated RBC adhesion to 40 µM heme-activated HUVECs compared to non-transfused patients (ANOVA). The dashed rectangles indicate the normal clinical values for healthy individuals, while all patients had total hemoglobin levels lower than normal. Scale bars represent 30 µm. Figure 1 Figure 1. Disclosures Little: Hemex Health: Equity Ownership. Gurkan: Hemex Health: Employment, Equity Ownership.

Culture system and long-term storage of culture media in the in vitro production of bovine embryos

The optimum culture system for in vitro matured and fertilised oocytes still remains to be clarified. Culture media (CM) for mammalian embryos are routinely prepared fresh for use and preserved under refrigeration during one or two weeks. The purposes of this work were (1) to compare the efficiency of a synthetic oviduct fluid (SOF) with two different bovine serum albumin (BSA) concentrations (3 and 8 g/L) for the in vitro production of bovine blastocysts, (2) to test the effect of timing on adding fetal calf serum (FCS) to the SOF, and (3) to evaluate the effects on bovine embryo development of freezing and lyophilisation as procedures for preserving the SOF. Supplementation of SOF with 3 g/L BSA increased Day-7 blastocyst expansion rates (18.3 ± 1.6 vs. 14.4 ± 0.7; P < 0.05), although no differences in hatching rates were found. Addition of FCS to SOFaa (SOF with amino acids) medium supplemented with sodium citrate (SOFaaci) at 48 and at 72 h post-insemination (PI) allowed obtaining higher Day-6 embryo development rates than when FCS was added at 18 or 96 h PI (Day-6 morulae + blastocyst rate: 30.0 ± 1.1, 40.8 ± 1.1, 43.9 ± 2.3 and 39.3 ± 0.5 for FCS addition at 18, 48, 72 and 96 h, respectively). Hatching rates were significantly improved when serum was added at 72 h PI. Finally, both refrigeration and lyophilisation appeared as useful cryopreservation procedures for SOFaaci, although a significant loss of its ability to support embryo development, compared to the control fresh culture medium, was observed.

Tomato culture filtrate stimulates hatching and activity of Meloidogyne incognita juveniles

Hydroponic culture media were tested after growing tomato, okra, cucumber and bean for their effect on hatching and mobility of Meloidogyne incognita second-stage juveniles (J2) in laboratory experiments. Tomato and okra culture media increased the numbers of J2 that hatched as compared to those in water or fresh culture medium. The tomato culture medium increased hatching even in the presence of fosthiazate, an organophosphate nematicide, and at a concentration that inhibited hatching in the absence of tomato culture medium. Neither heat treatment of the tomato culture medium nor change of pH abolished its hatching stimulatory activity. When active J2 were incubated in the tomato culture media, the percentages of nematodes that became quiescent were lower than those of nematodes incubated in water or in fresh culture medium for 3 and 7 days in two trials. Moreover, the sigmoid movement of J2 was faster in the tomato culture medium than in water. Quiescent J2 became active more rapidly in culture media of tomato, cucumber and okra than in water or fresh culture medium. In experiments using soil containing quiescent J2, more J2 were extracted with the Baermann funnel method from soil treated with tomato culture medium than from soil treated with water. The results confirm that nematode stimulants, which may serve as a potential means in nematode control, exist in the plant culture media.