Nuclear Status Research Articles

Polityka Francji i Niemiec wobec niepodległej Ukrainy. Wokół stabilizacji i bezpieczeństwa

This essay provides the shaping of the leading EU countries’ policy towards independent Ukraine. It examines that it was influenced by several factors: including the history of relations between each country with the Russian Empire and the USSR, and the lack of experience in cooperation with Ukraine as an independent state, and geopolitical interests of each state. The German political circles and the media as well as the French ones have been expressed their concern over the possible proliferation of nuclear weapons by Ukraine, which in turn has led to political and economic pressure on Ukraine’s stance on its nuclear status. The essay proves that the issue of nuclear technologies was not limited to the discussion of the former existence of a nuclear arsenal in Ukraine. France and Germany take an active part in the activities in the Chernobyl zone. Leading EU countries in the 1990s hoped there will be democratic changes in Russia, transparency and the development of civil society. Ukraine’s multi-vector policy was incomprehensible to Western countries, as was the security vacuum felt by Ukraine, which was gradually trapped in the geopolitical grip of two defense blocs: NATO in the West and Collective Security Treaty Organisation (Tashkent Pact) in the East. Since 2014, France and Germany have been mediators in the resolution of the Russian-Ukrainian conflict in Donbas in the framework of Normandy format, but so far there is no progress in resolving this low-intensity conflict.

133 Effect of cytokines on the quality bovine oocytes matured invitro

As has been shown previously, fibroblast growth factor 2 (FGF2), leukaemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1) in combination play a positive role in maintaining the quality of mammalian oocytes maturing invitro. In the present work, we studied the effects of these cytokines in optimal concentrations when added in combination to IVM medium on the nuclear status and development competence of bovine oocytes. Slaughterhouse-derived cumulus–oocyte complexes (COC) (n=1107 COC) were cultured for 22h in either IVM medium [TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2mM sodium pyruvate, 10μgmL−1 porcine FSH, and 10μgmL−1 ovine LH] (Control) or the same IVM medium supplemented with FGF2, LIF, and IGF1. The eight combinations of cytokines tested during maturation were (1) 20ngmL−1 LIF/10ngmL−1 IGF1/10ngmL−1 FGF2 (Group 1); (2) 20ngmL−1 LIF/10ngmL−1 IGF1/40ngmL−1 FGF2 (Group 2); (3) 20ngmL−1 LIF/20ngmL−1 IGF1/10ngmL−1 FGF2 (Group 3); (4) 20ngmL−1 LIF/20ngmL−1 IGF1/40ngmL−1 FGF2 (Group 4); (5) 5ngmL−1 LIF/10ngmL−1 IGF1/10ngmL−1 FGF2 (Group 5); (6) 5ngmL−1 LIF/10ngmL−1 IGF1/40ngmL−1 FGF2 (Group 6); (7) 5ngmL−1 LIF/20ngmL−1 IGF1/10ngmL−1 FGF2 (Group 7); and (8) 5ngmL−1 LIF/20ngmL− 1 IGF1/40ngmL−1 FGF2 (Group 8). After IVM, matured and denuded oocytes were activated by culturing in 5μM ionomycin solution for 5min followed by 4h in 2mM 4-dimethylaminopyridine (DMAP) and 10mgmL−1 cyclohexamide. Activated oocytes were cultured in CR1aa medium until Day 5 and then transferred to the same medium supplemented with 5% FCS and cultured until Day 7. All cultures were performed at 38.5°C and 5% CO2 in humidified air. At Days 2 and 7 after activation, the cleavage and blastocyst rates were determined. The data from 6 to 7 replicates (122–181 oocytes per treatment) were analysed by ANOVA. The rate of MII oocytes, assessed by the presence of the first polar body, did not differ between the groups and reached 67.1 to 84.5%. Cleavage rate was higher (84.5±2.9%, P<0.05) in Group 1 compared with Control (68.9±2.7%), Group 4 (67.1±4.9%) and Group 7 (66.6±2.0%), but not compared with Group 2 (76.5±4.8%), Group 3 (76.6±4.8%), Group 5 (78.2±3.1%), or Group 8 (79.9±3.1%). The percentage of blastocyst formation relative to the total number of MII oocytes in the control group was 19.6±2.4%. The culture of COC in Group 1 (20 ngmL−1 LIF/10ngmL−1 IGF1/10ngmL−1 FGF2) and Group 2 (20ngmL−1 LIF/10ngmL−1 IGF1/40ngmL−1 FGF2) caused the blastocyst yield to increase to 33.0±3.8 and 31.2±4.3%, respectively (P<0.05), whereas the culture COC in other cytokine-treated groups had no effect. In Groups 3 to 8, the blastocyst rates were 22.9±4.6, 19.2±3.0, 24.2±3.2, 27.8±1.7, 21.6±1.8, and 25.5±9.0%, respectively, and did not differ (except Group 4) compared with those of Group 1 and Group 2. In conclusion, LIF, FGF2, and IGF1 in optimal combinations can maintain competence for parthenogenetic development of bovine COC during their maturation invitro. This research was supported by RFBR (projects No. 18-29-07089) and the Ministry of Science and Higher Education of Russia.

