One-carbon metabolites supplementation and nutrient restriction alter the fetal liver metabolomic profile during early gestation in beef heifers.

Maternal nutrition is pivotal for proper fetal development, with one-carbon metabolites (OCM) playing a key role in fetal epigenetic programming through DNA and histone methylation. The study aimed to investigate the effects of nutrient restriction and OCM supplementation on fetal liver metabolomics in pregnant beef-heifers, focusing on metabolites and pathways associated with amino acid, vitamin and cofactor, carbohydrate, and energy metabolism at day 63 of gestation. Thirty-one crossbred Angus heifers were artificially inseminated and allocated to 4 nutritional treatments in a 2 × 2 factorial arrangement of treatments, with the 2 factors being dietary intake/rate of gain (control-diet [CON]; 0.60 kg/d ADG, vs. restricted-diet [RES]; -0.23 kg/d ADG) and OCM supplementation (supplemented [+OCM] vs. not supplemented [-OCM]). The resulting treatment groups-CON - OCM, CON + OCM, RES - OCM, and RES + OCM were maintained for 63 day post-breeding. Following this period, fetal liver tissues were collected and subjected to metabolomic analysis using UPLC-tandem mass-spectrometry. We identified 288 metabolites, with the majority (n = 54) being significantly influenced by the main effect of gain (P ≤ 0.05). Moreover, RES showed decreased abundances of most metabolites in pathways such as lysine metabolism; leucine, isoleucine, and valine metabolism; and tryptophan metabolism, compared to CON. Supplementation with OCM vs. no OCM supplementation, resulted in greater abundance of metabolites (P ≤ 0.05) affecting pathways associated with methionine, cysteine, S-adenosylmethionine and taurine metabolism; guanidino and acetamido metabolism; and nicotinate and nicotinamide metabolism. Notably, OCM supplementation with a moderate rate of gain increased the concentrations of ophthalmate, N-acetylglucosamine, and ascorbic-acid 3-sulfate, which are important for proper fetal development (P ≤ 0.05). Nutrient restriction reduced the majority of liver metabolites, while OCM supplementation increased a smaller number of metabolites. Thus, OCM supplementation may be protective of metabolite concentrations in key developmental pathways, which could potentially enhance fetal development under nutrient-restricted conditions.

Maternal nutrition is crucial for pregnancy outcomes, influencing offspring health and productivity. Poor nutrition during pregnancy can lead to fetal growth restrictions, impacting liver development. Such changes can increase the risk of metabolic syndromes and predispose them to impaired immune function. In cattle, optimal nutrition during early pregnancy is essential for reproductive efficiency and herd health. This period is critical for developmental programming through epigenetic changes triggered by environmental or genetic factors. These modifications are heritable which are influenced by maternal diet and play a critical role in determining health outcomes post-birth, relying significantly on the availability of one-carbon metabolites (OCM) like methionine, choline, folate, and vitamin B12. Supplementing these nutrients during early gestation may counteract the negative effects of poor nutrition. This study explores the impact of OCM supplementation and dietary restrictions on the fetal liver metabolism in beef heifers during early gestation. Our findings showed that dietary restrictions decrease fetal liver metabolites, whereas OCM supplementation increases certain metabolites, indicating a compensatory effect to support fetal development under nutrient-restricted conditions. Highlighting the importance of maternal nutrition, our findings provide valuable insights for developing nutritional strategies to enhance livestock efficiency and inform dietary guidelines during pregnancy for better health outcomes.

Surface and composition effects on the biphasic cytotoxicity of nanocomposites synthesized using leaf extracts.

