Design, synthesis and biological evaluation of 6-chloro-quinolin-2-one derivatives as novel FXIa inhibitors.

A series of 6-chloro-quinolin-2-one derivatives were designed and synthesized as FXIa inhibitors by exploration of P1, P1 prime and P2 prime groups. Each compound was accessed for inhibitory effect on FXIa and some of them were evaluated in the clotting assay. 14c demonstrated excellent in-vitro potency (FXIa IC50: 15 nM, 2 x aPTT: 6.8 µM) and good in-vivo efficacy (prolonged in-vivo aPTT by more than 1-fold but not PT). Moreover, the pharmacokinetics property of 14c were evaluated following intravenous administration in rats, which indicated that 14c probably will be a clinical candidate for intravenous administration.

DDT exposure induces tremor-like behavior and neurotoxicity in developmental stages of embryonic zebrafish.

Dichlorodiphenyltrichloroethane (DDT) is a broad-spectrum insecticide, widely detected in environments due to its high stability characteristic and long natural half-life period. The adverse impact of DDT exposure on organisms and humans has attracted great concern worldwide. The current study explored the developmental and neurobehavioral toxicity response of DDT in embryonic zebrafish. The embryos were treated with DDT (0, 0.1, 1, 2.5 and 5 µM) during 6 h post fertilization (hpf) to 144 hpf. Our result indicated that DDT exposures increased the embryo hatching rate at 48 and 60 hpf, the larval malformation rate at 120 hpf and mortality rate at 144 hpf. The manifested malformations included uninflated swim bladder, bent spine and tail, deformed liver, and pericardial edema. The 120 hpf larval organs size of the gut and swim bladder was decreased in higher exposed concentration groups. Besides, DDT exposure resulted in hyperactivity for the embryo spontaneous movement at 24 hpf and tremor like movement measured by the free larval activity at 72 hpf, as well as the larval activity at 96 hpf under light-dark transition stimulus. Mechanistic examinations at 120 hpf revealed DDT exposure elevated oxidative stress through MDA formation increase, ATP level decrease as well as antioxidant enzyme genes (sod1 and gpx1a) expression decrease. DDT exposure induced abnormal neurotransmitters expression with DA level increase, 5-HT and NOS level decrease. DDT exposure suppressed the gene expressions involved in axon development (rab33a and nrxn2a) and potassium channel (kcnq2 and kcnq3). Our results suggest that the hyperactivity and tremor like movement in DDT-exposed embryos/larvae may result from oxidative stress involved with neuronal damage.

Structure-based developmental toxicity and ASD-phenotypes of bisphenol A analogues in embryonic zebrafish.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that has become more prevalent in recent years. Environmental endocrine disruptor bisphenol A (BPA) has been linked to ASD. BPA analogues (BPs) are structure-modified substitutes widely used as safer alternatives in consumer products, yet few studies have explored the developmental neurotoxicity (DNT) of BPA analogues. In the present study, we used the larval zebrafish model to assess the DNT effects of BPA and its analogues. Our results showed that many BPA analogues are more toxic than BPA in the embryonic zebrafish assay regarding teratogenic effect and mortality, which may partially due to differences in lipophilicity and/or different substitutes of structural function groups such as CF3, benzene, or cyclohexane. At sublethal concentrations, zebrafish embryos exposed to BPA or BPs also displayed reduced prosocial behavior in later larval development, evidenced by increased nearest neighbor distance (NND) and the interindividual distance (IID) in shoaling, which appears to be structurally independent. An in-depth analysis of BPA, bisphenol F (BPF), and bisphenol S (BPS) revealed macrocephaly and ASD-like behavioral deficits resulting from exposures to sublethal concentrations of these chemicals. The ASD-like behavioral deficits were characterized by hyperactivity, increased anxiety-like behavior, and decreased social contact. Mechanistically, accelerated neurogenesis that manifested by increased cell proliferation, the proportion of newborn mature neurons, and the number of neural stem cells in proliferation, as well as upregulated genes related to the K+ channels, may have contributed to the observed ASD-like morphological and behavioral alterations. Our findings indicate that BPF and BPS may also pose significant risks to ASD development in humans and highlight the importance of a comprehensive assessment of DNT effects for all BPA analogues in the future.

Several non-salt and solid thienopyridine derivatives as oral P2Y12 receptor inhibitors with good stability.

A series of novel thienopyridine derivatives were designed and synthesized as P2Y12 receptor inhibitors. Several solid compounds were assessed for inhibitory effect where they exhibited stronger potency than clopidogrel. Compound 6b and 6g were evaluated for metabolism to verify that they could overcome clopidogrel resistance and for toxicity where they showed lower toxicity than prasugrel. Compound 6b exhibited lower risk of bleeding than prasugrel and showed good stability under stress testing. Overall, as a promising antiplatelet agent, representative compound 6b showed the following advantages: (1) no drug resistance for CYP2C19 poor metabolizers; (2) higher potency than clopidogrel; (3) lower toxicity than prasugrel; (4) lower risk of bleeding than prasugrel; (5) good stability as a non-salt solid.

Leachate from plastic food packaging induced reproductive and neurobehavioral toxicity in zebrafish.

The present study mimicked daily life exposure to plastic food package bags and evaluated its effects on the reproductive and neurobehavioral responses using zebrafish model. Gas chromatography-mass spectrometer (GC/MS) full scan analysis revealed that phthalic acid, isobutyl octyl ester (DEHP) and its metabolites were the main leachate from plastic bags. Our results demonstrated that during the eight weeks exposure, leaching from plastic bags treated with boiling water (P-high group) significantly affected the spawn egg production, embryo hatching and larval malformation rate. Cross-spawning trails between zebrafish collected from the controls and P-high group at the end of eight weeks showed that these adverse effects were more severe in the offspring derived from paternal exposure than those derived from the maternal exposure, suggesting leached chemicals may have a more pronounced effect in sperm than in eggs. In addition, P-high group male testis weight, sperm motility and sperm swimming velocities were decreased significantly. After eight weeks treatment, neurobehavioral tests demonstrated significant changes in the swimming speed during free swimming and light-dark stimulation in the adult zebrafish from P-high group, with the effects being more severe in the males than females. P-high group males also showed altered response in the light/dark explore and mirror attacks assays.

Nonlocality, Steering, and Quantum State Tomography in a Single Experiment.

We investigate whether paradigmatic measurements for quantum state tomography, namely mutually unbiased bases and symmetric informationally complete measurements, can be employed to certify quantum correlations. For this purpose, we identify a simple and noise-robust correlation witness for entanglement detection, steering, and nonlocality that can be evaluated based on the outcome statistics obtained in the tomography experiment. This allows us to perform state tomography on entangled qutrits, a test of Einstein-Podolsky-Rosen steering and a Bell inequality test, all within a single experiment. We also investigate the trade-off between quantum correlations and subsets of tomographically complete measurements as well as the quantification of entanglement in the different scenarios. Finally, we perform a photonics experiment in which we demonstrate quantum correlations under these flexible assumptions, namely with both parties trusted, one party untrusted and both parties untrusted.

"Super-Heisenberg" and Heisenberg Scalings Achieved Simultaneously in the Estimation of a Rotating Field.

The Heisenberg scaling, which scales as N^{-1} in terms of the number of particles or T^{-1} in terms of the evolution time, serves as a fundamental limit in quantum metrology. Better scalings, dubbed as "super-Heisenberg scaling," however, can also arise when the generator of the parameter involves many-body interactions or when it is time dependent. All these different scalings can actually be seen as manifestations of the Heisenberg uncertainty relations. While there is only one best scaling in the single-parameter quantum metrology, different scalings can coexist for the estimation of multiple parameters, which can be characterized by multiple Heisenberg uncertainty relations. We demonstrate the coexistence of two different scalings via the simultaneous estimation of the magnitude and frequency of a field where the best precisions, characterized by two Heisenberg uncertainty relations, scale as T^{-1} and T^{-2}, respectively (in terms of the standard deviation). We show that the simultaneous saturation of two Heisenberg uncertainty relations can be achieved by the optimal protocol, which prepares the optimal probe state, implements the optimal control, and performs the optimal measurement. The optimal protocol is experimentally implemented on an optical platform that demonstrates the saturation of the two Heisenberg uncertainty relations simultaneously, with up to five controls. As the first demonstration of simultaneously achieving two different Heisenberg scalings, our study deepens the understanding on the connection between the precision limit and the uncertainty relations, which has wide implications in practical applications of multiparameter quantum estimation.

Developmental co-exposure of TBBPA and titanium dioxide nanoparticle induced behavioral deficits in larval zebrafish.

Both tetrabromobisphenol A (TBBPA) and titanium dioxide nanoparticle (TiO2 NP) have widespread commercial applications, resulting in their ubiquitous co-presence in the environment and biota. Although environmental chemicals exist as mixtures, toxicity studies are nearly always conducted with single chemicals. Few studies explore potential interactions of different chemical mixtures. In this study, we employ the sensitive developing nerve system in zebrafish to assess the neurotoxicity of TBBPA/TiO2 NP mixtures. Specifically, zebrafish embryos were exposed to solvent control (0.1% DMSO), 2 µM TBBPA, 0.1 mg/L TiO2 NP, and their mixture from 8 to 120 h post fertilization (hpf), and motor/social behavioral assessments were conducted on embryos/larvae at different developmental stages. Our results showed that TBBPA/TiO2 NP single or co-exposures increased spontaneous movement, decreased touch response and swim speed, and affected social behaviors of light/dark preference, shoaling, mirror attack and social contact. In particular, many of these phenotypes were manifested with higher magnitude of changes from the mixture exposure. These behavioral deficits were also accompanied with increased cell death in olfactory region and neuromasts in the lateral line system, increased ROS in gallbladder, pancreas, liver, and intestine, as well as increased lipid peroxidation and decreased ATP levels in whole larval tissue homogenates. Further, genes coding for key cell apoptosis marker and antioxidant enzyme were significantly upregulated by these two chemicals, in particular to their mixture. Interestingly, the co-presence of TBBPA also increased the mean particle size of TiO2 NP in the exposure solutions and the TiO2 NP content in larval tissue. Together, our analysis suggests that TBBPA/TiO2 NP induced behavioral changes may be due to physical accumulation of these two chemicals in the target organs, and TiO2 NP may serve as carriers for increased accumulation of TBBPA. To conclude, we demonstrated that TBBPA/TiO2 NP together cause increased bioaccumulation of TiO2, and heightened responses in behavior, cell apoptosis and oxidative stress. Our findings also highlight the importance of toxicity assessment using chemical mixtures.

Cryopreservation disrupts lipid rafts and heat shock proteins in yellow catfish sperm.

Lipid rafts and associated membrane proteins (flotillin, caveolin) play important roles in cell signaling and sperm fertilization while heat shock proteins (Hsp) ensure properly protein folding to fulfill their physiological functions. The markedly reduced fertility in thawed sperm after cryopreservation could result from disrupted membrane lipid rafts and these proteins. To explore the effect of sperm cryopreservation on lipid rafts and heat shock proteins, we compared lipid raft integrity, and the expression levels of lipid raft associated proteins (Flot-1, Flot-2, Cav-1) as well as heat shock proteins (Hsp90, Hsp70) in fresh and thawed sperm cryopreserved under different scenarios in yellow catfish. We found higher lipid raft integrity, higher protein expression levels of Flot-1, Flot-2, Cav-1, Hsp90, and Hsp70 in fresh sperm samples than in thawed sperm samples, in thawed sperm samples cryopreserved with optimal cooling rate than those cryopreserved with sub-optimal cooling rate, and in thawed sperm samples cryopreserved with extenders supplemented with cholesterol than those supplemented with methyl-ß-cyclodextrin (for cholesterol removal). Our findings indicate that lipid raft integrity, and expression levels of Flot-1, Flot-2, Cav-1, Hsp90, and Hsp70 are clearly associated with sperm quality, and together they may play a cumulative role in reduced fertility associated with thawed sperm in aquatic species.

Neurodevelopmental toxicity assessments of alkyl phenanthrene and Dechlorane Plus co-exposure in zebrafish.

Alkyl phenanthrene (A-Phen) and Dechlorane Plus (DP) are ubiquitous environmental pollutants that widely co-exist in the environment. It has been established that both A-Phen and DP elicit neurotoxicity, but the potential interactive toxicity of these contaminants is not well-known. To determine whether a mixture of A-Phen and DP would exhibit interactive effects on neurodevelopment, we co-exposed 3-methylphenanthrene (3-MP), a representative of A-Phen, with DP. Our results illustrated that exposure to 5 or 20 µg/L 3-MP alone or in combination with 60 µg/L DP caused neurobehavioral anomalies in zebrafish. In accordance with the behavioral deficits, 3-MP alone or co-exposed with DP significantly decreased axonal growth of secondary motoneurons, altered intracellular Ca2+ homeostasis and induced cell apoptosis in the muscle of zebrafish. Additionally, 3-MP alone or co-exposed with DP significantly increased reactive oxygen species (ROS) and the mRNA levels of apoptosis-related genes. These findings indicate that 3-MP alone or co-exposed with DP induces neurobehavioral deficits through the combined effects on neuronal connectivity and muscle function. Chemical analysis revealed significant increases in 3-MP and DP bioaccumulation in zebrafish co-exposed with 3-MP and DP. Elevated bioaccumulation resulting from mixture exposure may represent a significant contribution of the synergistic effects observed in combined chemical exposure.

Tissue distribution of tetrabromobisphenol A and cadmium in mixture inhalation exposure.

Tetrabromobisphenol A (TBBPA) and cadmium chloride (CdCl2) are the typical representative pollutants of brominated flame retardants and heavy metals found in the air of e-waste recycling workshops. However, their metabolic kinetics through mixture inhalation is unknown. In the present study, 8-week old Institute of Cancer Research (ICR) male mice were whole-body exposed to TBBPA and CdCl2 mixtures by inhalation. Tissue samples were collected for TBBPA and cadmium (Cd) analysis at 2, 4, 6, and 8 weeks during exposure and at 4 and 8 weeks after the completion of the 8-week exposure period. TBBPA was mainly distributed to the lungs, liver, kidney, testis, and spleen, with a high amount accumulated in the brain, liver, and spleen. Cd was mainly distributed to the lungs, liver, and kidney, with a high amount accumulated in the liver, kidney, and testis and a low amount accumulated in brain and serum. Tissue burden of TBBPA and Cd in all organs increased in a dose- and time-dependent manner during the exposure period. However, 4 weeks after the completion of an 8-week exposure, TBBPA concentrations in the liver, testis, brain, and serum and Cd concentrations in the liver, testis, and kidney were higher than the corresponding tissue concentrations during the exposure period. The rapid accumulation of both TBBPA and Cd in the lungs after inhalation exposure indicated a high risk of the respiratory system diseases for workers in e-waste recycling workshops. In addition, the migration of both TBBPA and Cd from lungs to liver and testis may result in more complex toxic effects in vivo.

Selective extraction of bisphenol A from water by one-monomer molecularly imprinted magnetic nanoparticles.

One-monomer molecularly imprinted magnetic nanoparticles were prepared as adsorbents for selective extraction of bisphenol A from water in this study. A single bi-functional monomer was adopted for preparation of the molecularly imprinted polymer, avoiding the tedious trial-and-error optimizations as traditional strategy. Moreover, bisphenol F was used as the dummy template for bisphenol A to avoid the interference from residual template molecules. These nanoparticles showed not only large adsorption capacity and good selectivity to the bisphenol A but also outstanding magnetic response performance. Furthermore, they were successfully used as magnetic solid-phase extraction adsorbents of bisphenol A from various water samples, including tap water, river water, and seawater. The developed method was found to be much more efficient, convenient, and economical for selective extraction of bisphenol A compared with the traditional solid-phase extraction. Separation of these nanoparticles can be easily achieved with an external magnetic field, and the optimized adsorption time was only 15 min. The recoveries of bisphenol A in different water samples ranged from 85.38 to 93.75%, with relative standard deviation lower than 7.47%. These results showed that one-monomer molecularly imprinted magnetic nanoparticles had the potential to be popular adsorbents for selective extraction of pollutants from water.

Chronic co-exposure to low levels of brominated flame retardants and heavy metals induces reproductive toxicity in zebrafish.

Brominated flame retardants (BFRs) and heavy metals (HMs) are two main types of pollutants in electronic waste recycling sites, which are also ubiquitously detectable in environmental media and human tissues. However, the adverse health effects of exposure to the mixture of these types of pollutants are unknown. In this study, we investigated the reproductive toxicity of a mixture of decabromodiphenyl ether (BDE-209), tetrabromobisphenol A, cadmium chloride, and lead acetate (PbAc) at the environmental relevant levels. Zebrafish were waterborne and exposed to chemical mixtures for one generation. The reproductive effects were evaluated for F0 adults and F1 offspring. Chemical residues were also analyzed in the exposed adults and their eggs at the end of exposure. Our findings demonstrated that exposure to the chemical mixture for 150 days had no effect on the survival rate of zebrafish, but it decreased body length and weight in females and increased body weight and condition factor in males. The mixture exposure resulted in a female-biased sex ratio in adults and decreased sperm density and motility in males and egg production in females. For the F1 offspring, decreased fertilization, delayed hatching, and increased malformation were found in all exposure groups. In conclusion, chronic co-exposure to BFRs and HMs at the environmental relevant levels not only affected growth, sex ratio, and sperm quantity/quality and egg production in adults but also reduced the reproductive success in the offspring, implying that multi-pollutants in the environmental media may pose a public health risk to other exposed organisms or human beings.

Fragment-Based Lead Generation of 5-Phenyl-1H-pyrazole-3-carboxamide Derivatives as Leads for Potent Factor Xia Inhibitors.

FXIa is suggested as a major target for anticoagulant drug discovery because of reduced risk of bleeding. In this paper, we defined 5-phenyl-1H-pyrazole-3-carboxylic acid derivatives as privileged fragments for FXIa inhibitors' lead discovery. After replacing the (E)-3-(5-chloro-2-(1H-tetrazol-1-yl)phenyl)acrylamide moiety in compound 3 with 5-(3-chlorophenyl)-1H-pyrazole-3-carboxamide, we traveled from FXIa inhibitor 3 to a scaffold that fused the privileged fragments into a pharmacophore for FXIa inhibitors. Subsequently, we synthesized and assessed the FXIa inhibitory potency of a series of 5-phenyl-1H-pyrazole-3-carboxamide derivatives with different P1, P1' and P2'moiety. Finally, the SAR of them was systematically investigated to afford the lead compound 7za (FXIa Ki = 90.37 nM, 1.5× aPTT in rabbit plasma = 43.33 µM) which exhibited good in vitro inhibitory potency against FXIa and excellent in vitro coagulation activities. Furthermore, the binding mode of 7za with FXIa was studied and the results suggest that the 2-methylcyclopropanecarboxamide group of 7za makes 2 direct hydrogen bonds with Tyr58B and Thr35 in the FXIa backbone, making 7za binds to FXIa in a highly efficient manner.

Chronic PFOS Exposure Disrupts Thyroid Structure and Function in Zebrafish.

Perfluorooctane sulfonic acid (PFOS), as a potential endocrine disrupting chemical, is widely detected in the environment, wildlife and human. Currently few studies have documented the effects of chronic PFOS exposure on thyroid in aquatic organisms and the underlying mechanisms are largely unknown. The present study assessed the effect of chronic PFOS exposure on thyroid structure and function using zebrafish model. Zebrafish at 8 h post fertilization (hpf) were exposed to PFOS (250 µg/l) until 120 d post fertilization (dpf). Thyroid hormone (T3 and T4) level, thyroid morphology and thyroid function related gene expression were evaluated in zebrafish at 120 dpf. Our findings demonstrated that chronic PFOS exposure altered thyroid hormone level, thyroid follicular cell structure and thyroid hormone related gene expression, suggesting the validity of zebrafish as an alternative model for PFOS chronic toxicity screening.

Resolvin D1 stimulates alveolar fluid clearance through alveolar epithelial sodium channel, Na,K-ATPase via ALX/cAMP/PI3K pathway in lipopolysaccharide-induced acute lung injury.

Resolvin D1 (7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid) (RvD1), generated from ω-3 fatty docosahexaenoic acids, is believed to exert anti-inflammatory properties including inhibition of neutrophil activation and regulating inflammatory cytokines. In this study, we sought to investigate the effect of RvD1 in modulating alveolar fluid clearance (AFC) on LPS-induced acute lung injury. In vivo, RvD1 was injected i.v. (5 µg/kg) 8 h after LPS (20 mg/kg) administration, which markedly stimulated AFC in LPS-induced lung injury, with the outcome of decreased pulmonary edema. In addition, rat lung tissue protein was isolated after intervention and we found RvD1 improved epithelial sodium channel (ENaC) α, γ, Na,K-adenosine triphosphatase (ATPase) α1, ß1 subunit protein expression and Na,K-ATPase activity. In primary rat alveolar type II epithelial cells stimulated with LPS, RvD1 not only upregulated ENaC α, γ and Na,K-ATPase α1 subunits protein expression, but also increased Na+ currents and Na,K-ATPase activity. Finally, protein kinase A and cGMP were not responsible for RvD1's function because a protein kinase A inhibitor (H89) and cGMP inhibitor (Rp-cGMP) did not reduce RvD1's effects. However, the RvD1 receptor (formyl-peptide receptor type 2 [FPR2], also called ALX [the lipoxin A4 receptor]) inhibitor (BOC-2), cAMP inhibitor (Rp-cAMP), and PI3K inhibitor (LY294002) not only blocked RvD1's effects on the expression of ENaC α in vitro, but also inhibited the AFC in vivo. In summary, RvD1 stimulates AFC through a mechanism partly dependent on alveolar epithelial ENaC and Na,K-ATPase activation via the ALX/cAMP/PI3K signaling pathway.

Novel Anthranilamide-Based FXa Inhibitors: Drug Design, Synthesis and Biological Evaluation.

Factor Xa (FXa) plays a significant role in the blood coagulation cascade and it has become a promising target for anticoagulation drugs. Three oral direct FXa inhibitors have been approved by the FDA for treating thrombotic diseases. By structure-activity relationship (SAR) analysis upon these FXa inhibitors, a series of novel anthranilamide-based FXa inhibitors were designed and synthesized. According to our study, compounds 1a, 1g and 1s displayed evident FXa inhibitory activity and excellent selectivity over thrombin in in vitro inhibition activities studies. Compounds 1g and 1s also exhibited pronounced anticoagulant activities in in vitro anticoagulant activity studies.

A novel humanized anti-HER2 antibody conjugated with MMAE exerts potent anti-tumor activity.

Human epidermal growth factor receptor-2 (HER2) is a validated therapeutic target for breast cancer and trastuzumab (Herceptin), a humanized anti-HER2 antibody, has significant anti-cancer effects in the clinic. However, breast cancer patients often experience disease progression after prolonged Herceptin treatment. To develop a more effective therapy, we generated humanized monoclonal antibody hertuzumab and hertuzumab-drug conjugates as novel breast cancer therapies. The hertuzumab was conjugated with small molecule cytotoxic agents monomethylauristatin E (MMAE) or monomethylauristatin F (MMAF) with various linkers to generate antibody-drug conjugates (ADCs), which were evaluated for their in vitro and in vivo anti-cancer activities. Among these ADCs, hertuzumab-vc-MMAE can be effectively internalized and potently kill HER2 over-expressing tumor cells. In xenograft tumor models, hertuzumab-vc-MMAE showed a more potent anti-tumor activity than T-DM1, a FDA-approved ADC drug. More importantly, this novel ADC drug also showed superior anti-tumor activity than T-DM1 in trastuzumab- and lapatinib-resistant xenograft tumor models, suggesting its potential as an improved therapy for HER2-positive breast cancers. The novel ADC, hertuzumab-vc-MMAE, is an effective and selective agent for the treatment of HER2-positive breast tumors.

Upregulation of uncoupling protein Ucp2 through acute cold exposure increases post-thaw sperm quality in zebrafish.

Oxidative stress plays an important role in sperm damage during cryopreservation. Mild mitochondrial uncoupling has been shown to reduce excessive reactive oxygen species (ROS) and thus mitigate oxidative stress. Uncoupling protein (Ucp2) regulates mitochondrial uncoupling and can be induced by temperature fluctuation. In the present study, we explored a novel approach of acute cold exposure on Ucp2 activation and its association with oxidative damage and post-thaw sperm quality in zebrafish. Our study revealed that acute cold exposure of zebrafish at 18 °C for 24 h led to significant increase of ucp2 mRNA and Ucp2 protein in zebrafish fresh sperm as well as thawed sperm after cryopreservation. Although cold exposure had no effect on fresh sperm quality except for decreasing lipid peroxidation, sperm collected from cold-exposed zebrafish exhibited higher resistance to cryodamage, which was demonstrated by increased post-thaw motility, decreased lipid peroxidation, increased ATP production, and ultimately increased fertilization success. However, except for reduced lipid peroxidation, we did not observe any significant ROS reduction associated with increased Ucp2 activation in cold-exposed group, suggesting mechanisms other than mitochondrial uncoupling could have contributed to cold exposure associated benefits in post-thaw sperm survival. Nevertheless, our findings indicate that acute cold exposure prior to sperm cryopreservation is beneficial for post-thaw sperm survival in zebrafish, and this novel approach may be used to improve post-thaw sperm quality for other aquatic species.

Inhibition of ROS production through mitochondria-targeted antioxidant and mitochondrial uncoupling increases post-thaw sperm viability in yellow catfish.

Reactive oxygen species (ROS) are one of the main causes for decreased viability in cryopreserved sperm. Many studies have reported the beneficial effect of antioxidant supplements in freezing media for post-thaw sperm quality. In the present study, we explored two new approaches of ROS inhibition in sperm cryopreservation of yellow catfish, namely mitochondrial-targeted antioxidant and metabolic modulator targeting mitochondrial uncoupling pathways. Our study revealed that addition of MitoQ, a compound designed to deliver ubiquinone into mitochondria, significantly decreased ROS production, as well as lipid peroxidation, and increased post-thaw viability. Similarly, sperm incubated with 2,4-dinitrophenol (DNP), a chemical protonophore that induces mitochondrial uncoupling, also had reduced ROS production, as well as lipid peroxidation, and increased post-thaw sperm viability. Conversely, activation of uncoupling protein (UCP2) by 4-hydroxynonenal (HNE) neither reduced ROS production nor increased post-thaw sperm viability. Our findings indicate that ROS inhibition through mitochondrial-targeted antioxidant or mild mitochondrial uncoupling is beneficial for sperm cryopreservation in yellow catfish. Our study provides novel methods to mitigate oxidative stress induced damage in cryopreserved sperm for future applications.