85 The effects of heat exposure on the growth and developmental competence of oocytes derived from early antral follicles in dairy cows

Summer heat stress in dairy cows impairs the developmental competence of oocytes from antral follicles (2–8mm) which are used in conventional IVM and IVF systems. Moreover, summer heat stress is considered to impair the oocyte competence derived from smaller follicles; therefore, the impairment of oocyte competence possibly continues into the cooler autumn season. To investigate the thermosensitivity of early antral follicles (<1mm), we evaluated the effects of heat exposure on the growth and developmental competence of oocytes using invitro culture of oocyte–cumulus-granulosa complexes (OCGCs) derived from early antral follicles. OCGCs (n=315) were collected from early antral follicles (0.5–1mm) and cultured for 12 days. OCGCs in the heat shock group were cultured using a temperature cycle of 38.5°C for 5h, 39.5°C for 5h, 40.5°C for 5h, and 39.5°C for 9h, whereas those in the control group were cultured at a constant temperature of 38.5°C for 24h. The diameters of oocytes were measured before culture. Half of the culture medium was replaced every 4 days. Oestradiol-17β (E2) and progesterone (P4) production during the first, second, and third 4-day periods were measured by enzyme immunoassay; the viability of OCGCs was evaluated based on their morphology. Oocytes that survived after 12 days of culture (n=191) were subjected to IVM (38.5°C, 22h); their diameter and nuclear status were evaluated. Some oocytes (n=71) were subjected to IVF (38.5°C, 18h) and embryo culture (39.0°C, 150h). Cleavage and blastocyst rates were evaluated at 48h and 168h after IVF. Effects of treatment groups and culture periods on E2 and P4 production and diameters of oocytes were evaluated by two-way ANOVA followed by Tukey-Kramer or Student’s t-test. The viability of OCGCs, nuclear maturation, cleavage and blastocyst rates between two groups were compared by the chi-squared or Fisher’s exact test. E2 and P4 production and the viability of OCGCs were not different between the 2 groups. Although mean oocyte diameters before culture did not differ between the 2 groups, the mean diameters after IVM were significantly smaller in the heat shock group (108.0µm, n=56) than in the control group (111.7µm, n=61; P<0.05). The nuclear maturation rate in the heat shock group (36.4%, n=55) was significantly lower than in the control group (60.3%, n=58; P<0.05). Cleavage rates were similar between the control (54.5%, n=33) and heat shock groups (45.7%, n=35). However, no oocytes developed to blastocysts in the heat shock group (0%, n=35), whereas 30.3% (n=33) oocytes developed to blastocysts (cell number±s.d.; 92.4±28.4) in the control group (P<0.05). These findings suggest that summer heat stress in dairy cows impairs the growth, nuclear maturation, and developmental competence of oocytes derived from early antral follicles. This experimental model could be used to explore the mechanisms by which heat stress subsequently impairs oocyte competence during the cooler autumn season.

Computational docking and in vitro analysis identifies novel arylidene analogue FPMXY-14 against renal cancer cells by attenuating Akt.

Targeted therapies are gaining global attention to tackle Renal Cancer (RC). This study aims to screen FPMXY-14 (novel arylidene analogue) for Akt inhibition by computational and in vitro methods. FPMXY-14 was subjected to proton NMR analysis and Mass spectrum analysis. Vero, HEK-293, Caki-1, and A498 cell lines were used. Akt enzyme inhibition was studied with the fluorescent-based kit assay. Modeller 9.19, Schrodinger 2018-1, LigPrep module, and Glide docking were used in computational analysis. The nuclear status was assessed by PI/Hoechst-333258 staining, cell cycle, and apoptosis assays were performed using flow cytometry. Scratch wound and migrations assays were performed. Western blotting was applied to study key signalling proteins. FPMXY-14 selectively inhibited kidney cancer cell proliferation with GI50 values of 77.5 nM and 101.40 nM in Caki-1 cells and A-498 cells, respectively. The compound dose-dependently inhibited Akt enzyme with an IC50 value of 148.5 nM and bound efficiently at the allosteric pocking of the Akt when computationally analyzed. FPMXY-14 caused nuclear condensation/fragmentation, increased the sub G0/G1, G2M populations, and induced early, late phase apoptosis in both cells when compared to controls. Treatment of the compound inhibited wound healing and migration of tumor cells, while proteins like Bcl-2, Bax, and caspase 3 were also altered. FPMXY-14 effectively inhibited the phosphorylation of Akt in these cancer cells, while total Akt was unaltered. FPMXY-14 exhibited anti-proliferative and anti-metastatic activities in kidney cancer cells by attenuating the Akt enzyme. Further pre-clinical research on animals with a detailed pathway elucidation is recommended.