The Malus sylvestris L. (LE1), Pinus sylvestris L. (LE2), and Sorbus aucuparia L. (LE3) leaves` extracts were used for the synthesis of silver (Ag) nanocomposites containing different amounts of silver chloride (AgCl), silver metal (Agmet), and silver phosphate (Ag3PO4). These nanocomposites were capped with the organic functional groups in the leaf extract. Notably, the nanocomposites caused biphasic cytotoxic response on cells; first attributed to the inhibition of cell growth and second to cell death. The nanocomposites were biocompatible with normal embryonic kidney (HEK293) cells in the cytotoxic range for cancer cells. [25(±1) °C synthesis] nanocomposites exhibited the highest cytotoxicity towards HeLa (lethal concentration- LC50 value of 11.4 µg mL-1) and A549 (LC50 value of 14.7 µg mL-1) after 24-h incubation and its efficiency was shown also for the more resistant MCF-7 and MDA-MB-231, however, their respective LC50 values were larger. For the HeLa cell line, this designed nanocomposite exhibited an LC50 value similar to the effective concentration (EC50) value of Cisplatin and about 3 times larger than Doxorubicin. nanocomposite contained Ag3PO4 in the composite and P on the surface, higher AgCl content, smaller crystallite size of all nanoparticle phases, and carbon-rich oxygen-deficient surface compared to all other nanocomposites.

One-carbon metabolite supplementation increases vitamin B12, folate, and methionine cycle metabolites in beef heifers and fetuses in an energy dependent manner at day 63 of gestation.

One-carbon metabolites (OCM) are metabolites and cofactors which include folate, vitamin B12, methionine, and choline that support methylation reactions. The objectives of this study were to investigate the effects of moderate changes in maternal body weight gain in combination with OCM supplementation during the first 63 d of gestation in beef cattle on (1) B12 and folate concentrations in maternal serum (2) folate cycle intermediates in maternal and fetal liver, allantoic fluid (ALF), and amniotic fluid (AMF) and (3) metabolites involved in one-carbon metabolism and related metabolic pathways in maternal and fetal liver. Heifers were either intake restricted (RES) and fed to lose 0.23 kg/d, or fed to gain 0.60 kg/d (CON). Supplemented (+ OCM) heifers were given B12 and folate injections weekly and fed rumen-protected methionine and choline daily, while non-supplemented (-OCM) heifers were given weekly saline injections. These two treatments were combined in a 2 × 2 factorial arrangement resulting in 4 treatments: CON-OCM, CON + OCM, RES-OCM, and RES + OCM. Samples of maternal serum, maternal and fetal liver, ALF, and AMF were collected at slaughter on day 63 of gestation. Restricted maternal nutrition most notably increased (./ ≤ 0.05) the concentration of vitamin B12 in maternal serum, 5,10-methylenetetrahydrofolate and 5,10-methenyltetrahydrofolate in maternal liver, and cystathionine in the fetal liver; conversely, maternal restriction decreased (P = 0.05) 5,10-methylenetetrahydrofolate concentration in fetal liver. Supplementing OCM increased (P ≤ 0.05) the concentrations of maternal serum B12, folate, and folate intermediates, ALF and AMF 5-methyltetrahydrofolate concentration, and altered (P ≤ 0.02) other maternal liver intermediates including S-adenosylmethionine, dimethylglycine, cystathionine Glutathione reduced, glutathione oxidized, taurine, serine, sarcosine, and pyridoxine. These data demonstrate that OCM supplementation was effective at increasing maternal OCM status. Furthermore, these data are similar to previously published literature where restricted maternal nutrition also affected maternal OCM status. Altering OCM status in both the dam and fetus could impact fetal developmental outcomes and production efficiencies. Lastly, these data demonstrate that fetal metabolite abundance is highly regulated, although the changes required to maintain homeostasis may program altered metabolism postnatally.

Maternal stresses that occur during pregnancy, such as restricted nutrition, can impact the developmental outcomes of the offspring in a process known as developmental programming. This programming can occur through epigenetics, which involves changes in fetal gene expression and can occur through the addition of methyl groups to DNA. These changes regulate gene transcription in the offspring and can alter offspring health, efficiency, and life-long outcomes. One-carbon metabolites (OCM), which are nutrients like the amino acid methionine and the vitamins B12, folate, and choline, act as intermediates or cofactors for the donation of methyl groups to DNA. This study investigated the effects of differing maternal rates of gain along with OCM supplementation during early gestation on OCM and related metabolite concentrations in the dam and fetus. We found that supplementing OCM to beef heifers increased maternal OCM and related metabolite concentrations and fetal fluid OCM concentrations. We also found that low maternal gain increased maternal serum and liver OCM concentrations. We can conclude from these findings that both maternal rate of gain and OCM supplementation can impact maternal OCM concentrations at day 63 of gestation and further research is needed to see if those maternal impacts will affect the developing fetus or calf later in its life.

