Innovative development of COVID-19 mRNA vaccine using the gold nanoparticle delivery platform

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Possible mechanisms by which the PCB mixture aroclor 1242 stimulates pregnant rat uterine oscillatory contraction.

Polychlorinated biphenyl (PCB) exposures are associated with decreased gestation length and increased spontaneous abortion. Uterine quiescence is necessary to maintain pregnancy to term. Because PCBs release the known uterotonic agents inositol 1,4,5-trisphosphate and arachidonic acid and also increase intracellular calcium concentration ( (Ca\rm\sp{2+}\rbrack\sb{i}), it is proposed that PCB mixtures: (1) increase uterine spontaneous oscillatory contraction, (2) stimulate uterine contraction by releasing arachidonic acid through activation of phospholipase A\sb2 (PLA\sb2) or phospholipase C (PLC), and (3) stimulate uterine contraction by increasing \rm\lbrack Ca\sp{2+}\rbrack\sb{i} due to activation of voltage-operated calcium channels. The initial study examined whether PCBs could stimulate contractions of pregnant rat uteri. The PCB mixtures Aroclor (A) 1242, 1248, 1254, and bacterial dechlorinated products of A1242 and A1254 stimulated uterine contractions in a concentration-dependent manner, with the trend that less chlorinated mixtures were more potent. A1242 released arachidonic acid in a concentration- and time-dependent manner from myometrial cells prelabeled with \sp3H-arachidonic acid. HPLC data showed that most of the liberated arachidonic acid remained as arachidonic acid, suggesting that arachidonic acid itself, rather than a metabolite, may mediate A1242 action. In contrast, A1242 did not increase inositol phosphate release from myo- (\sp3H) inositol labeled myometrial cells. In accordance, pretreatment with PLA\sb2 inhibitors prevented A1242-induced stimulation of contraction, whereas PLC inhibitors did not block the A1242 response. Pretreatment with depletors of intracellular calcium pools did not prevent the uterotonic effect of A1242. However, A1242 was unable to increase contraction without extracellular calcium present or in the presence of the voltage-operated calcium channel blocker nifedipine. A1242 depolarized the membrane of myometrial cells as determined by a potential-sensitive dye. Using the fluorescent calcium-sensitive probe, fura-2, an increase of \rm\lbrack Ca\sp{2+}\rbrack\sb{i} by A1242 was observed in myometrial cells, and this increase was completely blocked in the absence of extracellular calcium or in the presence of nifedipine. In conclusion, this study demonstrated that PCB mixtures stimulated uterine contractility, and this response is dependent on PLA2-mediated arachidonic acid release and increased \rm\lbrack Ca\sp{2+}\rbrack\sb{i} via voltage-operated calcium channels.Ph.D.Health and Environmental SciencesToxicologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/130898/2/9825165.pd

Disturbed relaxin signaling pathway and testicular dysfunction in mouse offspring upon maternal exposure to simazine.

Simazine is a triazine herbicide that is being widely applied worldwide and commonly detected in surface and groundwater. Despite its popular use in controlling weeds and algae, very limited information is available regarding its toxicity. In the present study, pregnant mice were orally exposed to low doses (0, 5, 50, or 500 µg/kg body weight per day) of simazine during gestation and lactation, during which no overt maternal toxic response was detected, and their offspring was assessed. Simazine-exposed male offspring showed decreased body, testicular, and epididymis weight, increased testicular apoptosis, and decreased sperm concentrations. Differentially-expressed genes in the testes of male offspring exposed to simazine were identified by DNA microarray, revealing 775 upregulated and 791 downregulated genes; among these, the relaxin-family peptide receptor 1 (Rxfp1), which is the receptor for relaxin hormone, was significantly downregulated. In addition, the expression of target genes in the relaxin pathway, including nitric oxide synthase 2 (Nos2) and Nos3, was significantly decreased in simazine-exposed F1 testes. Moreover, simazine inhibited NO release, and knockdown of Rxfp1 blocked the inhibitory action of simazine on NO production in testicular Leydig cells. Therefore, the present study provides a better understanding of the toxicities associated with the widely used herbicide simazine at environmentally relevant doses by demonstrating that maternal exposure interferes with the pleotropic relaxin-NO signaling pathway, impairing normal development and reproductive activity of male offspring

Simazine-induced decreased Rxfp1 and Nos2 expression and reduced NO production in testicular cells <i>in vitro</i>.

<p>Rat Leydig cells (LC540) were exposed to simazine (0, 0.01, 0.1, or 1 µM) for 36 h, and the mRNA levels of Rxfp1 (A) and Nos2 (B) were analyzed by qRT-PCR. The relative expression levels of Rxfp1 and Nos2 normalized to β-actin are shown. (C) LC540 cells were exposed to various concentrations of simazine for 36 h, and the levels of NO produced were determined by measuring nitrite concentration. (D) LC540 knockdown cells were prepared by transfection with a scrambled sequence or siRNAs for Rxfp1, and reduced Rxfp1 expression was demonstrated by western blotting. Subsequently, knocked-down cells were treated with simazine for 36 h, and the levels of NO produced were determined. For all experiments (A-D), three independent experiments were performed in triplicate, and different letters denote significant values (<i>p<</i>0.05).</p

MCL-1ES Induces MCL-1L-Dependent BAX- and BAK-Independent Mitochondrial Apoptosis

<div><p>MCL-1 (myeloid cell leukemia-1), a member of the BCL-2 family, has three splicing variants, antiapoptotic MCL-1L, proapoptotic MCL-1S, and MCL-1ES. We previously reported cloning MCL-1ES and characterizing it as an apoptotic molecule. Here, we investigated the molecular mechanism by which MCL-1ES promotes cell death. MCL-1ES was distinct from other proapoptotic BCL-2 members that induce apoptosis by promoting BAX or BAK oligomerization, leading to mitochondrial outer membrane permeabilization (MOMP), in that MCL-1ES promoted mitochondrial apoptosis independently of both BAX and BAK. Instead, MCL-1L was crucial for the apoptotic activity of MCL-1ES by facilitating its proper localization to the mitochondria. MCL-1ES did not interact with any BCL-2 family proteins except for MCL-1L, and antiapoptotic BCL-2 members failed to inhibit apoptosis induced by MCL-1ES. The BCL-2 homology 3 (BH3) domain of MCL-1ES was critical for both MCL-1ES association with MCL-1L and apoptotic activity. MCL-1ES formed mitochondrial oligomers, and this process was followed by MOMP and cytochrome <i>c</i> release in a MCL-1L-dependent manner. These findings indicate that MCL-1ES, as a distinct proapoptotic BCL-2 family protein, may be useful for intervening in diseases that involve uncontrolled MCL-1L.</p></div

Increased testicular and ovarian apoptosis of mouse offspring exposed to simazine.

<p>(A) TUNEL-positive control section pretreated with DNase I (a), TUNEL-negative control section without terminal deoxynucleotidyl transferase treatment (b). The TUNEL assay was conducted on the cross-sections of 8-week-old mice testes (c and d) prepared from groups exposed to corn oil (c) or simazine (50 µg/kg) (d). (B) Real-time PCR analysis of Bcl-2 family members in the testes of control and simazine-exposed offspring was shown. The data were normalized to the expression level of β-actin and are presented as relative fold changes. The results are the mean ± SEM of six independent testicular analyses performed in triplicate for each group.</p

Decreased expression of Rxfp1 in the testis of F1 male mice exposed to simazine.

<p>(A) Testicular sections of young adult F1 male control (a) or 500 µg/kg simazine-exposed (b) mice were immunostained for Rxfp1. (B) Six independent testicular lysates of the control and the 500 µg/kg groups were prepared, and western blot analyses were performed using anti-Rxfp1 antibodies (left panel). Equal loading of each sample was confirmed by immunoblotting of the same membrane with anti-β-actin antibody. The results of a quantitative analysis of the Rxfp1 expression shown in the left panel using Multi Gauge V3.0 software are presented in the right panel. The asterisk indicates a significant difference compared with the control (<i>p</i><0.05).</p

BAK- and BAX-independent apoptosis induced by MCL-1ES via its mitochondrial oligomerization.

<p>(A) A time course of cell viability was performed in wild-type (WT), <i>bak^−/−</i>, <i>bax^−/−</i>, and <i>bax^−/−bak^−/−</i> knockout MEF cells after transfection with WT or BH3 mutant (BH3M) MCL-1ES. (B) A flow cytometry analysis of Annexin V-positive apoptotic cells was performed, and (C) MMP, (D) cytochrome <i>c</i> release to cytosol, (E) caspase-3 activity, and (F) formation of apoptosome complexes were determined in <i>bax^−/−bak^−/−</i> MEF cells transfected with WT or BH3M MCL-1ES as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079626#s2" target="_blank">Materials and Methods</a>. The values (mean ± SEM) were determined from three independent experiments. (G) The lack of MCL-1ES-induced BAK or BAX oligomerization was verified in 293T cells. (H) Oligomer formation of MCL-1ES in 293T cells and (I) in <i>bax^−/−bak^−/−</i> MEF cells was assessed. The heavy membrane fraction was obtained and cross-linked with glutaraldehyde. Oligomer formation was determined by western blot analysis using the appropriate antibodies.</p