Effect of roscovitine pretreatment on in vitro maturation of oocytes and their subsequent developmental after chemical activation in dromedary camel (Camelus dromedarius)

Studies were conducted to evaluate an optimal concentration of roscovitine needed to maintain abattoir origin oocytes at germinal vesicle stage in experiment 1 and their subsequent maturation and developmental competence after chemical activation in experiments 2 and 3, respectively. The cumulus-oocyte complexes (COCs) aspirated from ovaries collected from a local slaughterhouse were cultured in TCM-199 based pre-maturation medium supplemented with 25, 50 or 75 μM roscovitine, depending on the experimental group. After 24 h, the COCs were denuded of cumulus, fixed and stained with aceto-orcein and examined for their nuclear status. They were classified as germinal vesicle, diakinesis, metaphase-I, metaphase-II and those with degenerated, fragmented, scattered, activated or without visible chromatin as others. In experiment 2, the COCs pre-matured in media supplemented with 50 μM roscovitine for 24 h were washed and kept for in vitro maturation along with another group of freshly collected COCs for 30 h. All the oocytes were fixed and stained to evaluate their nuclear status as described above. In experiment 3, all mature oocytes obtained from the COCs pre-matured in media supplemented with 50 μM roscovitine and those obtained from freshly collected group were activated by 5 mM ionomycin. Activated oocytes were cultured in embryo culture medium for a period of 7 days to evaluate their developmental potential. The proportion of oocytes at GV stage in the group pre-matured in media with 50 μM-was significantly (P < 0.01) higher when compared with the group having 25 μM of roscovitine. No difference was found in the proportion of GV stage oocytes in this group when compared with the freshly collected COCs. None of the oocytes reached to M-II stage in any of the three treatment groups. In experiment 2, no difference was observed in the proportion of oocytes reaching M-II stage between the groups after 30 h of in vitro culture; however, higher proportion of oocytes (P < 0.05) were classified as others in the pre-maturation group when compared with the group having freshly collected oocytes. In experiment 3, no difference was observed in the proportion of oocytes cleaving and those developing to the blastocyst stage between the pre-matured and freshly matured groups. In conclusion, the present study, for the first time, demonstrates the possible use of roscovitine as a meiotic inhibitor for camel oocytes. Keeping in view the ability of these oocytes to mature and develop to the blastocyst stage at par with the fresh oocytes, more flexible schedules for maturation and manipulation of such oocytes could be developed.

Progesterone Prolongs Viability and Anti-inflammatory Functions of Explanted Preterm Ovine Amniotic Membrane.

Amniotic membrane (AM) is considered an important medical device with many applications in regenerative medicine. The therapeutic properties of AM are due to its resistant extracellular matrix and to the large number of bioactive molecules released by its cells. An important goal that still remains to be achieved is the identification of cultural and preservation protocols able to maintain in time the membrane morphology and the biological properties of its cells. Recently, our research group demonstrated that progesterone (P4) is crucial in preventing the loss of the epithelial phenotype of amniotic epithelial cells in vitro. Followed by this premise, it has been evaluated whether P4 may also affect AM properties in a short-term culture. Results confirm that P4 preserves AM integrity and architecture with respect to untreated AM, which showed alterations in morphology. Transmission electron microscopy (TEM) analyses demonstrate that P4 also maintains unaltered cell–cell junctions, nuclear status, and intracellular organelles. On the contrary, an untreated AM experienced an extensive cell death and a strong reduction of immunomodulatory properties, measured in terms of anti-inflammatory cytokine expression and secretion. Overall, these results could open to new strategies to ameliorate the protocols for cryopreservation and tissue culture, which represent preliminary stages of AM application in regenerative medicine.

Ядерний статус Франції як інструмент реалізації її зовнішньополітичних інтересів

The article reveals the significance of the usage of France’s nuclear status as a tool for the realisation of its foreign policy interests in the International Arena, especially in the context of necessity to ensure its security. A well-formulated and scientifically grounded, clear and reasonably transparent strategy for Foreign Policy ensures state security and is an inalienable attribute of a civilised, contemporary International Actor. One of the most critical aspects of France’s Defence strategy is based on its nuclear status. Therefore, it is essential to understand its role and function in the formulation of the foreign policy strategy. In particular, the concept of «nuclear diplomacy» is explored on the example of the French Republic. This concept describes the possibility of using nuclear status in various fields of foreign policy to achieve the interests of the state in the world arena. The survey describes political, economic and security aspects of the disposition of nuclear diplomacy in foreign policy. Also, the historical experience of exercising the nuclear status as a tool has been analysed. Relying on the analysis made it has been found that the practice of using nuclear status as a tool for conducting international dialogue has its several advantages, the main of which is the efficient protection of the national interests of the state. The author emphasises the fact that the wise usage of all nuclear status benefits, in the present conditions of growing instability and turbulence in international relations is a vital requirement for the development of France, especially as for nuclear-weapon state and a significant actor in the international arena. It has been argued that the nuclear status is inextricably associated not only with the formation of a state security strategy but also with the development of the image and geopolitical component of security, aimed at providing the necessary conditions for the realisation of French national interests in the International Arena, as one of the most effective methods of guaranteeing security and independence in the modern world. The modern world is the world of pervasive transformations and breakthroughs connected with the degeneration of traditional and modern structures. Although today the world is interconnected and united more than ever before, it is necessary to respect and estimate the idea of boundaries, sovereignty, and independence of a state, the right of a state to lead its policy. So, the best guarantee of directing separate political line it is wise using of one of the most effective and impressive tools, in a Foreign Policy too, that is nuclear status.