EvIPqPCR, Target Circulating Tumorous Extracellular Vesicles for Detection of Pancreatic Cancer.

Pancreatic cancer patients predominantly present with advanced disease at diagnosis, contributing to its high mortality. A noninvasive, fast screening method to detect this disease is an unmet need. Tumor-derived extracellular vesicles (tdEVs) bearing information from parental cells have emerged as a promising cancer diagnostic biomarker. However, most tdEV-based assays have impractical sample volumes and time-consuming, complex, and costly techniques. To overcome these limitations, we developed a novel diagnostic method for pancreatic cancer screening. Our approach utilizes the mitochondrial DNA to nuclear DNA ratio of EVs as a collective cell-specific characteristic. We introduce EvIPqPCR, a fast method that combines immunoprecipitation (IP) and qPCR quantification to detect tumor-derived EVs directly from serum. Importantly, our method employs DNA isolation-free and duplexing probes for qPCR, saving at least 3 h. This technique has the potential to serve as a translational assay for cancer screening with a weak correlation to prognosis biomarkers and sufficient discriminatory power among healthy controls, pancreatitis, and pancreatic cancer cases.

Peptide Selection of MMP-1 for Electrochemical Sensing with Epitope-Imprinted Poly(TPARA-co-EDOT)s.

Instead of molecularly imprinting a whole protein molecule, imprinting protein epitopes is gaining popularity due to cost and solubility issues. Belonging to the matrix metalloproteinase protein family, MMP-1 is an interstitial collagenase that degrades collagen and may be involved in cell migration, cell proliferation, the pro-inflammatory effect, and cancer progression. Hence, it can serve as a disease protein biomarker and thus be useful in early diagnosis. Herein, epitopes of MMP-1 were identified by screening its crystal structure. To identify possible epitopes for imprinting, MMP-1 was cleaved in silico with trypsin, pepsin at pH = 1.3, and pepsin at pH > 2.0 using Peptide Cutter, generating peptide fragments containing 8 to 12 amino acids. Five criteria were applied to select the peptides most suitable as potential epitopes for MMP-1. The triphenylamine rhodanine-3-acetic acid (TPARA) functional monomer was synthesized to form a stable pre-polymerization complex with a selected template epitope. The complexed functional monomer was then copolymerized with 3,4-ethoxylenedioxythiophene (EDOT) using potentiodynamic electropolymerization onto indium−tin−oxide (ITO) electrodes. The composition of the molecularly imprinted poly(TPARA-co-EDOT) (MIP) was optimized by maximizing the film's electrical conductivity. Cyclic voltammetry was used to determine MMP-1 concentration in the presence of the Fe(CN)63−/Fe(CN)64− redox probe actuating the "gate effect." A calibration curve was constructed and used to determine the usable concentration range and the limit of detection as ca. 0.001 to 10.0 pg/mL and 0.2 fg/mL MMP-1, respectively. Finally, the MMP-1 concentration in the A549 human lung (carcinoma) culture medium was measured, and this determination accuracy was confirmed using an ELISA assay.

Valorizing the Unexplored Filtration Waste of Brewing Industry for Green Silver Nanocomposite Synthesis.

