Cell Number Ratio Research Articles

Effect of bovine oviductal epithelial cell lysate on the developmental competence and quality of bovine in vitro fertilized embryos.

In vitro fertilization (IVF) technology for embryo production has been applied in basic research, animal husbandry and medicine. However, the developmental efficiency and quality of embryos produced by IVF are inferior to those produced in vivo. In this study, we investigated the effects of supplementing bovine oviductal epithelial cells (BOEC) lysate during the in vitro culture period on the developmental competence and quality of bovine embryos. IVF embryos were cultured for 4 days post-IVF in medium supplemented with 10 % BOEC lysate at various concentrations (1.0×105, 2.0×105, and 4.0×105cells/mL) or 10 % PBS (-), which was used to adjust the lysate concentration (control). BOEC lysate at 2.0×105cells/mL significantly increased the blastocyst formation rate compared to that in the control group. Blastocysts from BOEC lysate supplemented groups showed significantly lower apoptosis rate than that in the control group. The ratio of inner cell mass cell number in blastocysts was significantly higher in all BOEC lysate supplemented groups than in the control group. The survival rate after vitrification/thawing was improved in the 1.0×105 and 2.0×105cells/mL BOEC lysate supplemented groups. In addition, gene expression analysis of blastocysts showed that 2.0×105cells/mL of BOEC lysate supplementation significantly enhanced the expression of anti-apoptotic genes (BCL2 and BIRC5), antioxidant-related genes (GPX1 and SOD2), and cell differentiation-related genes (SOX2 and OCT4). In conclusion, supplementation with 2.0×105cells/mL BOEC lysate during early in vitro culture improved the developmental competence and quality of bovine IVF embryos.

Segregation of endosymbionts in complex symbiotic system of cicadas providing novel insights into microbial symbioses and evolutionary dynamics of symbiotic organs in sap-feeding insects

The most extraordinary systems of symbiosis in insects are found in the suborder Auchenorrhyncha of Hemiptera, which provide unique perspectives for uncovering complicated insect-microbe symbiosis. We investigated symbionts associated with bacteriomes and fat bodies in six cicada species, and compared transmitted cell number ratio of related symbionts in ovaries among species. We reveal that Sulcia and Hodgkinia or a yeast-like fungal symbiont (YLS) are segregated from other host tissues by the bacteriomes in the nymphal stage, then some of them may migrate to other organs (i.e., fat bodies and ovaries) during host development. Particularly, YLS resides together with Sulcia in the “symbiont ball” of each egg and the bacteriomes of young-instar nymphs, but finally migrates to the fat bodies of adults in the majority of Hodgkinia-free cicadas, whereas it resides in both bacteriome sheath and fat bodies of adults in a few other species. The transmitted Sulcia/YLS or Sulcia/Hodgkinia cell number ratio in ovaries varies significantly among species, which could be related to the distribution and/or lineage splitting of symbiont(s). Rickettsia localizes to the nuclei of bacteriomes and fat bodies in some species, but it was not observed to be transmitted to the ovaries, indicating that this symbiont may be acquired from environments or from father to offspring. The considerable difference in the transovarial transmission process of symbionts suggests that cellular mechanisms underlying the symbiont transmission are complex. Our results may provide novel insights into insect-microbe symbiosis.

Bark structure is coordinated with xylem hydraulic properties in branches of five Cupressaceae species.

The properties of bark and xylem contribute to tree growth and survival under drought and other types of stress conditions. However, little is known about the functional coordination of the xylem and bark despite the influence of selection on both structures in response to drought. To this end, we examined relationships between proportions of bark components (i.e. thicknesses of tissues outside the vascular cambium) and xylem transport properties in juvenile branches of five Cupressaceae species, focusing on transport efficiency and safety from hydraulic failure via drought-induced embolism. Both xylem efficiency and safety were correlated with multiple bark traits, suggesting that xylem transport and bark properties are coordinated. Specifically, xylem transport efficiency was greater in species with thicker secondary phloem, greater phloem-to-xylem thickness ratio and phloem-to-xylem cell number ratio. In contrast, species with thicker bark, living cortex and dead bark tissues were more resistant to embolism. Thicker phellem layers were associated with lower embolism resistance. Results of this study point to an important connection between xylem transport efficiency and phloem characteristics, which are shaped by the activity of vascular cambium. The link between bark and embolism resistance affirms the importance of both tissues to drought tolerance.

Mathematical analysis identifies the optimal treatment strategy for epidermal growth factor receptor-mutated non-small cell lung cancer.

In Asians, more than half of non-small cell lung cancers (NSCLC) are induced by epidermal growth factor receptor (EGFR) mutations. Although patients carrying EGFR driver mutations display a good initial response to EGFR-Tyrosine Kinase Inhibitors (EGFR-TKIs), additional mutations provoke drug resistance. Hence, predicting tumor dynamics before treatment initiation and formulating a reasonable treatment schedule is an urgent challenge. To overcome this problem, we constructed a mathematical model based on clinical observations and investigated the optimal schedules for EGFR-TKI therapy. Based on published data on cell growth rates under different drugs, we found that using osimertinib that are efficient for secondary resistant cells as the first-line drug is beneficial in monotherapy, which is consistent with published clinical statistical data. Moreover, we identified the existence of a suitable drug-switching time; that is, changing drugs too early or too late was not helpful. Furthermore, we demonstrate that osimertinib combined with erlotinib or gefitinib as first-line treatment, has the potential for clinical application. Finally, we examined the relationship between the initial ratio of resistant cells and final cell number under different treatment conditions, and summarized it into a therapy suggestion map. By performing parameter sensitivity analysis, we identified the condition where osimertinib-first therapy was recommended as the optimal treatment option. This study for the first time theoretically showed the optimal treatment strategies based on the known information in NSCLC. Our framework can be applied to other types of cancer in the future.

Effects of chlorpyrifos toxicity on brain, pseudobranchial neurosecretory system and swimming performance of a catfish, Heteropneustes fossilis

In the present study, it was aimed to evaluate the adverse effects of CPF on the histopathology of the optic tectum and cerebellum, pseudobranchial neurosecretory system (PNS), biochemical assays of brain tissue, and locomotory behavior in catfish, Heteropneustes fossilis. The fishes were exposed to an environmentally relevant concentration of 0.09 and 0.192 mg/L of CPF for 7, 15, and 30 d. The CPF toxicity induced degenerative changes with significantly decreased cell size, number, and nucleo-cytoplasmic (N/C) ratio of the PNS; and altered neuro-architectural pattern of optic tectum with degenerative changes in mononuclear and granular cells and necrotic variation in granular and Purkinje cells of the cerebellum. The Acetylcholinesterase (AChE) and Catalase (CAT) activity in the CPF-exposed brain was significantly decreased, whereas Superoxide dismutase (SOD) and Malondialdehyde (MDA) level was significantly increased in comparison with control. In CPF-exposed fishes, the respiratory movements and locomotory behavioral pattern like swimming speed, total distance traveled, time mobile, absolute turn angle, head: distance traveled, maximum speed were significantly decreased, whereas time immobile and time freezing episodes were significantly increased as compared to control fishes. The present study concludes that environmentally relevant concentration of CPF may induce histopathological, biochemical, physiological, and behavioral disturbances in a non-target organism, H. fossilis.

Construction of a conjugation method for the transformation of Cobetia sp. IU180733JP01 (5-11-6-3) that accumulates poly(3-hydroxybutyrate) from seaweeds

Cobetia bacteria are considered useful hosts for industrial applications owing to their fast growth, high cell density, and halophilicity. Here, we constructed an efficient conjugation method to obtain Cobetia sp. IU180733JP01 (5-11-6-3) transformants, which can produce bioplastics from alginate or seaweed waste. Lysogeny Broth medium containing 2% NaCl was used to co-cultivate the 5-11-6-3 strain (plasmid recipient) with Escherichia coli S17-1 (plasmid donor). Transformants with the highest conjugation efficiency [(2.92 ± 1.37) × 10-3] were obtained at a donor:recipient cell number ratio of 5:1. This is the first study reporting the creation of recombinant strains in the genus Cobetia. This method will contribute to creating a platform strain for the production of bioplastics and other useful materials from marine biomass.

Economical Optimization of Industrial Medium Culture for Bacterial Cellulose Production.

The competitiveness of bacterial cellulose (BC) production with plant cellulose can be achieved by production on cost-effective media. It was found that the bacterial cell number ratio of BC to culture medium increases over time so that from the fourth day, the entrapped cell number in the cellulose network exceeds the suspended cells. Optimization based on 23-full factorial showed that inoculum development at 50rpm and the main culture process under static conditions significantly increases BC production. A cost-effective culture medium containing molasses (ML) and corn steep liquor (CSL) was developed based on the same C/N ratio to HS medium, with 7.24g/l cellulose at C/N ratio 12.6 is competitive with maximum production 8.7g/L in HS medium. The BC production cost was reduced about 94% using the proposed cheap and locally available medium containing ML and CSL, while BC mechanical properties increased by about 50%.

Oviduct Epithelial Cell-Derived Extracellular Vesicles Improve Porcine Trophoblast Outgrowth.

Porcine species have a great impact on studies on biomaterial production, organ transplantation and the development of biomedical models. The low efficiency of in vitro-produced embryos to derive embryonic stem cells has made achieving this goal a challenge. The fallopian tube plays an important role in the development of embryos. Extracellular vesicles (EVs) secreted by oviductal epithelial cells play an important role in the epigenetic regulation of embryo development. We used artificially isolated oviductal epithelial cells and EVs. In this study, oviductal epithelial cell (OEC) EVs were isolated and characterized through transmission electron microscopy, nanoparticles tracking analysis, western blotting and proteomics. We found that embryo development and blastocyst formation rate was significantly increased (14.3% ± 0.6% vs. 6.0% ± 0.6%) after OEC EVs treatment. According to our data, the inner cell mass (ICM)/trophectoderm (TE) ratio of the embryonic cell number increased significantly after OEC EVs treatment (43.7% ± 2.3% vs. 28.4% ± 2.1%). Meanwhile, the attachment ability of embryos treated with OEV EVs was significantly improved (43.5% ± 2.1% vs. 29.2% ± 2.5%, respectively). Using quantitative polymerase chain reaction (qPCR), we found that the expression of reprogramming genes (POU5F1, SOX2, NANOG, KLF4 and c-Myc) and implantation-related genes (VIM, KRT8, TEAD4 and CDX2) significantly increased in OEC EV-treated embryos. We report that OEC EV treatment can improve the development and implantation abilities of embryos.

Effect of Curcumin as Feed Supplement on Immune Response and Pathological Changes of Broilers Exposed to Aflatoxin B1.

In this study, we examined the protective effects of curcumin against the AFB1-induced immune response of and pathological changes in broilers. Histopathology examinations showed that at day 28, AFB1 (5 mg/kg) exposure leads to severe histological changes in the spleen, thymus and bursa of Fabricius with a decrease in the number and karyoplasmic area ratio of plasma cells. Curcumin alleviated the AFB1-induced immune organs’ damage as well as the changes in plasma cells in a dose-dependent manner. RT-PCR data showed that AFB1 significantly downregulated the IL-2 and IFN-γ mRNA expression levels in the thymus, spleen and bursa of Fabricius. However, curcumin supplementation improved the AFB1-induced immune organs’ damage via upregulated cytokines’ expression. Intriguingly, similar trends were noticed in abnormal morphological changes and the immune response at day 35 after the withdrawal of AFB1 and curcumin from the diet, suggesting the protective effects and immunomodulatory function against AFB1 in broilers. The current study provides a scientific experimental basis for the application of curcumin as a therapeutic drug or additive in animal husbandry productive practice.

Exogenous Melatonin in the Culture Medium Does Not Affect the Development of In Vivo-Derived Pig Embryos but Substantially Improves the Quality of In Vitro-Produced Embryos.

Cloned and transgenic pigs are relevant human disease models and serve as potential donors for regenerative medicine and xenotransplantation. These technologies demand oocytes and embryos of good quality. However, the current protocols for in vitro production (IVP) of pig embryos give reduced blastocyst efficiency and embryo quality compared to in vivo controls. This is likely due to culture conditions jeopardizing embryonic homeostasis including the effect of reactive oxygen species (ROS) influence. In this study, the antioxidant melatonin (1 nM) in the maturation medium, fertilization medium, or both media was ineffective in enhancing fertilization or embryonic development parameters of in vitro fertilized oocytes. Supplementation of melatonin in the fertilization medium also had no effect on sperm function. In contrast, the addition of melatonin to the embryo culture medium accelerated the timing of embryonic development and increased the percentages of cleaved embryos and presumed zygotes that developed to the blastocyst stage. Furthermore, it increased the number of inner mass cells and the inner mass cell/total cell number ratio per blastocyst while increasing intracellular glutathione and reducing ROS and DNA damage levels in embryos. Contrarily, the addition of melatonin to the embryo culture medium had no evident effect on in vivo-derived embryos, including the developmental capacity and the quality of in vivo-derived 4-cell embryos or the percentage of genome-edited in vivo-derived zygotes achieving the blastocyst stage. In conclusion, exogenous melatonin in the embryo culture medium enhances the development and quality of in vitro-derived embryos but not in in vivo-derived embryos. Exogenous melatonin is thus recommended during embryo culture of oocytes matured and fertilized in vitro for improving porcine IVP efficiency.

Association of HER-2/CEP17 Ratio and HER-2 Copy Number With pCR Rate in HER-2-Positive Breast Cancer After Dual-Target Neoadjuvant Therapy With Trastuzumab and Pertuzumab.

ObjectiveTo explore the correlation between HER-2 status and pathological complete response (pCR) in HER-2-positive breast cancer after dual anti-HER-2 neoadjuvant therapy with trastuzumab and pertuzumab.MethodsA total of 57 HER-2-positive breast cancer patients admitted to the Second Affiliated Hospital of Anhui Medical University and Xijing Hospital Affiliated to Air Force Military Medical University, between January 1, 2019 and September 30, 2020, were enrolled in this multicenter retrospective study. HER-2 status, including HER-2/CEP17 ratio and HER-2/cell number ratio, was detected by FISH. The correlation between HER-2 status/clinicopathological data and pCR was analyzed. The ROC curve was drawn to determine the cutoff value.ResultsIHC assessment revealed 40 (70.18%) patients with IHC 3+ and 17 (29.82%) with IHC 2+. 41/57 (71.93%) patients achieved pCR. FISH revealed that the ratio of HER-2/chromosome 17 centromere (HER-2/CEP17) (p<0.001) and the ratio of HER-2/cell number (p<0.001) was significantly correlated with the pCR rate. ROC analysis showed that patients with an HER-2/CEP17 ratio ≥4.495 or HER-2/cell number ≥11.650 have a high pCR rate after dual anti-HER-2 neoadjuvant therapy, suggesting its predictive significance.ConclusionThe response to dual-targeted neoadjuvant therapy with trastuzumab and pertuzumab was adequate in hormone receptor-negative, HER-2-positive breast cancer patients. HER-2/CEP17 ratio and HER-2/cell number ratio were crucial for predicting efficacy.

The Expression and Function of NK Cells in Patients with Acute Myeloid Leukemia

To explore the expression characteristics of antigens and functional markers of natural killer (NK) cells in patients with acute myeloid leukemia (AML). Multi-parameter flow cytometry was used to detect NK cell surface markers and their functional indicators in 56 newly diagnosed AML patients and 24 healthy controls, including activating receptors NKG2D, NKP46, DNAM-1, and killing indicators granzyme B, perforin. Referring to the WHO hematopoiesis and lymph tissue tumor classification criteria, 56 cases were roughly divided into three types: AML M1, M2, and M4/M5. However, there was no differences about NK cells among the three types, so it was no longer subdivided. NK cells were divided into two groups: CD3-CD56hiCD16- (CD56hiNK) and CD3-CD56dimCD16+ (CD56dimNK). Compared with CD56dimNK cell population, except for NKP46, the positive expression levels of NKG2D and other receptors of CD56hiNK cells in AML patients decreased (P<0.001). Compared with healthy controls, the proportion of CD56hiNK cells in AML patients increased, while the number and proportion of NK cells and proportion of CD56dimNK cells significantly decreased (P<0.05). The proportion of perforin in CD56hiNK cells significantly increased (P<0.05). The expression of DNAM-1 in CD56hiNK cells, NKG2D, DNAM-1, and perforin in CD56dimNK cells decreased significantly (P<0.05). There was no statistically significant difference in expression of other functional indexes in AML patients compared with corresponding indexes of healthy controls. In addition, the proportion of CD56hiNK cells was positively correlated with the expression of CD34+ in AML (r=0.303). Compared with CD56dimNK, the ratio of CD56hiNK and the expression of functional markers in AML patients are lower. Compared with healthy controls, the number and expression ratio of NK cells in AML patients decrease and the expression of functional markers is abnormal, indicating that its function is impaired.

Abstract 13258: Transcriptomic Remodeling of Brugada Syndrome Arises During in vitro Cardiac Development

Introduction: Recent genetic data suggest that abnormal cardiac development participate to the pathogenesis of Brugada Syndrome (BrS), a rare inherited arrhythmia responsible for sudden cardiac death in young adults. In vitro cardiac differentiation of human induced pluripotent stem cells (hiPSCs) mimics cardiac development at the cellular level up to a prenatal stage. Objective: This study aims at defining whether BrS impairs cardiac differentiation of hiPSCs. Methods &amp; Results: Transcriptomic kinetics (daily bulk 3’RNA-seq from day 0 to day 30 of in vitro cardiac differentiation) were generated in triplicate for 2 control hiPSC lines and 2 BrS-patient hiPSC lines. First, global analysis unveiled that BrS and control kinetics start to diverge as early as day 8, coinciding with the emergence of beating cells. The 500 most differentially expressed genes between BrS and control kinetics revealed 7 main distinct expression profiles. Interestingly, in one of the clusters (Cluster 2), enriched in genes involved in ventricular development ( e.g. IRX4 , NKX2-5 ), the expression levels were higher in BrS as compared to control, starting at day 8. Inversely, another cluster (Cluster 4), enriched in genes involved in atrial development ( e.g. TBX18 , PITX2 ), displayed an opposite expression profile. Cell-type annotation of single-cell RNA-seq data obtained at day 30 of cardiac differentiation for 1 control (n=2; 11,499 cells) and 1 BrS hiPSCs line (n=2; 12,142 cells) confirmed this ventricular-to-atrial imbalance with an average ventricular-to-atrial cell number ratio of 0.97 and 8.27 for control and BrS lines, respectively. Conclusion: This first transcriptomic kinetic study supports the hypothesis of an early developmental defect in BrS. Altogether, our data show that BrS hiPSCs are more prone to ventricular specification as compared to control cells. This suggests that an abnormal cell fate during cardiac differentiation may participate to BrS pathogeny.

An Effective Simulation Scheme for Predicting the Aerodynamic Heat of a Scramjet-Propelled Vehicle

Currently, aerothermal research into scramjet-propelled vehicles characterized by a wedge-shaped section is relatively sparse. Based on the Mach number, grid strategy, and numerical method, an effective simulation scheme for predicting the aerodynamic heat of a scramjet-propelled vehicle during flight is proposed in this paper. At different Mach numbers, the appropriate grid strategy and numerical method were determined by validation tests. Two-dimensional external flow field models based on wedge sections were established and, unlike in blunt bodies, the tests showed that at the high supersonic stage, the ideal cell Reynolds number should be no larger than 16. At the hypersonic stage, the ideal cell Reynolds number and aspect ratio of wall cells near the shock should be no larger than 40, and the AUSM+ flux type performs better than Roe’s FDS flux type at the above stages. The aerothermal prediction indicates that during a flight time of about 34 s, the temperature change reaches about 1913.35 °C, and the maximum average temperature change rate reaches 115 °C/s.

Exposure to Pulsed Electromagnetic Fields Improves the Developmental Competence and Quality of Somatic Cell Nuclear Transfer Buffalo (Bubalus bubalis) Embryos Produced Using Fibroblast Cells and Alters Their Epigenetic Status and Gene Expression.

We examined the effects of treatment with pulsed electromagnetic fields (PEMFs) on cumulus cells and buffalo somatic cell nuclear transfer (SCNT) embryos. PEMF treatment (30 μT for 3 hours) of cumulus cells increased (p < 0.05) the relative cell viability and cell proliferation and the expression level of OCT4, NANOG, SOX2, P53, CCNB1, and GPX, but decreased (p < 0.05) that of DNMT1, DNMT3a, GSK3b, and BAX, whereas the expression level of DNMT3b, GLUT1, BCL2, CASPASE3, SOD1, and CATALASE was not affected. PEMF treatment of SCNT embryos at the beginning of in vitro culture increased (p < 0.05) the blastocyst rate (51.4% ± 1.36% vs. 42.8% ± 1.29%) and decreased (p < 0.01) the apoptotic index to the level in in vitro fertilization blastocysts, but did not significantly alter the total cell number and the inner cell mass:trophectoderm cell number ratio of blastocysts compared to the controls. PEMF treatment increased the expression level of NANOG, SOX2, CDX2, GLUT1, P53, and BCL2 and decreased that of BAX, CASPASE3, GSK3b, and HSP70, but not OCT4, DNMT1, DNMT3a, DNMT3b, HDAC1, and CCNB1 in blastocysts. It increased (p < 0.001) the global level of H3K27me3 but not H3K18ac. These results suggest that PEMF treatment of SCNT embryos improves their developmental competence, reduces the level of apoptosis, and alters the expression level of several important genes related to pluripotency, apoptosis, metabolism, and stress.

Accelerated T Cell Senescence after Allogeneic Hematopoeitic Stem Cell Transplantation

Long-term survivors of allogeneic hematopoietic stem cell transplantation (HSCT) remain susceptible to opportunistic infections, relapse of primary malignancy and development of secondary malignancy, features that are consistent with continued immune deficiency. Reconstitution of T cell populations following HSCT and the preceding conditioning regimens occurs via two main routes: a thymic-dependent generation of naive T cells from progenitors via thymopoeisis, and a thymic-independent peripheral expansion of adoptively transferred, mature T cells in the graft. We hypothesized that dependence on peripheral expansion for reconstitution in individuals over forty years old leads to T cell populations that are prematurely senescent. In this retrospective study of patients who underwent matched, related allogeneic transplantation, we compared PBMCS from a cohort of long-term (4-5 years) survivors of transplant and their original donor infusion products (n=13), evaluating the expession of markers of immunosenescence and aging (CD28, KLRG1, CD57 and PD-1) at each time point. Using multi-parameter flow cytometry, we determined that CD8 cells expressed significantly higher levels of markers of T cell terminal differentiation and senescence post-transplant. Both the frequency and absolute number of Effector Memory and TEMRA, as well as the incidence of cells expressing senescence markers increased. Although there was no significant change in frequency of CD8+ T-cells actively proliferating (% Ki-67+), TREC analysis revealed that the frequency of recent thymic emigrants in the peripheral T-cell pool was reduced long-term. Additionally, we assessed the telomere lengths of CD8+ T-cells using Flow-FISH and, in line with our hypothesis that reliance on peripheral expansion results in replicative senescence, found that they were significantly shorter post-transplant. In conclusion, our study suggests that a contributory factor underlying the disposition for opportunistic infections in long-term survivors of transplant may be premature T cell dysfunction resulting from immune reconstitution-related senescence.Figure Captions:Figure I. Immune reconstitution post-transplant(A) Absolute T, B, and NK cell counts were measured at the donor infusion and post-transplant time points in 13 donor-recipient pairs. Lines indicate median values for each time point. (B) Individual CD4+ and CD8+ T cell changes post-transplant relative to the donor infusion time point. (C) Inversion of the normal CD4:CD8 ratio at the post-transplant time point. (D) Median values for absolute CD4 and CD8 cell counts per uL for the patient cohort throughout the post-transplant time period.Figure II. Alterations in T cell subsets post-transplant(A) Frequencies of Naive (CCR7+ CD45RA+), Central Memory (CCR7+ CD45RA-), Effector Memory (CCR7- CD45RA-), and Effector Memory cells that re-express CD45RA (TEMRA; CCR7- CD45RA+) subsets for both CD4 and CD8 cells at the donor infusion and post-transplant time points. Boxes indicate the 25th quartile, median, and 75th quartile values; whiskers indicate the minimum and maximum values. (B) Absolute cell numbers per uL for each CD4 and CD8 T cell subset. (C) Ratio of absolute cell number for Naïve/Central Memory + Effector Memory populations at the donor infusion and post-transplant time points. (D) TREC analysis of the CD4 and CD8 populations assessing thymic generation of naive T cells at the donor infusion and post-transplant time points (n=6). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

1182-P: Trans Fatty Acid Intake Induces Impaired Glucose Tolerance via Dysbiosis

Trans fatty acids (TFAs) are known to increase the risk for various metabolic syndromes. The effect of TFA on dysbiosis and intestinal inflammation, especially caused by modifying innate immunity, were evaluated. Eight-week-old C57BL6/J male mice were treated with a normal diet (ND), high saturated fatty acid high sucrose diet (HFHSD), and high TFA high sucrose diet (HTHSD) for 12 weeks (A). There was no significant difference in body weight, oral intake, liver or visceral fat weight between the HFHSD and HTHSD groups (B-E). On the other hand, the HTHSD group showed more impaired glucose tolerance than the HFHSD group (p=0.015) (F and G). Multicolor flow cytometry analysis revealed that the T-bet-positive ILC3/CD45-positive cell number ratio and the M1/M2 macrophage ratio in the small intestinal lamina propria were significantly increased in the HTHSD group (p Disclosure T. Okamura: None. R. Bamba: None. Y. Hashimoto: None. T. Senmaru: None. M. Hamaguchi: Research Support; Spouse/Partner; AstraZeneca K. K. M. Fukui: None. Funding The Japan Food Chemical Research Foundation

BPA, BPAF and TMBPF Alter Adipogenesis and Fat Accumulation in Human Mesenchymal Stem Cells, with Implications for Obesity.

Bisphenol A (BPA) is an endocrine-disrupting chemical used in the production of plastics, and is linked to developmental, reproductive, and metabolic disorders including obesity. Manufacturers have begun using ‘BPA-free’ alternatives instead of BPA in many consumer products. However, these alternatives have had much less testing and oversight, yet they are already being mass-produced and used across industries from plastics to food-contact coatings. Here, we used human female adipose-derived stem cells (hASCs), a type of adult mesenchymal stem cell, to compare the effects of BPA and BPA alternatives on adipogenesis or fat cell development in vitro. We focused on two commonly used BPA replacements, bisphenol AF (BPAF) and tetramethyl bisphenol F (TMBPF; monomer of the new valPure V70 food-contact coating). Human ASCs were differentiated into adipocytes using chemically defined media in the presence of control differentiation media with and without 17β-estradiol (E2; 10 μM), or with increasing doses of BPA (0, 0.1 and 1 μM), BPAF (0, 0.1, 1 and 10 nM), or TMBPF (0, 0.01 and 0.1 μM). After differentiation, the cells were stained and imaged to visualize and quantify the accumulation of lipid vacuoles and number of developing fat cells. Treated cells were also examined for cell viability and apoptosis (programmed cell death) using the respective cellular assays. Similar to E2, BPA at 0.1 μM and BPAF at 0.1 nM, significantly increased adipogenesis and lipid production by 20% compared to control differentiated cells (based on total lipid vacuole number to cell number ratios), whereas higher levels of BPA and BPAF significantly decreased adipogenesis (p < 0.005). All tested doses of TMBPF significantly reduced adipogenesis and lipid production by 30–40%, likely at least partially through toxic effects on stem cells, as viable cell numbers decreased and apoptosis levels increased throughout differentiation. These findings indicate that low, environmentally-relevant doses of BPA, BPAF, and TMBPF have significant effects on fat cell development and lipid accumulation, with TMBPF having non-estrogenic, anti-adipogenic effects. These and other recent results may provide a potential cellular mechanism between exposure to bisphenols and human obesity, and underscore the likely impact of these chemicals on fat development in vivo.

Effect of sulfur dioxide exposure on histopathology and morphometry of pancreatic islet cells and glycemic indices in Wistar rats.

Sulfur dioxide (SO2) is a ubiquitous air pollutant. Recent studies suggest that SO2 is a momentous risk factor for diabetes mellitus (DM). The present investigation aimed to evaluate the effects of SO2 exposure on histopathology and morphometry of pancreatic islet cells and serum glycemic indices in rats. Sixteen male Wistar rats were divided equally into SO2 and control groups. SO2 group was exposed to 10 parts per million (ppm) SO2 for 5 weeks (6 days a week, 3 h/day) and control group to filtered air for the same time as SO2 group. Blood serums were collected and pancreatic tissue isolated. Glycemic indices were measured. Pathological and morphometric changes were studied in the pancreatic tissues. Exposure to SO2 caused a significant increase in blood glucose but did not significantly change insulin and HbA1c serum levels and HOMA-IR. There were significant differences in vascular congestion (p= 0.02) and insulitis (p= 0.04) between the groups. SO2 inhalation significantly reduced beta cell number and beta-alpha cell ratio compared with the control group (p=0.03 and p<0.0001, respectively). These findings suggest that SO2 exposure damages pancreatic tissue which subsequently influences either the incidence of DM or the trend of diabetic complications.

Effect of Dickkopf-1 and colony stimulating factor-2 on the developmental competence, quality, gene expression and live birth rate of buffalo (Bubalus bubalis) embryos produced by hand-made cloning

A functional canonical WNT signaling pathway exists in preimplantation embryos and inhibits embryonic development. Recent studies suggest that this pathway is over-expressed in nuclear transferred (NT), compared to IVF embryos. The present study investigated the effects of Dickkopf-1 (DKK1), an inhibitor of canonical WNT signaling pathway and colony stimulating factor-2 (CSF2), an embryokine, on the developmental competence, quality, gene expression and live birth rate of NT buffalo embryos produced by Hand-made cloning (HMC). Following supplementation of the in vitro culture medium on day 5 with DKK1 (100 ng/mL), CSF2 (10 ng/mL), DKK1+CSF2 or no supplementation (control), the blastocyst rate was higher (P < 0.05) with DKK1 and DKK1+CSF2 (42.6 ± 1.4% and 46.6 ± 0.9%, respectively) than with CSF2 or controls (40.6 ± 1.3% and 39.0 ± 1.3%, respectively). The apoptotic index of the blastocysts was lower (P < 0.05) for DKK1, CSF2 and DKK1+CSF2 groups (3.44 ± 0.14, 3.39 ± 0.11 and 3.11 ± 0.22, respectively) compared to controls (6.64 ± 0.25), and was similar to that of the IVF blastocysts (3.67 ± 0.18). Although the total cell number was similar for the DKK1, CSF2, DKK1+CSF2 and control groups (200.4 ± 3.05, 196.4 ± 3.73, 204.7 ± 3.71 and 205 ± 4.03, respectively), the inner cell mass:trophectoderm cell number ratio of DKK1, CSF2 and DKK1+CSF2 groups (0.21 ± 0.01, 0.17 ± 0.01 and 0.22 ± 0.02, respectively) was higher (P < 0.05) than controls (0.13 ± 0.01) and was similar to that of IVF blastocysts (0.19 ± 0.01). Treatment with DKK1 or CSF2 or both increased (P < 0.05) the expression level of OCT4, NANOG,SOX2, GATA6, BCL2, PTEN, P53, FGF4, GLUT1 and IFN-τ, and decreased that of C-MYC, CDX2, CASPASE, DNMT3a, TCF7 and LEF1 in blastocysts, compared to controls. Transfer of DKK1-treated embryos to 13 recipients resulted in 4 pregnancies (30.8%; 2 live births, one abortion and one currently at 9 months of pregnancy) whereas, transfer of DKK1+CSF2-treated embryos to 16 recipients, resulted in 4 pregnancies (25.0%), all of which resulted in live births. No pregnancy was obtained after transfer of control and CSF-treated embryos to 12 and 16 recipients, respectively. These results suggest that DKK1 treatment of NT embryos increases the blastocyst, conception and live birth rate, and improves their quality whereas, CSF2 treatment, does not affect the blastocyst, conception and live birth rate despite improvement in embryo quality.