In vitro matured oocytes have a higher developmental potential than in vivo matured oocytes after hormonal ovarian stimulation in Callithrix jacchus.

BACKGROUND: The common marmoset, Callithrix jacchus, is an invaluable model in biomedical research. Its use includes genetic engineering applications, which require manipulations of oocytes and production of embryos in vitro. To maximize the recovery of oocytes suitable for embryo production and to fulfil the requirements of the 3R principles to the highest degree possible, optimization of ovarian stimulation protocols is crucial. Here, we compared the efficacy of two hormonal ovarian stimulation approaches: 1) stimulation of follicular growth with hFSH followed by triggering of oocyte maturation with hCG (FSH + hCG) and 2) stimulation with hFSH only (FSH-priming). METHODS: In total, 14 female marmosets were used as oocyte donors in this study. Each animal underwent up to four surgical interventions, with the first three performed as ovum pick-up (OPU) procedures and the last one being an ovariohysterectomy (OvH). In total, 20 experiments were carried out with FSH + hCG stimulation and 18 with FSH-priming. Efficacy of each stimulation protocol was assessed through in vitro maturation (IVM), in vitro fertilization (IVF) and embryo production rates. RESULTS: Each study group consisted of two subgroups: the in vivo matured oocytes and the oocytes that underwent IVM. Surprisingly, in the absence of hCG triggering some of the oocytes recovered were at the MII stage, moreover, their number was not significantly lower compared to FSH + hCG stimulation (2.8 vs. 3.9, respectively (ns)). While the IVM and IVF rates did not differ between the two stimulation groups, the IVF rates of in vivo matured oocytes were significantly lower compared to in vitro matured ones in both FSH-priming and FSH + hCG groups. In total, 1.7 eight-cell embryos/experiment (OPU) and 2.1 eight-cell embryos/experiment (OvH) were obtained after FSH + hCG stimulation vs. 1.8 eight-cell embryos/experiment (OPU) and 5.0 eight-cell embryos/experiment (OvH) following FSH-priming. These numbers include embryos obtained from both in vivo and in vitro matured oocytes. CONCLUSION: A significantly lower developmental competence of the in vivo matured oocytes renders triggering of the in vivo maturation with hCG as a part of the currently used FSH-stimulation protocol unnecessary. In actual numbers, between 1 and 7 blastocysts were obtained following each FSH-priming. In the absence of further studies, FSH-priming appears superior to FSH + hCG stimulation in the common marmoset under current experimental settings.

Non-communicable diseases and their risk factors in Ukraine: analysis of the global burden of disease 2019 study.

OBJECTIVE: Aim: To analyse the burden and risk factors of Non-Communicable diseases (NCDs) in Ukraine to determine the ways to prevent them. PATIENTS AND METHODS: Materials and Methods: Using a statistical method, NCDs DALYs (Disability-Adjusted Life Years) in Ukraine were analyzed in dynamics for 1990-2019 and in comparison, with European and EU countries based on the data from "Global Burden of Disease, 2019" research. RESULTS: Results: The burden of NCDs in Ukraine is 1.5 time higher than in European and EU countries. The most negative dynamic trends and significant differences between indicators in Ukraine and EU countries (with an excess of 2 or more times) were identified for DALYs due to cardiovascular diseases, digestive diseases and substance use disorders. In Ukraine the burden of NCDs can be reduced on 25.9% by normalization of systolic blood pressure, on 21.2% by optimizing diet, on18.5% by quitting smoking, on 17.6% by lowering LDL cholesterol, on 16.5% by normalizing body weight and on 9.2% by quitting alcohol abuse. CONCLUSION: Conclusions: Ukraine should develop and implement a modern system for monitoring and assessing the NCDs burden and their risk factors; strengthen the capacity of public health institutions and their ability to attract communities to implement interventions to control NCDs modified risk factors, increase awarnes and the population's responsible attitude towards their health; strengthen the ability and motivate primary health care to provide quality primary prevention, screening and timely diagnosis and treatment of chronic NCDs.

Association between gut health and gut microbiota in a polluted environment.

Animals host complex bacterial communities in their gastrointestinal tracts, with which they share a mutualistic interaction. The numerous effects these interactions grant to the host include regulation of the immune system, defense against pathogen invasion, digestion of otherwise undigestible foodstuffs, and impacts on host behaviour. Exposure to stressors, such as environmental pollution, parasites, and/or predators, can alter the composition of the gut microbiome, potentially affecting host-microbiome interactions that can be manifest in the host as, for example, metabolic dysfunction or inflammation. However, whether a change in gut microbiota in wild animals associates with a change in host condition is seldom examined. Thus, we quantified whether wild bank voles inhabiting a polluted environment, areas where there are environmental radionuclides, exhibited a change in gut microbiota (using 16S amplicon sequencing) and concomitant change in host health using a combined approach of transcriptomics, histological staining analyses of colon tissue, and quantification of short-chain fatty acids in faeces and blood. Concomitant with a change in gut microbiota in animals inhabiting contaminated areas, we found evidence of poor gut health in the host, such as hypotrophy of goblet cells and likely weakened mucus layer and related changes in Clca1 and Agr2 gene expression, but no visible inflammation in colon tissue. Through this case study we show that inhabiting a polluted environment can have wide reaching effects on the gut health of affected animals, and that gut health and other host health parameters should be examined together with gut microbiota in ecotoxicological studies.

POLYMORPHISM OF ACE AND AT2R1 GENES AS A GENETIC BACKGROUND FOR DIFFERENT TYPES OF ENCEPHALOPATHIES.

OBJECTIVE: The aim: To study the prevalence of ACE I/D and AT2R1 A1166C gene polymorphisms in patients with CTE, SVD, AIE, and PIE and to assess the influence of the presence of a particular genotype of the studied genes on the occurrence and/or progression of encephalopathies. PATIENTS AND METHODS: Materials and methods: A total of 96 patients with encephalopathies of various genesis (chronic traumatic encephalopathy (CTE) n=26; chronic alcohol-induced encephalopathy (AIE) n=26; microvascular ischemic disease of the brain (or cerebral small vessel disease, (SVD)) n=18; post-infectious encephalopathy (PIE) n=26) were involved in the study. The molecular genetic study was performed in the molecular genetics laboratory of the State Institution «Reference Center for Molecular Diagnostics of the Ministry of Health of Ukraine¼, Kyiv. Statistical processing of the results was performed using the STATISTICA 10.0 program. RESULTS: Results: In patients with various types of encephalopathies, probable changes in the frequency distribution of genotypes of polymorphic variants I/D of the ACE gene were established (11.11% vs. 33.33% - carriers of the I/I genotype, 27.78% vs. 50.00% - carriers of the I/D genotype and 61.11% vs. 16.67% - carriers of the D/D genotype) and A1166C of the AT2R1 gene (22.22% vs. 66.67% - carriers of the A/A genotype, 50.00% vs. 25.00% - carriers A/C genotype, 27.78% versus 8.33% - carriers of the C/C genotype) compared to individuals of the control group only in patients with SVD. The presence of the D allele and the D/D genotype of the ACE gene is associated with a statistically significant increase in the risk of SVD development and progression (respectively, 4.2 times (95% CI (1.39-12.72)) and 7.9 (95% CI ( 1.31-47.05)) times). A similar trend was established for the carrier of the C allele of the A1166C polymorphic variant of the AT2R1 gene in patients with SVD: a 4.3-fold increase in the risk of development and progression (95% CI (1.30-13.86). In addition, there is a probable dependence between carrier genotype A/C of the AT2R1 gene and increased risk of PIE and AIE by 4.8 and 5.7 times, respectively. CONCLUSION: Conclusions: Therefore, results suggest the reasonability to include the I/D of the ACE gene polymorphism investigation in the genetic panel of encephalopathies.

Insulin-like growth factor 2 is produced by antral follicles and promotes preantral follicle development in macaques†.

Insulin-like growth factors (IGFs) are known for their involvement in endocrine and paracrine regulation of ovarian function. Although IGF2 is the predominant circulating and intraovarian form of IGFs in primate species, the stage-specific follicular expression, action, and regulation of IGF2 are not well defined. Therefore, experiments were conducted to investigate the follicular IGF production in response to steroid hormone regulation and the direct IGF actions on follicular development and function in vitro. Preantral follicles were isolated from rhesus macaque ovaries and cultured to the antral stage in media supplemented with follicle-stimulating hormone and insulin. Follicles were randomly assigned to treatment groups: (a) control, (b) trilostane (a steroid synthesis inhibitor), (c) trilostane + estradiol, (d) trilostane + progesterone, and (e) trilostane + dihydrotestosterone. Media was analyzed for IGF concentrations, which were correlated to follicle growth. Follicles produced IGF2, but not IGF1, at the antral stage. Steroid depletion decreased, whereas steroid replacement increased, IGF2 production by antral follicles. Media IGF2 levels correlated positively with antral follicle diameters. Macaque preantral follicles and granulosa cells were subsequently cultured without (control) and with recombinant human IGF2 supplementation. Follicle survival, growth, and paracrine factor production, as well as granulosa cell proliferation and gonadotropin receptor gene expression, were assessed. IGF2 addition increased follicle survival rates, diameters and inhibin B production, as well as granulosa cell proliferation. These data demonstrate that IGF2 produced by antral follicles, in response to steroid hormone regulation, could act as a paracrine factor that positively impacts preantral follicle development and function in primates.

Stage-dependent actions of antimüllerian hormone in regulating granulosa cell proliferation and follicular function in the primate ovary.

OBJECTIVE: To study the direct action and physiological role of antimüllerian hormone (AMH) in regulating ovarian follicular development and function in vivo in primates. DESIGN: Animals were assigned to six treatment sequences in a crossover design study. Intraovarian infusion was performed during the follicular phase of the menstrual cycle with agents including: control vehicle; recombinant human AMH (rhAMH); and neutralizing anti-human AMH antibody (AMHAb). Before ovariectomy after the final treatment, the animals received intravenous injections of bromodeoxyuridine (BrdU). SETTING: National primate research center. ANIMALS: Adult female rhesus macaques (Macaca mulatta). INTERVENTIONS: None. MAIN OUTCOME MEASURES: Cycle length, follicle cohorts, and serum steroid levels were assessed. Ovarian histology, as well as granulosa cell (GC) proliferation and oocyte viability, were evaluated. RESULTS: In vehicle-infused ovaries, a dominant follicle was observed at midcycle E2 peak. However, rhAMH-treated ovaries exhibited an increased number of small antral follicles, whereas AMHAb-treated ovaries developed multiple large antral follicles. Serum E2 levels in the follicular phase decreased after rhAMH infusion and increased after AMHAb infusion. The rhAMH infusion increased serum T levels. Whereas early-growing follicles of rhAMH-treated ovaries contained BrdU-positive GCs, antral follicles containing BrdU-positive GCs were identified in AMHAb-treated ovaries. Autophagy was observed in oocytes of early-growing and antral follicles exposed to AMHAb and rhAMH, respectively. CONCLUSIONS: AMH enhanced early-stage follicle growth, but prevented antral follicle development and function via its stage-dependent regulation of GC proliferation and oocyte viability. This study provides information relevant to the pathophysiology of ovarian dysfunction and the treatment of infertility.

Formation of a long-lived cyclic isomer of ethylenedione.

A century of unsuccessful attempts to identify the neutral ethylenedione molecule combined with the results of quantum-chemical computations resulted in the conclusion on the instability of this molecule. In this article, we demonstrate that although the lowest energy isomer of ethylenedione with linear geometry is indeed unstable, a higher energy three-membered cyclic isomer can be stabilized, and at low temperature has a life-time longer than one millisecond. In our study, the ethylenedione C2O2 molecule was synthesized in the low-temperature reaction CO2 + C → C2O2 inside liquid helium nanodroplets. To study the reaction, a newly developed calorimetric technique was applied. Single pairs of reactants were incorporated into tiny helium droplets having a temperature of 0.37 K. The reaction energy was transferred to liquid helium stabilizing an intermediate gas-phase reaction product. The energy transfer also resulted in the evaporation of helium atoms. Therefore, the change of the helium droplets' size allowed precise calorimetry on a molecular scale.

Support of acute lymphoblastic leukemia cells by nonmalignant bone marrow stromal cells.

The present report describes work examining the manner in which nonmalignant bone marrow stromal cells prevent acute lymphoblastic leukemia (ALL) cell death. The initial focus was on the role of stromal cell-derived C-X-C motif chemokine 12 (CXCL12). Interference with CXCL12 production by stroma or blockade of its interactions with ALL by plerixafor did increase ALL cell death and in sensitive ALLs there was synergistic effect with conventional chemotherapy drugs. However, in contrast to most reports, there was considerable heterogeneity regarding the effect between 7 unique primary ALLs, with several exhibiting no sensitivity to CXCL12 blockade. The diversity in effect was not explained by differences in the expression of ALL cell surface receptors for CXCL12. The modest and variable effects of interference with CXCL12 on ALL led to the assessment of gene expression profiles of stromal cells and ALL cells. Gene set enrichment analysis identified pathways associated with metabolism and redox reactions as potentially important in the stromal cell: leukemia cell interaction. Exploratory imaging studies demonstrated bidirectional transfer of intracellular calcien-labelled molecules and also bidirectional transfer of mitochondria between stromal cells and ALL cells, providing potential means of metabolic interdependence of stromal cells and ALL cells.

Developmental and family history-based analysis of congenital fused labia phenotype in the captive common marmoset (Callithrix jacchus).

BACKGROUND: Congenital fused labia (CFL) is defined as a failure or significant delay in the opening of the juvenile sealed labia majora. This phenotype is known to be variably common in adult captive female marmosets but has never been investigated in detail before. MATERIALS AND METHODS: Here, we define, describe and quantify the variations in the degree of closure of the vulva in 122 captive marmosets (Callithrix jacchus) from 1.2 to 42 months old and include colony analysis. RESULTS: There was a negative correlation between the degree of labial fusion and animal age after prepubertal period (P < 0.05). CFL females had higher number CFL relatives (4.3 ± 0.6 vs 2.4 ± 0.5 for non-CFL, P < 0.05) and more external ancestors compared to non-CFL (P < 0.05). CONCLUSIONS: Our results therefore suggest that CFL phenotype is most likely associated with epigenetic effects induced by the captive environment and colony management strategy of extensive crossing of family lines to promote heterozygosity.

Vitamin D3 Regulates Follicular Development and Intrafollicular Vitamin D Biosynthesis and Signaling in the Primate Ovary.

There is an increasing recognition that vitamin D plays important roles in female reproduction. Recent studies demonstrated that 1α,25-dihydroxyvitamin D3 (VD3), the biologically active form of vitamin D, improved ovarian follicle survival and growth in vitro. Therefore, we investigated the direct effects of VD3 at the specific preantral and antral stages of follicular development, and tested the hypothesis that vitamin D receptor (VDR) and enzymes critical for vitamin D biosynthesis are expressed in the primate ovary. Fourteen adult rhesus macaques provided ovarian tissue. Secondary and antral follicles were isolated for PCR analysis on VDR, vitamin D3 25-hydroxylase, and 25-hydroxyvitamin D3-1α-hydroxylase. VDR protein localization was determined by immunohistochemistry on ovarian sections. Isolated secondary follicles were cultured under conditions of control and VD3 supplementation during the preantral or antral stage. Follicle survival, growth, steroid and anti-Müllerian hormone (AMH) production, as well as oocyte maturation were evaluated. In vivo- and in vitro-developed follicles were also assessed for genes that are critical for vitamin D biosynthesis and signaling, gonadotropin signaling, steroid and paracrine factor production, and oocyte quality. The mRNA encoding VDR, 25-hydroxylase, and 1α-hydroxylase was detectable in in vivo- and in vitro-developed preantral and antral follicles. The 25-hydroxylase was elevated in cultured follicles relative to in vivo-developed follicles, which further increased following VD3 exposure. VD3 treatment increased 1α-hydroxylase in in vitro-developed antral follicles. The absence of VD3 during culture decreased VDR expression in in vitro-developed antral follicles, which was restored to levels comparable to those of in vivo-developed antral follicles by VD3 supplementation. Positive immunostaining for VDR was detected in the nucleus and cytoplasm of granulosa cells and oocytes. While only survival was improved in preantral follicles treated with VD3, VD3 supplementation promoted both survival and growth of antral follicles with increased estradiol and AMH production, as well as oocyte maturation. Thus, Vitamin D biosynthesis and signaling systems are expressed in primate ovarian follicles. Our findings support a role for VD3 in regulating follicular development in a stage-dependent manner, as well as the intrafollicular vitamin D biosynthesis and signaling, directly in the ovary.

Anti-Müllerian hormone is a survival factor and promotes the growth of rhesus macaque preantral follicles during matrix-free culture.

Anti-Müllerian hormone (AMH) plays a key role during ovarian follicular development, with local actions associated with a dynamic secretion profile by growing follicles. While results for AMH effects on antral follicle growth and function are consistent among studies in various species, any effects on preantral follicle development remain controversial. Therefore, experiments were conducted to investigate the direct actions and role of AMH during follicle development at the preantral stage. Macaque-specific short-hairpin RNAs (shRNAs) targeting AMH mRNA were incorporated into adenoviral vectors to decrease AMH gene expression in rhesus macaque follicles. Secondary follicles were isolated from adult macaque ovaries and cultured individually in the ultra-low-attachment dish containing defined medium supplemented with follicle-stimulating hormone and insulin for 5 weeks. Follicles were randomly assigned to treatment groups: (a) control, (b) nontargeting control shRNA-vector, (c) AMH shRNA-vector, (d) AMH shRNA-vector + recombinant human AMH, and (e) recombinant human AMH. Follicle survival and growth were assessed. Culture media were analyzed for steroid hormone and paracrine factor concentrations. For in vivo study, the nontargeting control shRNA-vector and AMH shRNA-vector were injected into macaque ovaries. Ovaries were collected 9 days postinjection for morphology and immunohistochemistry assessment. Decreased AMH expression reduced preantral follicle survival and growth in nonhuman primates. Supplemental AMH treatment in the culture media promoted preantral follicle growth to the small antral stage in vitro with increased steroid hormone and paracrine factor production, as well as oocyte maturation. These data demonstrate that AMH is a critical follicular paracrine/autocrine factor positively impacting preantral follicle survival and growth in primates.

Use of the over-the-scope-clip (OTSC) in non-variceal upper gastrointestinal bleeding in patients with severe cardiovascular comorbidities: a retrospective study.

INTRODUCTION: The over-the-scope-clip (OTSC) can potentially overcome limitations of standard clips and achieve more efficient and reliable hemostasis. Data on OTSC use for non-variceal upper gastrointestinal bleeding (NVUGIB) in patients with cardiovascular comorbidities are currently limited. PATIENTS AND METHODS: We prospectively collected and retrospectively analyzed our database from February 2009 to September 2015 from all patients who underwent emergency endoscopy for high-risk NVUGIB in 2 academic centers and were treated with OTSC as first-line (n = 81) or second-line therapy (n = 19). RESULTS: One hundred patients mean age 72 (range 27 - 97 years) were included in this study. Fifty-one percent (n = 51) had severe cardiovascular co-morbidity (ischemic heart disease, congestive heart failure, hypertension, valvular heart disease, peripheral arterial occlusive disease and atrial fibrillation) and 73 % (n = 73) were on antiplatelet or/and anticoagulation therapy. The median size of the treated ulcers was 3 cm (range 1 - 5 cm). In 94 % (n = 94) primary hemostasis with OTSC was achieved. Clinical long-term success during a mean 6-month follow-up without rebleeding was 86 % (n = 86). CONCLUSIONS: In this cohort OTSC was demonstrated to be a safe and effective first- or second-line treatment for NVUGIB in high-risk patients with cardiovascular disease and complex, large ulcers.

Acute lymphoid leukemia cells with greater stem cell antigen-1 (Ly6a/Sca-1) expression exhibit higher levels of metalloproteinase activity and are more aggressive in vivo.

Stem cell antigen-1 (Ly6a/Sca-1) is a gene that is expressed in activated lymphocytes, hematopoietic stem cells and stem cells of a variety of tissues in mice. Despite decades of study its functions remain poorly defined. These studies explored the impact of expression of this stem cell associated gene in acute lymphoid leukemia. Higher levels of Ly6a/Sca-1 expression led to more aggressive leukemia growth in vivo and earlier death of hosts. Leukemias expressing higher levels of Ly6a/Sca-1 exhibited higher levels of matrix metalloproteinases. The results suggest the hypothesis that the more aggressive behavior of Ly6a/Sca-1 expressing leukemias is due at least in part to greater capacity to degrade microenvironmental stroma and invade tissues.

Evidence of B cell immune responses to acute lymphoblastic leukemia in murine allogeneic hematopoietic stem cell transplantation recipients treated with donor lymphocyte infusion and/or vaccination.

These experiments explored mechanisms of control of acute lymphoblastic leukemia (ALL) following allogeneic hematopoietic stem cell transplantation using a murine model of MHC-matched, minor histocompatibility antigen-mismatched transplantation. The central hypothesis examined was that addition of active vaccination against leukemia cells would substantially increase the effectiveness of allogeneic donor lymphocyte infusion (DLI) against ALL present in the host after transplantation. Although vaccination did increase the magnitude of type I T cell responses against leukemia cells associated with DLI, it did not lead to substantial improvement in long-term survival. Analysis of immunologic mechanisms of leukemia progression demonstrated that the failure of vaccination was not because of antigen loss in leukemia cells. However, analysis of survival provided surprising findings that, in addition to very modest type I T cell responses, a B cell response that produced antibodies that bind leukemia cells was found in long-term survivors. The risk of death from leukemia was significantly lower in recipients that had higher levels of such antibodies. These studies raise the hypothesis that stimulation of B cell responses after transplantation may provide a novel way to enhance allogeneic graft-versus-leukemia effects associated with transplantation.

Synthesis of substituted thienopyrimidine-4-ones.

The parallel solution-phase synthesis of more than 3000 substituted thienopyrimidin-4-ones has been accomplished. Key reactions include assembly of the 2-thioxopyrimidin-4-one ring by condensation of isomeric aminothiophenecarboxylates or their appropriate reactive derivatives (isothiocyanates or dithiocarbamates) with the corresponding isothiocyanates or amines. The libraries from libraries were then obtained in good yields and purities using solution-phase alkylation and acylation methodologies. Simple manual techniques for parallel reactions using special CombiSyn synthesizers were coupled with easy purification procedures (crystallization from the reaction mixtures) to give high-purity final products. The scope and limitations of the developed approach are discussed.