Корея в начале XXI века и интересы России

The article is a response to a new, pioneering book Modern Korea. Metamorphoses of Turbulent Years (2008–2020) written by a group of leading Russian experts on Korea from MGIMO University: Anatoly Torkunov, Georgy Toloraya, and Ilya Dyachkov. The book is a valuable addition to the existing literature and a product of a unique approach to modern Korean studies conducted at MGIMO. The article examines and develops the comprehensive analysis provided by MGIMO professors, point out pressing issues on the Korean Peninsula and assess Russia’s potential role in solving them. The fact that the goal of denuclearizing the Korean Peninsula based on Pyonyang’s rejection of nuclear weapons not only has not been achieved, but became even more distant than before, leads us to questioning the logic behind the authors' recommendation for Russia to withdraw its support from the international system of sanctions against Pyongyang and to move closer to North Korean. We argue that easing the sanctions may mean recognizing that Russia does not believe in this goal and wishes to encourage North Korea's refusal to comply with the demands of the international community. Moreover, such an approach could be perceived as evidence of Russia’s support of some new academic theories which claim that the very system of nonproliferation has become outdated and can even be abolished altogether. At the same time, this position would reduce the role of Russia in the Korean settlement, which would inevitably have a negative impact on DPRK's renunciation of nuclear weapons in the foreseeable future. The article argues that such views, especially that the non-proliferation strategy is outdated, come into a direct contradiction with Russia's interests. The recognition of DPRK's nuclear status may spark a chain reaction in the quest for nuclear weapons in the region which in turn would create a serious security threat for Russia's eastern regions. In addition, an increase in the number of nuclear states would devalue Russia’s status as a nuclear power, thus, the policy of nonproliferation of nuclear weapons should remain a priority of Russia’s foreign policy. Overall, we conclude that Moscow's policy towards the Korean peninsula should be based on careful evaluation of the current international situation as well as Russia’s interests, and not on outdated and often counterproductive Soviet tradition.

ECONOMIC COMPONENT OF FRENCH NUCLEAR STATUS

The article reveals the significance of the economic component of the French Republic’s nuclear status notion in the context of ensuring its security and support of military power, and especially the financial support of this concept, as one of the most effective methods of guaranteeing security and independence in the modern world. The author emphasizes the fact that in present conditions, considering growing instability and international relations turbulence, the economic component tends to be a vital requirement for the development of state nuclear safety, on the example of France. Relying on the analysis of the economic component and the definition of the French nuclear power notion, it is possible to predict and assess the main trends in the development of the state security system and its defence strategy, as well as to formulate ideas about threats and national interests. It has been argued that the economic component of nuclear status is inextricably associated not only with the formation of a state security strategy but also with the development of the image and geopolitical component of security, aimed at providing the necessary conditions for the realization of French national interests.

37 In vitro maturation and fertilization in white-tailed deer (Odocoileus virginianus) oocytes vitrified with trehalose or sucrose

White-tailed deer (WTD) invitro embryo production is not well documented, due to the seasonality of the wildlife species and available hunting permits. The objective of the present study was to conduct IVM and IVF using WTD oocytes vitrified with trehalose (TH) or sucrose (SC). A total of 121 immature oocytes were obtained from ovaries (n=18) using the slicing technique from hunter-slaughtered deer, not more than 2h after death. The WTD oocytes were vitrified using a solid surface vitrification technique in two different groups: 1) TH (n=60) and 2) SC (n=61). Oocyte warming (OW) was done using four concentrations (OW1 1 M, OW2 0.5 M, OW3 0.25 M, and OW4 0 M) in the oocytes after thawing. Then, a sample was used to evaluate viability for TH (n=5) and SC (n=5) (MTT stain (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), 0.5mgmL−1) and nuclear status (NS) for TH (n=4) and SC (n=5) with 4′,6-diamidino-2-phenylindole (1μgmL−1), defining the stage as germinal vesicle (GV), MI, and unable to evaluate (NE). The remaining oocytes (n=88) were used for IVM for 36h in tissue culture medium-199 supplemented with human menopausal hormone (75 UImL−1) and epidermal growth factor (10ngmL−1). The evaluation of NS was defined (GV, MI considered immature and MII matured). The IVF (n=15) took place in Tyrode's albumin lactate pyruvate media supplemented with heparin (10μgmL−1), penicillamine (0.075mgmL−1), hypotaurine (10M), epinephrine (1μM), and bovine serum albumin fraction V (0.4%), with a final semen concentration of 3×106 spermmL−1. Frozen straws of conventional WTD semen were used; the straws were split into 3 parts, with one fraction thawed and capacitated using “swim-up” technique. After 24h of incubation at 38.5°C with 5% CO2, and humidified air, nuclear evaluation was made as fertilized (F), not fertilized (NF), or NE. A Fisher exact test was used (SAS, 9.0 version for Windows), α=0.05. After warming, for the TH group (n=5), viability was 60% and nuclear status (n=4) was 50% GV, 50% MI, and 0% NE. For the SC group (n=5), viability was 40% and nuclear status (n=5) was 60% GV, 20% MI, and 20% NE. After 36h IVM, NS evaluation in the TH group (n=38) was 66% GV, 24% MI, 0% MII, and 10% NE; for the SC group (n=50), 84% GV, 10% MI, 2% MII, and 4% NE, not a statistically significant difference (P&amp;gt;0.05). For IVF after 24h, the NS evaluation in the TH treatment (n=10) was 0% F, 60% NF, and 40% NE versus the SC treatment (n=5) with 20% F, 40% NF, and 40% NE, not a statistically significant difference (P&amp;gt;0.05). A statistically significant difference (P&amp;gt;0.05) was not found between the TH and SC groups with regard to post-thaw viability, IVM, and IVF. Future research with larger numbers of immature or mature oocytes is suggested for further evaluation of both TH and SC for WTD oocyte vitrification for use with invitro-production techniques as a model for other endangered cervid species.

Mancozeb exposure during development and lactation periods results in decreased oocyte maturation, fertilization rates, and implantation in the first-generation mice pups: Protective effect of vitamins E and C.

This study aimed to evaluate the mancozeb (MNZ) impact on oocyte maturation of first-generation mice pups as well as their fertilization rate, embryo development, and implantation along with the preventative effect of vitamins E and C. Pregnant mice were randomly divided into six groups: control, vehicle, and MNZ (500 mg/kg body weight (BW)), vitamin E (200 mg/kg BW), MNZ plus vitamin E, MNZ plus vitamin C (100 mg/kg BW), and MNZ plus two vitamins. All treatments were conducted by oral gavage every 2 days from the second day of gestation until the end of lactation. Vitamin treatment was initiated 30 min before receiving MNZ. After birth, first-generation mice pups were kept until adulthood (8-10 W). Adult female mice pups superovulated and then the collected oocytes were examined for nuclear maturity status. After in vitro fertilization of metaphase II oocytes with sperm of the first-generation male mice pups, fertilization rate and embryo development were evaluated over 24 h. Also, the fecundity rate and the number of implanted embryos in vivo were studied on the eighth day of pregnancy. MNZ exposure during embryo development and lactation significantly decreased the total number of collected oocytes, oocyte maturation, fertilization rate, implantation rate, fecundity rate, and embryo development compared with the control group in the first-generation pups. In contrast, vitamin treatments significantly increased these parameters compared to the MNZ group. Reduction in the quality of oocyte, the rate of fertilization, embryo implantation, and development following MNZ exposure could decrease female reproductive success, while coadministration of vitamins E and C could prevent these complications.

Production of collared peccary (Pecari tajacu Linnaeus, 1758) parthenogenic embryos following different oocyte chemical activation and in vitro maturation conditions

To optimize the protocols for assisted reproductive techniques (ARTs) in collared peccary (Pecari tajacu Linnaeus, 1758), we evaluated various conditions for oocyte in vitro maturation (IVM) and chemical activation. Initially, we assessed the IVM rates, cumulus-oocyte complex (COC) quality, and oocyte morphometry in the absence or presence of epidermal growth factor (EGF). There was no difference between the COCs matured in absence or presence of EGF for the expansion of cumulus cells (97.6% ± 1.2 vs. 100% ± 0.0), presence of first polar body (65.9% ± 1.2 vs. 70.5% ± 1.8), nuclear status in second metaphase (62.5% ± 11.6 vs. 68.4% ± 4.9), cytoplasmic maturation (100.0% ± 0.7 vs. 75.0% ± 0.7), reactive oxygen species levels (0.5 ± 0.2 vs. 0.3 ± 0.1), and mitochondrial membrane potential (1.1 ± 0.2 vs. 1.1 ± 0.1). However, the zona pellucida thickness of matured COCs was reduced in the presence of EGF. Thus, the EGF group was used for further experiments. The oocytes were artificially activated with ionomycin and four secondary activator combinations [6-dimethylaminopurine (6D), 6D and cytochalasin B (6D + CB), cycloheximide (CHX), and CHX and CB (CHX + CB)]. The effect of immature COCs based on cumulus cell layers and cytoplasm homogeneity (GI and GII or GIII COCs) on embryonic development and quality was evaluated. There was no difference in the cleavage rates among the groups of secondary activators. The cleavage rates of embryos derived from GI/GII and GIII COCs were greater than 72.2% and 25.0%, respectively. Moreover, treatment with CHX showed a reduction in the cleavage rate of embryos derived from GIII COCs when compared to the cleavage rate of embryos derived from GI/GII COCs (P < 0.05). Nevertheless, higher rates of blastocyst/total GI and GII COCs were observed in the 6D group (27.6% ± 0.3) compared to CHX group (6.9% ± 0.3). Additionally, only 6D treatment resulted in the production of embryos derived from GIII COCs (25.0% ± 0.2). The percentage of the ICM/total cell ratio was also greater in blastocysts derived from 6D (42.5% ± 19.0), 6D + CB (37.9% ± 21.9), and CHX + CB (43.8% ± 19.6) groups when compared to CHX (3.6% ± 0.1) group. Thus, the combination of ionomycin and 6D could produce collared peccary embryos by activation of both GI/GII COCs and GIII COCs. These optimized IVM conditions using EGF and chemical activation using ionomycin and 6D in collared peccaries form the first steps for establishing ARTs to conserve this species.

Law as discursive resource: the politics of the nuclear/non-nuclear distinction in the Non-Proliferation Treaty

Realist approaches to international law conceptualize the law as epiphenomenal to state interest, whereas liberal institutionalist approaches theorize the ability of law to curb state power. Through the example of the Treaty on the Non-Proliferation of Nuclear Weapons, this article challenges these approaches by arguing that law’s power comes from its productive and constitutive effects. Despite perennial conflict, the Treaty on the Non-Proliferation of Nuclear Weapons endures because it has ordered nuclear politics by constituting a legal distinction between “nuclear weapon states” and “non-nuclear weapon states.” Instead of assuming that this distinction reflects self-evident material differences, this article shows how states actively construct nuclear status through international law. The dynamics of this construction reflect significant actions on the behalf of conventionally disempowered states and not merely great powers. An analysis of the meeting documents of the Eighteen Nation Disarmament Committee finds that the participants used the forum to perform a burgeoning “non-nuclear” identity. The politics of this distinction also generated the discourse of “nuclear apartheid,” which was subsequently used by states outside the Treaty on the Non-Proliferation of Nuclear Weapons regime to justify their pursuit of nuclear weapons. Taken together, the role of non-nuclear diplomacy and the discourse of nuclear apartheid demonstrate that the Treaty on the Non-Proliferation of Nuclear Weapons does not simply endure because the powerful have sanctioned it, but because it created a space for the disempowered to expand their influence from below. Though the article builds on existing sociological approaches to the law, it also moves beyond conflicts over legal and textual interpretation to demonstrate the diplomatic practices around the constitution of legal categories.

Theca cells can support bovine oocyte growth in vitro without the addition of steroid hormones

Theca cells (TCs) are essential to folliculogenesis by contributing to steroidogenesis. However, the in vitro growth (IVG) of oocytes co-cultured with TCs has not yet been examined. In the present study, we investigated the feasibility of the IVG of bovine oocyte-cumulus-granulosa cell complexes (OCGCs) co-cultured with TCs and the developmental competence of co-cultured oocytes. OCGCs and TCs were co-cultured without steroid hormone addition for 12 days. Steroidogenesis, the viability of OCGCs, and TC numbers during co-culture were assessed every 4 days. After IVG, oocytes were matured and the nuclear status was evaluated. Some oocytes were inseminated and cultured to examine blastocyst development. During the co-culture, androstenedione production by TCs was only observed during the first 4 days (1.1 ng/well) while estradiol-17β was continuously produced, peaking during the second 4 days (0.5 ng/well). The number of TCs decreased to ∼60% of the seeding number (4.0 × 104 cells/well) during the first 4 days, and was maintained thereafter. The majority of co-cultured OCGCs (82.7%) survived after 12-day IVG. Only a few OCGCs (6.2%) survived in the OCGC culture without TCs (p < 0.01); however, the addition of androstenedione to the culture medium markedly improved survivability to 80.1%, which was similar to that in the co-culture with TCs. In the subsequent development of oocytes derived from the co-culture, 58.3% reached metaphase II stage, 58.7% cleaved, and 17.3% developed to blastocysts, which were similar values to those of oocytes cultured with the addition of androstenedione. In conclusion, TC-produced androgen contributes to OCGC growth and the acquisition of subsequent embryonic developmental competence.

(2E)-2-Benzylidene-4,7-dimethyl-2,3-dihydro-1H-inden-1-one (MLT-401), a novel arylidene indanone derivative, scavenges free radicals and exhibits antiproliferative activity of Jurkat cells

Abstract Background The arylidene indanone scaffold has contributed many lead molecules in chemotherapeutic anticancer agent research. Objectives To determine the oxidant-scavenging activities and antiproliferative activity of (2E)-2-benzylidene-4,7-dimethyl-2,3-dihydro-1H-inden-1-one (MLT-401), an arylidene indanone derivative. Methods Jurkat cells, primary lymphocytes, and Vero cells were treated with MLT-401. Antioxidant properties of MLT-401 were determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH)-based, 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS)-based, and ferric-reducing antioxidant potential (FRAP) assays. Inhibition of cell proliferation was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based assay. Nuclear status was determined using a DNA fragmentation assay, and cell cycle stage was analyzed by flow cytometry. Mitochondrial membrane enzyme activities were measured using colorimetric methods. Results The antioxidant assays gave MLT-401 half maximal inhibitory concentration (IC50) values of 1611 nM (DPPH-based assay), 2115 nM (ABTS-based assay), and 1586 nM (FRAP assay). MLT-401 inhibited proliferation of Jurkat cells with a concentration for 50% of maximal inhibition of cell proliferation (GI50) of 341.5 nM, being 12- and 9-fold less than GI50 concentrations for normal lymphocytes and Vero cells, respectively. MLT-401 caused nuclear fragmentation and DNA laddering as seen by electrophoresis. Jurkat cells showed a time-dependent accumulation of sub G0/G1 cells after MLT-401 treatment. Mitochondrial membrane-bound Na+/K+ ATPase, Ca2+ ATPase, and Mg2+ ATPase activities were inhibited by MLT-401 in a dose-dependent manner. Conclusion MLT-401 possesses significant antiproliferative activity and scavenges free radicals released through mitochondrial membrane damage in a Jurkat cell line model of cancer cells. Further investigation of MLT-401 as a chemotherapeutic anticancer agent and development of other arylidene indanone analogues are warranted. A detailed elucidation of mechanistic pathways is required for further development.

Transformation of ram sperm nuclei in oocytes cytoplasm during in vitro fertilization

The aim of present study was to understand the transformation of ram sperm nuclei within oocyte cytoplasm during in vitro fertilization. The oocytes were collected from slaughterhouse ovaries. Before fertilization, the oocytes were maturated in vitro for 24 hours in the incubator with 5% CO2 at 38.5°C. Then the oocytes (n= 635) was fertilized by incubating the oocytes with sperm (5x106 spermatozoa/ ml) for 3, 6, 9, 12, and 15 hours. At the end of incubating period, the oocytes were fixed and stained with aceto-orcein 2% before evaluated under phase contrast microscope. Sperm nuclear transformation was evaluated according to sperm nuclear status of sperm, such as condensation, decondensation, and formation of prepronuclei and pronuclei. Sperm condensation and decondensation were seen at 3 hours after incubation. Prepronuclei and pronuclei were found at 6 hours of incubation. Pronuclei formation was significantly increased in the 9 hours after incubation (P&lt;0.05). The incidence of polyspermia was significantly increased at 12-15 hours after incubation (P&lt;0.05). In conclusion penetration of sperm into oocytes has been occurred at 3 hour of fertilization period. The formation of pronuclei was found at 6 hours after incubation and the incidence of polyspermia was increased when the fertilization period prolonged.

Hydrogen peroxide concentration and DNA fragmentation of buffalo oocytes matured in sericin-supplemented maturation medium

This study was conducted to investigate the effects of sericin on meiotic maturation, hydrogen peroxide (H 2 O 2 ) concentration and deoxyribonucleic acid (DNA) fragmentation in buffalo oocytes. Oocytes were matured in vitro in tissue culture medium (TCM199) with in vitro maturation (IVM) groups under several conditions, namely without bovine serum albumin (BSA), (+BSA), and 0.025%, 0.05%, 0.1% and 0.25% (w/v) sericin. The results showed that supplementation of the maturation medium with 0.05% sericin significantly increased the rate of oocytes that reach metaphase II compared with other groups, except for the 0.025% sericintreated group. Intracellular H 2 O 2 concentrations in oocytes of all groups were measured using 2',7'dichlorodihydrofluorescein diacetate (DCHFDA). The concentration of H 2 O 2 in matured oocytes treated with 0.05% sericin was lower than in other groups. DNA fragmentation and the nuclear status of oocytes were examined using the terminal deoxyribonucleotidyl transferase-mediated dUTP digoxigenin nick endlabelling (TUNEL) method. The total proportion of TUNELpositive oocytes at the MII stage was lower in the 0.05% sericin group. The results indicate that addition of 0.05% sericin to the maturation medium may improve nuclear maturation and may inhibit DNA fragmentation in oocytes by decreasing H2O2 concentrations. Keywords: antioxidant, apoptosis, in vitro maturation, reactive oxygen species

ЯДЕРНЕ РОЗЗБРОЄННЯ УКРАЇНИ: ЧИ БУЛА АЛЬТЕРНАТИВА?

The article is devoted to the actual problems of nuclear disarmament in Ukraine and its consequences.The basic legal acts of the mentioned problem are analyzed, it highlightsthe historiography of the issue of nuclear disarmament, it grounded conclusions about the impossibility of restoring Ukraine's nuclear potential.The process of nuclear disarmament in Ukraine is being explored, besides the article analyzes the events that preceded the process of abandoning nuclear potential, identifies the main causes and consequences of the elimination of Ukraine's nuclear weapons, and determines an alternative to Ukraine. From the very beginning of the existence, the Ukrainian authorities have been pursuing a course of nuclear refusal by the state. Inheriting nuclear weapons and remaining nuclear power, Ukraine would have been tightly tied to the military-industrial complex of the USSR, because nuclear weapons were developed and created beyond its borders. Its control, maintenance and application centers would belocated outside of Ukraine’s borders. If Ukraine had left this weapon, our country would continue to be part of this complex, which would mean political, economic, and military ties to Russia. The author indentifies two main reasons for the Ukrainian nuclear establishment's consent to nuclear-free status and it is the fear of political pressure of Russia and the USА, as well as possible international isolation, and an idealistic vision of a future world without nuclear weapons. In recent years, discussions on the issue of nuclear disarmament in Ukraine and the implementation of the Budapest memorandum have intensified in Ukrainian society. The systematic violation of the Russian guarantees assumed by the Budapest memorandum has given rise to an active discussion about the possibility of reinstatement of the nuclear status that became practical after the Ukrainian-Russian conflict over the Tuzla Island in 2003. Cause of the fact that Russia is one of the countries of the Budapest Memorandum, which should guarantee Ukraine's sovereignty and security, doubts were raised about the real effectiveness of this agreement and the fulfillment of its conditions by the participating States.

Abstract P1-13-07: Effect of adjuvant chemotherapy for patients with ER-positive/HER2-negative breast cancer assessed by the propensity score matching: Significance of nuclear grade and nodal status

Abstract Purpose: Although a survival benefit with chemotherapy (CT) is obtained over all among patients with estrogen receptor-positive (ER+) breast cancer, the degree of benefit differs among the subtypes. The 21-gene Recurrence Score assay is a validated prognostic/predictive tool for early ER+ breast cancer. However, genomic assays have not been approved in Japan and most other countries, except in some developed countries. Based on the St. Gallen 2015, 1-3 positive nodes are not an absolute indication for CT among patients with ER+/HER2-negative breast cancer. In contrast, it is better for CT to be performed except for low-risk groups, based on genomic tests according to the St. Gallen 2017. In fact, no prospective study has proven the survival benefit of CT specifically for luminal-A breast cancer. Therefore, we analyzed the survival benefit of CT for ER+/HER2-negative breast cancer by propensity score matching (PSM). Patients and methods: Between 2000 and 2015, 895patients with stage I-III ER+/HER2-negative breast cancer who had underwent surgery in our hospital were examined. Patients with bilateral breast cancer, ER&amp;lt;10%, and preoperative treatment were excluded. The primary end point was the 5-year recurrence-free survival (RFS) and overall survival (OS) in patients matched by propensity score that estimated by a logistic regression model that included factors likely to influence the decision of whether or not to administer CT (tumor size, nuclear grade [NG], progesterone receptor status and nodal status). Results: In the entire cohort, the median age was 59 (28-95) years; 223 patients (24.9%) were node positive and 126 patients (14.1%)had NG3 disease. Overall, all patients received endocrine therapy, and 24.1% received additional CT. After a median follow-up of 68.8 months, the 5-year RFS rate was 94.3% in CT-untreated patients (non-CT group) and 90.1% in CT-treated patients (CT group; hazard ratio [HR] for recurrence, 1.47; 95% confidence interval [CI], 0.90 -2.33; p=0.106). The 5-year OS rate was 97.5% in the non-CT group and 95.6% in the CT group (HR for death, 1.80; 95% CI, 0.99-3.21; p=0.047). Using PSM, 236 patients were selected (1:1 matching between non-CT and CT groups). After matching, the 5-year RFS rate was higher in the CT group than in the non-CT group (96.8% vs. 82.7%; HR for recurrence, 0.29; 95% CI, 0.11-0.68; p=0.003), and the 5-year OS rate was higher in the CT group than in the non-CT group (100% vs. 91.9%; HR for death, 0.06; 95% CI, 0.003-0.35; p&amp;lt;0.001). Among PSM patients, with node-negative/NG3 and 1-3 node positive/NG2 disease, the 5-year RFS rate was significantly higher in the CT group than in the non-CT group (p=0.041 and p=0.006, respectively). Conclusion: No significant benefit of CT was observed when considering the entire cohort because of the treatment bias. When clinical and tumor features were matched by propensity score, the addition of CT significantly improved both RFS and OS of ER+/HER2-negative breast cancer patients, especially for patients with node- negative/NG3 and 1-3 node positive/NG2 disease. Citation Format: Tanaka N, Ogura K, Kamimura M, Hattori A, Inoue H, Yukawa H, Sakaguchi S, Matsuoka A, Kodera A, Hirano A. Effect of adjuvant chemotherapy for patients with ER-positive/HER2-negative breast cancer assessed by the propensity score matching: Significance of nuclear grade and nodal status [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-13-07.

Abstract PD4-08: Relationship between FDG-uptake and expression level of PD-L1 in primary ER positive/HER2 negative breast cancer

Abstract Background: 18F-Fluorodeoxyglucose-positron emission tomography (FDG-PET) is used to evaluate the glucose metabolic rates of cancers. Several studies have reported that high FDG uptake is predictive of poor prognosis and aggressive features in patients with breast cancer (BC). FDG-uptake is influenced by many factors including inflammation. In this study, we investigated the relationship between FDG uptake and immunological factors, including degrees of stromal tumor-infiltrating lymphocytes (TILs), CD8 and programmed cell death ligand 1 (PD-L1) in ER positive/HER2 negative (ER+/HER2-) BC. No published study, to our knowledge, has assessed the association between FDG uptake and PD-L1 in BC cases, even though both represent prognosis. Methods: Invasive carcinoma tissues of 79 ER+/HER2- BC patients who underwent surgery without preoperative therapy were examined. PD-L1, CD8 and TILs expression were evaluated by immunohistochemically (IHC) method. The evaluation of PET was determined by standardized uptake value max (SUVmax). Multivariate linear regression analysis, including expression of PD-L1, CD8 and TILs, was performed to identify independent variable correlation with SUVmax. Results: Among the 79 ER+/HER2- BC (T1-4, N0-2, M0) patients, the analysis revealed that PD-L1 (P=0.005), lymphovascular invasion (P=0.011), large tumor size (p=0.001), lymph node meatstasis (P=0.010), and high nuclear grade (P=0.011) and premenopasal status (P=0.014) were significantly associated with high SUVmax in the primary tumor. To define predictive value for the expression of PD-L1, SUVmax cut offwas determined using receiver operating characteristic (ROC) analysis; low (&amp;lt;3.0) and high (≥3.0). Among the 30 cases with high SUVmax, 10 cases (33.3%) had PD-L1 positive expression in the primary tumor, while only 2 (4.1%) of the 49 cases with low SUVmax had PD-L1 positive expression in the primary tumor. Conclusions: The present study demonstrated that the finding of preoperative FDG uptake is associated with the expression of PD-L1 in ER+/HER2- BC. In light of our results, FDG uptake may be predictive of the expression of PD-L1 and may be reflective of immunological features as well as prognostic features among patients with ER+/HER2- BC. Citation Format: Hirakata T, Fijii T, Kaira K, Kurozumi S, Katayama A, Yajima R, Obayashi S, NaKazawa Y, Tokuda S, Yanai K, Shirabe K. Relationship between FDG-uptake and expression level of PD-L1 in primary ER positive/HER2 negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD4-08.