The brewing industry generates a substantial amount of by-products rich in polyphenols, carbohydrates, sugars, sulfates, nitrogen compounds, organic carbon, and several elements, including chlorine, magnesium, and phosphorus. Although limited quantities of these by-products are used in fertilizers and composts, a large amount is discarded as waste. Therefore, it is crucial to identify different ways of valorizing the by-products. Research regarding the valorization of the brewery by-products is still in its nascent stage; therefore, it still has high potential. Herein, we report the valorization of the brewery by-product from the filtration stage of the brewing process (BW9) to synthesize silver nanocomposites as this waste has remained largely unexplored. The BW9 nanocomposites have been compared to those obtained from the brewery product B. The chemical composition analysis of BW9 and B revealed several organic moieties capable of reducing metal salts and capping the formed nanoparticles. Therefore, the brewery waste from stage 9 was valorized as a precursor and added to silver-based precursor at various temperatures (25, 50, and 80 °C) and for various time periods (10, 30, and 120 min) to synthesize silver nanocomposites. The nanocomposites obtained using BW9 were compared to those obtained using the main product of the brewing industry, beer (B). Synthesized nanocomposites composed of AgCl as a major phase and silver metal (Agmet) was incorporated in minor quantities. In addition, Ag3PO4 was also found in B nanocomposites in minor quantities (up to 34 wt.%). The surface morphology depicted globular nanoparticles with layered structures. Small ball-like aggregates on the layer representative of Ag3PO4 were observed in B nanocomposites. The surface of nanocomposites was capped with organic content and functional groups present in the brewery products. The nanocomposites demonstrated high antibacterial activity against Escherichia coli (E. coli), with BW9 nanocomposites exhibiting a higher activity than B nanocomposites.

Selective Impedimetric Chemosensing of Carcinogenic Heterocyclic Aromatic Amine in Pork by dsDNA-Mimicking Molecularly Imprinted Polymer Film-Coated Electrodes.

Inspired by the easy intercalation of quinoxaline heterocyclic aromatic amines (HAAs) in double-stranded DNA (dsDNA), we synthesized a nucleobase-functionalized molecularly imprinted polymer (MIP) as the recognition unit of an impedimetric chemosensor for the selective determination of a 2-amino-3,7,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (7,8-DiMeIQx) HAA. HAAs are generated in meat and fish processed at high temperatures. They are considered to be potent hazardous carcinogens. The MIP film was prepared by potentiodynamic electropolymerization of a pre-polymerization complex of two adenine- and one thymine-substituted bis(2,2'-bithien-5-yl)methane functional monomer molecules with one 7,8-DiMeIQx template molecule, in the presence of the 2,4,5,2',4',5'-hexa(thiophene-2-yl)-3,3'-bithiophene cross-linking monomer, in solution. The as-formed MIP chemosensor allowed for the selective impedimetric determination of 7,8-DiMeIQx in the 47 to 400 µM linear dynamic concentration range with a limit of detection of 15.5 µM. The chemosensor was successfully applied for 7,8-DiMeIQx determination in the pork meat extract as a proof of concept.

Tyr192 Regulates Lymphocyte-Specific Tyrosine Kinase Activity in T Cells.

TCR signaling critically depends on the tyrosine kinase Lck (lymphocyte-specific protein tyrosine kinase). Two phosphotyrosines, the activating pTyr394 and the inhibitory pTyr505, control Lck activity. Recently, pTyr192 in the Lck SH2 domain emerged as a third regulator. How pTyr192 may affect Lck function remains unclear. In this study, we explored the role of Lck Tyr192 using CRISPR/Cas9-targeted knock-in mutations in the human Jurkat T cell line. Our data reveal that both Lck pTyr394 and pTyr505 are controlled by Lck Tyr192 Lck with a nonphosphorylated SH2 domain (Lck Phe192) displayed hyperactivity, possibly by promoting Lck Tyr394 transphosphorylation. Lck Glu192 mimicking stable Lck pTyr192 was inhibited by Tyr505 hyperphosphorylation. To overcome this effect, we further mutated Tyr505 The resulting Lck Glu192/Phe505 displayed strongly increased amounts of pTyr394 both in resting and activated T cells. Our results suggest that a fundamental role of Lck pTyr192 may be to protect Lck pTyr394 and/or pTyr505 to maintain a pool of already active Lck in resting T cells. This provides an additional mechanism for fine-tuning of Lck as well as T cell activity.

The effects of maternal nutrition during the first 50 d of gestation on the location and abundance of hexose and cationic amino acid transporters in beef heifer uteroplacental tissues.

We hypothesized that maternal nutrition during the first 50 d of gestation would influence the abundance of hexose transporters, SLC2A1, SLC2A3, and SLC2A5, and cationic amino acid transporters, SLC7A1 and SLC7A2, in heifer uteroplacental tissues. Angus-cross heifers (n = 43) were estrus synchronized, bred via artificial insemination, and assigned at breeding to 1 of 2 dietary intake groups (CON = 100% of requirements to achieve 0.45 kg/d of BW gain or RES = 60% of CON intake) and ovariohysterectomized on day 16, 34, or 50 of gestation (n = 6 to 9/d) in a completely randomized design with a 2 × 3 factorial arrangement of treatments. Uterine cross-sections were collected from the horn ipsilateral to the corpus luteum, fixed in 10% neutral buffered formalin, sectioned at 5 µm, and stained via immunofluorescence for transporters. For each image, areas of fetal membrane (FM; chorioallantois), luminal epithelium (ENDO), superficial glands (SG), deep glands (DG), and myometrium (MYO) were analyzed separately for relative intensity of fluorescence as an indicator of transporter abundance. Analysis of FM was only conducted for days 34 and 50. No transporters in target areas were influenced by a day × treatment interaction (P ≥ 0.06). In ENDO, all transporters were differentially abundant from days 16 to 50 of gestation (P ≤ 0.04), and SLC7A2 was greater (P = 0.05) for RES vs. CON. In SG, SLC7A1 and SLC7A2 were greater (P ≤ 0.04) at day 34 vs. day 16. In DG, SLC2A3 and SLC7A1 were greater (P ≤ 0.05) for CON vs. RES heifers; furthermore, SLC7A1 was greater (P < 0.01) at day 50 vs. days 16 and 34 of gestation. In MYO, SLC7A1 was greater (P < 0.01) for CON vs. RES and was greater (P = 0.02) at days 34 and 50 vs. day 16. There were no differences in FM (P ≥ 0.06). Analysis of all uterine tissues at day 16 determined that SLC2A1, SLC2A3, and SLC7A2 were all differentially abundant across uterine tissue type (P < 0.01), and SLC7A1 was greater (P = 0.02) for CON vs. RES. Analysis of all uteroplacental tissues at days 34 and 50 demonstrated that all transporters differed (P < 0.01) across uteroplacental tissues, and SLC7A1 was greater (P < 0.01) for CON vs. RES. These data are interpreted to imply that transporters are differentially affected by day of gestation, and that hexose and cationic amino acid transporters are differentially abundant across utero-placental tissue types, and that SLC7A1 is responsive to maternal nutritional treatment.

Adaptor proteins: Flexible and dynamic modulators of immune cell signalling.

To maintain homeostasis, all cells respond to environmental cues via a multitude of surface receptors. In order to act appropriately in their environment, cells are dependent on the transduction of the incoming signal through tightly regulated and interconnected signalling pathways to the cell nucleus. In particular, cells implicated in the immune system greatly depend on such systems to respond in a flexible and dynamic manner to environmental challenges. One major group of intracellular proteins that are involved in these signalling pathways are adaptor proteins. Although adaptor proteins are essential for normal immune cell operation, the functional role of this group of signalling proteins remains to be fully appreciated. So far, research on adaptor proteins has revealed their unique potential in building transient complexes in a reversible, dynamic and inducible manner. In this review, we explore the roles of adaptor proteins - in space and time of intracellular signalling - and their associations with human disease. Examples of adaptor proteins expressed in hematopoietic cells highlight their crucial role in the immune system. Lastly, we present challenges faced in elucidating roles of adaptor proteins, as illustrated by the T cell-specific adaptor (TSAd) protein encoded by the SH2D2A gene.

The effects of maternal nutrient restriction and day of early pregnancy on the location and abundance of neutral amino acid transporters in beef heifer utero-placental tissues.

We hypothesized that maternal nutrition and day of gestation would influence the abundance of the neutral amino acid transporters SLC1A1, SLC1A5, SLC7A5, SLC38A2, and SLC38A7 in heifer utero-placental tissues. Angus-cross heifers (n = 43) were estrus synchronized and bred via AI. At breeding, heifers were assigned to one of two dietary intake groups (CON = 100% of requirements to achieve 0.45 kg/d gain or restricted heifers (RES) = 60% of CON intake) and ovariohysterectomized on day 16, 34, or 50 of gestation (n = 6 to 9/d). Thus, the experimental design was a completely randomized design with a 2 × 3 factorial arrangement of treatments. