Effects of abamectin and deltamethrin to the foragers honeybee workers of Apis mellifera jemenatica (Hymenoptera: Apidae) under laboratory conditions

This study aimed at evaluating the toxicity of some insecticides (abamectin and deltamethrin) on the lethal time (LT₅₀) and midgut of foragers honeybee workers of Apis mellifera jemenatica were studied under laboratory conditions. The bees were provided with water, food, natural protein and sugar solution with insecticide (concentration: 2.50 ppm deltamethrin and 0.1 ppm abamectin). The control group was not treated with any kind of insecticides. The mortality was assessed at 1, 2, 4, 6, 12, 24, 48, and 72 hour (h) after insecticides treatment and period to calculate the value of lethal time (LT₅₀). But the samples the histology study of midgut collected after 24 h were conducted by Scanning Electron Microscope. The results showed the effects of insecticides on the current results show that abamectin has an adverse effect on honeybees, there is a clear impact on the lethal time (LT₅₀) was the abamectin faster in the death of honeybee workers compared to deltamethrin. Where have reached to abamectin (LT₅₀ = 21.026) h, deltamethrin (LT₅₀ = 72.011) h. However, abamectin also effects on cytotoxic midgut cells that may cause digestive disorders in the midgut, epithelial tissue is formed during morphological alterations when digestive cells die. The extends into the internal cavity, and at the top, there is epithelial cell striated border that has many holes and curves, abamectin seems to have crushed the layers of muscle. Through the current results can say abamectin most toxicity on honeybees colony health and vitality, especially foragers honeybee workers.     

Analysis of digestion of rice planthopper by Pardosa pseudoannulata based on CO-I gene

In order to systematically study the predatory behavior and digestion regularity of spiders, real-time fluorescence quantification PCR technique was used to detect the number of CO-I genes in Pardosa pseudoannulata after it preyed on rice planthoppers in different temperatures within different periods. At 28 °C, 0, 1, 2, 4, 8, 16, and 24 h after P. pseudoannulata preyed on rich planthopper, DNA was extracted from cephalothorax and abdomen of P. pseudoannulata. Routine PCR and real-time fluorescence PCR techniques were employed for CO-I gene amplification. The results show that: The prey liquid was temporarily stored in the sucking stomach of the spider head within 2 h after prey, and gradually transferred to the midgut of the abdomen with the prolongation of time. After 4 h, CO-I gene residues of rice planthopper in the cephalothorax gradually decreased. The CO-I gene of rice planthopper was basically transferred to the abdomen after 16 h. During 0–1 h, food contained in abdominal midgut and other digestive organs was very small, CO-I gene detection was not obvious. Over time, food entered into the midgut from the sucking stomach for digestion. During 2–4 h, CO-I gene amount increased, at 2–4 h, detected CO-I gene residue reached the peak; but rapidly declined after 8, 16, and 24 h, even it is still detectable. The results at different temperatures reveal that: As the temperature increased from 26 °C to 32 °C, CO-I gene residues of rich planthopper in cephalothorax and abdomen of P. pseudoannulata gradually decreased, which indicated that the digestion rate increased with the increase of temperature with some range. However, when the temperature continued to increase to 34 °C, the digestion rate decreased.     

Insecticidal and developmental inhibitory properties of monoterpenes on Culex pipiens L. (Diptera: Culicidae)

Twelve monoterpenes were evaluated for larvicidal and adulticidal activities towards Culex pipiens. Geraniol and cuminaldehyde were the most toxic monoterpenes to larvae, with LC₅₀ values of 38.6 and 38.9 mg/l after 24 h of treatment, respectively, whereas cuminaldehyde was the most potent compound after 48 h of treatment, followed by geraniol and thymol. In fumigant toxicity experiments, (R)-carvone and geraniol were the most toxic monoterpenes against the adults at all three tested concentrations and after both 24 and 48 h. When tested at sublethal concentrations (0.5 LC₅₀), (R)-carvone, (S)-limonene and cuminaldehyde decreased hatchability, pupation and adult emergence and induced high larval mortality. Our results suggest that geraniol, cuminaldehyde and (R)-carvone are promising toxicants against Culex pipiens and could be useful in the search for new natural insecticides.     

Entomotoxic properties of Dioclea violacea lectin and its effects on digestive enzymes of Anagasta kuehniella (Lepidoptera)

Entomotoxic plant lectins have been extensively studied in the past two decades, yet the exact mechanisms underlying their toxic effects remain unknown. This study investigated the effects of Dioclea violacea lectin (DVL) on larval development in Anagasta kuehniella. Chronic exposure of larvae (from neonates to the fourth instar) demonstrated that DVL interfered with larval growth, retarding development and decreasing larval mass without affecting survival. DVL decreased trypsin-like, chymotrypsin-like, and α-amylase activities and proved resistant to proteolysis by midgut proteases up to 24 h. Shorter exposures to dietary DVL had no effect on midgut enzyme activity. Feeding fourth-instar larvae with fluorescently-labeled DVL revealed lectin binding to the peritrophic membrane.     

Target and non-target effects of Foeniculum vulgare and Matricaria chamomilla combined extract on Culex pipiens mosquitoes

The present study focused on extracting green larvicides from extracts of the combination of Foeniculum vulgare and Matricaria chamomilla using different solvents of increasing polarity in a Soxhlet extractor and evaluating their ovicidal, larvicidal, and cytotoxic activities. The most promising among all tested extracts was hexane extract. The ovicidal activity of the hexane PH2 extract resulted in a significant (p < 0.05) decrease in egg hatchability from 95.00 ± 6.16% to 15 ± 9.04% at doses ranging from 62.5 to 500 µg/mL. The larval mortality with the hexane extract ranged from 13.33 ± 3.3% to 93.33 ± 3.3% at doses ranging from 31.25 to 250 µg/mL, respectively. The LC₅₀ and LC₉₀ values of the larvicidal activity of the hexane extract were estimated to be 148.3 and 242.17 µg/mL, respectively, after 24 h of exposure. Similarly, the LC₅₀ values after 48 and 72 h of exposure were 124.93 and 100.3 µg/mL, respectively, against the third instar of Cx. pipiens. PH2 treatment of larvae resulted in histopathological changes such as degenerated epithelial cells and destruction of microvilli on the epithelial cells. The PH2 extract achieved a dose-dependent decrease in the rate of cell survival. The IC₅₀ value of PH2-treated HUVECs was 192.07 µg/mL after 24 h of incubation. The cells showed changes in cellular and nuclear morphology. In conclusion, the hexane extract of PH2 could be used in mosquito management programs.     

The effect of Achyranthes japonica extract on larval survival and development and oviposition behavior of Plutella xylostella L. (Lepidoptera: Plutellidae)

The extract of Achyranthes japonica was tested for effects on larval survival and development and the oviposition behavior of the diamondback moth, Plutella xylostella L. Chinese cabbage dipped in A. japonica extract solution showed 51–80% antifeedant activity for 5 days against P. xylostella larvae, and more larvae were also on untreated cabbage leaves 24 h after release. The mortality of P. xylostella larvae increased proportionally to the duration of dipping time in the extract, and both pupation and emergence rates of larvae feeding only on treated cabbage were lower than those for larvae raised on untreated or with a choice of cabbage. The 20-hydroxyecdysone (20E) concentration in leaves was approximately 549, 1232, 1275, and 1426 μg/g at 6, 12, 24, and 48 h after dipping treatment, respectively. Notably, naive females laid more eggs on untreated cabbage than on treated cabbage, and females from larvae raised on treated Chinese cabbage also preferred the non-treated leaves. Our results are in contrast to those from earlier studies using various insect models that confirmed most females prefer to lay eggs on the host type that was eaten in the larval stage (Hopkins host selection principle). Cabbage dipped in the A. japonica solution for 24 h caused 59% larval mortality and inhibited both pupation and emergence rates of the larvae when exposed to plants 15 and 22 days after planting in the field, with the 20E concentration in the treated cabbage leaves at 1600.9 ± 122.36 and 1386.8 ± 24.69 μg/g, respectively. Therefore, the biological effectiveness could be attributed to the 20E in the treated cabbage leaves.     

Mechanical stress regulates osteogenic differentiation and RANKL/OPG ratio in periodontal ligament stem cells by the Wnt/β-catenin pathway

BackgroundThe balance between osteoblastic and osteoclastic activity is critical in orthodontic tooth movement (OTM). Mesenchymal stem cells (MSCs) play an important role in maintaining bone homeostasis, and periodontal ligament stem cells (PDLSCs) are tissue-specific MSCs in the periodontal ligament. However, whether PDLSCs are required for periodontal tissue remodeling during OTM is not fully understood.MethodsHere, we used PDGFRα and Nestin to trace PDLSCs during OTM in rats. We treat human PDLSCs with 100 kpa static pressure for 1 h or 12 h in vitro, and examined the phenotypic changes and expression of RANKL and OPG in these cells.ResultsIn vivo, we found that positive signals of PDGFRα and Nestin in the PDL gradually increased and then decreased on the pressure side to which pressure was applied. In vitro, the osteogenic differentiation of PDLSCs was significantly increased after force treatment for 1 h relative to 12 h. In contrast, the expression ratio of RANKL/OPG was reduced at 1 h and significantly increased at 12 h. Furthermore, we found that the Wnt/β-catenin pathway was dynamically activated in the PDL and in PDLSCs after mechanical stimulation. Importantly, the canonical Wnt pathway inhibitor DKK1 blocked the osteogenesis effect and rescued the ratio of RANKL/OPG in PDLSCs under force treatment for 1 h.ConclusionsOur findings reveal that PDLSCs participate in OTM and that the Wnt/β-catenin pathway maintains bone homeostasis during tooth movement by regulating the balance between osteoblastic and osteoclastic activity.General significanceWe describe a novel potential mechanism related to tooth movement.     

Lethal effects of ichthyotoxic raphidophytes,Chattonella marina,C. antiqua,and Heterosigma akashiwo,on post-embryonic stages of the Japanese pearl oyster,Pinctada fucata martensii

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5 × 10² cells ml⁻¹), C. antiqua (10³ cells ml⁻¹), and H. akashiwo (5 × 10³ cells ml⁻¹) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5 × 10³ cells ml⁻¹ and C. marina at 8 × 10³ cells ml⁻¹. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 10⁵ cells ml⁻¹ more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8 × 10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (10⁴ cells ml⁻¹, 24 h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (5 × 10⁴ cells ml⁻¹, 24 h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5 × 10²–8 × 10³ cells ml⁻¹, 48–86 h) and C. antiqua (10³–8 × 10³ cells ml⁻¹, 72–86 h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.     

Fixed-time post-cervical artificial insemination in sows receiving porcine luteinising hormone at oestrus onset

Fixed-time post-cervical artificial insemination (FTPCAI) allows a wider use of high indexing boars and a considerable reduction in labour requirements in swine production. The aim of this study was to evaluate fixed-time artificial insemination (FTAI) efficiency using different artificial insemination protocols and porcine luteinising hormone (pLH) to induce ovulation. A total of 597 weaned sows in which oestrus detection was performed once daily (08:00 am) was allocated to three groups: FTPCAI1 (n = 199) – sows received a 5-mg (4 ml) intramuscular injection of pLH at oestrus onset, and were inseminated 24 h later; FTPCAI2 (n = 199) – sows received 5 mg of pLH and were inseminated at oestrus onset (0 h) and 24 h after; and MultPCAI (n = 199) – sows did not receive pLH, and the first AI was performed at oestrus onset (0 h) and repeated every 24 h during oestrus. Homospermic doses (1.5 × 10⁹ total sperm cells/50 ml) were used in post-cervical artificial insemination (PCAI) in all the treatments. Hormonal treatment did not affect (P > 0.05) the interval between oestrus onset and ovulation (overall 32.4 h) and there were no differences (P > 0.05) in farrowing rate (overall 91.6%) or litter size (overall 12.6 piglets born) among treatments. In sows treated with pLH at oestrus onset, a single PCAI with 1.5 billion sperm cells did not compromise reproductive performance compared with a double PCAI at 24 h intervals.     

Phenoloxidase and its zymogen are required for the larval–pupal transition in Bactrocera dorsalis (Diptera: Tephritidae)

Phenoloxidases (POs) play a key role in melanin production, are involved in invertebrate immune mechanisms, and are considered important enzymes in the insect development process. In the present study, we report the developmental stage and tissue-specific expression patterns of BdPPO1 and PO activity from Bactrocera dorsalis. The results showed that the activity of PO and its zymogen expression were closely related to the development of B. dorsalis during the larval–pupal transition, particularly in the integument. Additionally, biochemical characterization showed that PO from different developmental stages and tissues all had maximum activity at pH 7.5 and 37 °C. After feeding a metal ion-containing artificial diet, the activity of PO and expression of BdPPO1 were significantly increased, indicating that PO was a metalloprotein and it could be activated by Zn²⁺, Mg²⁺, Ca²⁺, and Cu²⁺. The functional analysis showed that the expression of BdPPO1 could be regulated by 20-hydroxyecdysone (20E) after injection. Furthermore, injection of the double-stranded RNA of BdPPO1 into the 3rd instar larvae significantly reduced mRNA levels after 24 h and 48 h, and resulted in a lower pupation rate and abnormal phenotype. These results expand the understanding of the important role of PO and its zymogen in the growth of B. dorsalis.     

Combined effect of bone marrow derived mesenchymal stem cells and nitric oxide inducer on injured gastric mucosa in a rat model

AimTo study the effect of intravenous injection of bone marrow mesenchymal stem cells (BMMSCs), alone and combined with NO inducer in gastric ulcer healing in a rat model.MethodsRats were divided into controls, gastric ulcer, gastric ulcer receiving mesenchymal stem cells (MSCs), gastric ulcer receiving NO inducer (l-Arginine), gastric ulcer receiving MSCs plus NO inducer (l-Arginine) groups. MSCs were given in a dose of (10⁶cells) by intravenous injection. l-Arginine was given 300 mg/kg body weight intraperitoneally. 24 h and 7 days after BMMSCs and NO inducer injection, VEGF, PGE, TNF-α were assessed by ELISA. Gene expression of HGF, caspase-3, eNOS and BAX/Bcl-2 in gastric tissues were studied by real time PCR. Histopathology staining of gastric tissues was performed.ResultsInjection of MSCs or NO inducer or both to the gastric ulcer group significantly decreased caspase-3 and BAX genes expression (apoptotic factors) and increased Bcl-2 gene expression (anti-apoptotic factor) compared to that of the gastric ulcer group after both 24 h and 7 days with more significant results in the gastric group received both MSCs and NO inducer. HGF gene expression was significantly increased in the groups injected with MSCs or NO inducer or both compared with the corresponding gastric ulcer group (p < 0.05, p < 0.05 & p < 0.001 respectively). There was a significant decrease in the mean PGE2 and TNF-α levels in the gastric ulcer group receiving MSCs, the gastric ulcer group receiving NO and the gastric ulcer group receiving both MSCs and  NO compared to the gastric ulcer group after both 24 h and 7 days. Histopathological examination of gastric tissue of groups that received stem cells or NO alone, showed mucosal regenerative changes with increased thickness together with reduced inflammatory cellular infiltrate in the submucosa and decreased congestion. There was complete restoration in gastric mucosa in the group that received both stem cells and NO.ConclusionAdministration of MSCs, NO, or MSCs plus NO may exert a therapeutic effect on the mucosal lesion in gastric ulcer through their anti-inflammatory, angiogenic and antiapoptotic actions.     

Time course analysis of ABA and non-ionic osmotic stress-induced changes in water status,chlorophyll fluorescence and osmotic adjustment in Arabidopsis thaliana wild-type (Columbia) and ABA-deficient mutant (aba2)

In the present study, we investigated time course changes of water status including relative water content (RWC), leaf osmotic potential (Ψ_Π), stomatal conductance (g_s), proline (Pro), chlorophyll fluorescence (F_v/F_m) and total chlorophyll content in the Arabidopsis thaliana under PEG-induced drought stress after exogenous ABA treatment. To a better explanation for the role of ABA in the water status of A. thaliana to drought stress, wild-type (Columbia) and ABA-deficient mutant (aba2) of A. thaliana were used in the present study. Moreover, three weeks old Arabidopsis seedlings were applied exogenously with 50 μM ABA and exposed to drought stress induced by 40% PEG8000 (−0.73 MPa) for 6 h, 12 h and 24 h (hours). Our findings indicate that RWC of wild-type and aba2 started to decrease in the first 12 h and 6 h of PEG-induced drought stress, respectively. However, exogenous treatment of 50 μM ABA increased their RWC under drought stress. On the other hand, while Ψ_Π of both genotypes started to decrease in the first 6 h of drought stress, these declines in Ψ_Π were prevented by ABA treatment under stress throughout the experiment; it was more pronounced in aba2 at 24 h. While the highest increase in g_s was obtained in aba2 after 24 h stress, ABA-induced highest decrease in g_s was obtained in the same genotype during 12 h, as compared to PEG-treated group alone. On the other hand, Pro content increased in all treatment groups of ABA-deficient mutant aba2 at 12 h and 24 h. However, Pro content in ABA + PEG treated aba2 plants was higher than in PEG- and ABA-treated plants alone at the end of the 24 h. Drought stress decreased F_v/F_m and total chlorophyll contents of both genotypes while 50 μM ABA alleviated these reductions during drought stress, as compared to PEG stressed plants. On the other hand, 50 μM ABA treatment alone did not create any remarkable effect on F_v/F_m and total chlorophyll contents.These findings indicate that exogenous ABA showed an alleviative effect against damage of drought stress on relative water content, osmotic potential, stomatal conductance, proline, chlorophyll fluorescence and total chlorophyll content of both genotypes during 24 h of drought stress treatment.     

Thermal and physical stresses induce a short-term immune priming effect in Galleria mellonella larvae

Exposure of larvae of Galleria mellonella larvae to mild physical (i.e. shaking) or thermal stress for 24 h increased their ability to survive infection with Aspergillus fumigatus conidia however larvae stressed in a similar manner but incubated for 72 h prior to infection showed no elevation in their resistance to infection with A. fumigatus. Stressed larvae demonstrated an elevated haemocyte density 24 h after initiation of the stress event but this declined at 48 and 72 h. Larval proteins such as apolipophorin, arylophorin and prophenoloxidase demonstrated elevated expression at 24 h but not at 72 h. Larvae maintained at 37 °C showed increased expression of a range of antimicrobial and immune-related proteins at 24 h but these decreased in expression thereafter. The results presented here indicate that G. mellonella larvae are capable of altering their immune response following exposure to mild thermal or physical stress to mount a response capable of counteracting microbial infection which reaches a peak 24 h after the initiation of the priming event and then declines by 72 h. A short-term immune priming effect may serve to prevent infection but maintaining an immune priming effect for longer periods may be metabolically costly and unnecessary while living within the colony of another insect.     

Cell division in Lemna minor roots treated with lead

Treatment of Lemna minor L. roots with 15 μM Pb²⁺ supplied as Pb(NO₃)₂ in 50-fold diluted Wang medium caused a progressive reduction in mitotic activity in the root tip. The percentage of dividing nuclei after 1, 6, 12 and 12 h of lead treatment was 6.25, 4.4, 3.4 and 0.3, respectively as compared to 7.1–7.7% in the control. After 6 h of lead treatment the number of cells in metaphase and anaphase was reduced by four- and nine-fold, respectively and after 12 h these phases were not detected. There were 3- and 10-fold fewer cells in telophase after 6 and 24 h, while those in prophase were reduced only in the 24 h treatment (a 30-fold reduction). These effects were associated with an increase in the number of cells exhibiting disturbances including lagging chromosomes, chromosome bridges, micronuclei, and nuclei with more condensed chromatin. The formation of micronuclei in root cells of L. minor cells at a very low dose of lead indicates that roots of this aquatic plant may be more sensitive to lead than those of terrestrial plants.     

The impact of weight loss on the 24-h profile of circulating peptide YY and its association with 24-h ghrelin in normal weight premenopausal women

Peptide YY (PYY) and ghrelin exhibit a reciprocal association and antagonistic physiological effects in the peripheral circulation. Research has yet to clarify the effect of weight loss on the 24 h profile of PYY or its association to 24 h ghrelin. We sought to determine if diet- and exercise-induced weight loss affects the 24 h profile of PYY and its association with 24 h ghrelin in normal weight, premenopausal women. Participants (n = 13) were assessed at baseline (BL) and after a 3-month diet and exercise intervention (post). Blood samples obtained q10 min for 24 h were assayed for total PYY and total ghrelin q60 min from 0800 to 1000 h and 2000 to 0800 h and q20 min from 1000 to 2000 h. The ghrelin/PYY ratio was used as an index of hormonal exposure. Statistical analyses included paired t-tests and linear mixed effects modeling. Body weight (−1.85 ± 0.67 kg; p = 0.02), and body fat (−2.53 ± 0.83%; p = 0.01) decreased from BL to post. Ghrelin AUC (5252 ± 2177 pg/ml/24 h; p = 0.03), 24 h mean (216 ± 90 pg/ml; p = 0.03) and peak (300 ± 134 pg/ml; p = 0.047) increased from BL to post. No change occurred in PYY AUC (88.2 ± 163.7 pg/ml; p = 0.60), 24 h mean (4.8 ± 6.9 pg/ml; p = 0.50) or peak (3.6 ± 6.4 pg/ml; p = 0.58). The 24 h association between PYY and ghrelin at baseline (p = 0.04) was weakened at post (p = 0.14); however, the ghrelin/PYY lunch ratio increased (p = 0.01) indicating the potential for ghrelin predominance over PYY in the circulation. PYY and ghrelin are reciprocally associated during a period of weight stability, but not following weight loss. An “uncoupling” may have occurred, particularly at lunch, due to factors that modulate ghrelin in response to weight loss.     

Prevention of cell death by the zinc ion chelating agent TPEN in cultured PC12 cells exposed to Oxygen–Glucose Deprivation (OGD)

To elucidate the role of Zn²⁺-associated glutamate signaling pathway and voltage-dependent outward potassium ion currents in neuronal death induced by hypoxia–ischemia, PC12 cells were exposed to Oxygen–Glucose Deprivation (OGD) solution mimicking the hypoxic–ischemic condition in neuron, and the effect of N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a specific Zn²⁺ chelating agent on OGD-induced neuronal death was assessed in the present study. The cell survival rate, apoptosis status, potassium channel currents, intracellular free glutamate concentration and GluR2 expression in PC12 cells exposed to OGD in the absence or presence of TPEN for different time were investigated. The results showed that OGD exposure increased apoptosis, reduced the cell viability (P < 0.01 at 3 h, 6 h and 24 h, respectively compared to control), changed the voltage-dependent outward potassium ion current (increase at 1 h, but decrease at 3 h) and decreased the concentration of intracellular glutamate (P < 0.05 at 3 h and 6 h, P < 0.01 at 24 h respectively compared to control) and GluR2 expression (P < 0.05 at 3 h, 6 h and 24 h, respectively compared to control) in PC12 cells. TPEN partially reversed the influence resulted from OGD. These results suggest that OGD-induced cell apoptosis and/or death is mediated by the alteration in glutamate signaling pathway and the voltage-dependent outward potassium ion currents, while TPEN effectively prevent cell apoptosis and/or death under hypoxic–ischemic condition.     

Maximizing mouse embryonic stem cell production in a stirred tank reactor by controlling dissolved oxygen concentration and continuous perfusion operation

One of the key challenges in stem cell bioprocessing is the large-scale cultivation of stem cells in order to meet the demanding meaningful cell numbers needed for biomedical applications, especially for clinical settings. Mouse embryonic stem cells [1], used as a model system herein, were cultivated on microcarriers in a fully controlled stirred tank reactor (STR) [2]. The impact of varying the concentration of dissolved oxygen (at 5%, 10%, 20% and 30% DO) and operating under a continuous perfusion mode on cell growth and pluripotency maintenance was investigated. In addition, in order to further optimize the feeding strategy of the STR operating under continuous perfusion toward maximal cell production, the influence of different medium residences times (12 h, 24 h, 32 h, 48 h and 96 h) was evaluated. Overall, the maximal cell concentration of 7.9–9.2 × 10⁶ cells/mL were attained after 11 days, with no passaging required, under a DO of 10–20% in the continuous perfused bioreactor with cell retention and medium residences times of 32–48 h. Importantly, mESC expanded under these conditions, retained the expression of pluripotency markers (Oct4, Nanog and Ssea-1), as well as their differentiation potential into cells of the three embryonic germ layers.The STR-based cultivation platform optimized herein represents a major contribution toward the development of large-volume production systems of differentiated cell derivatives for a wide range of biomedical applications.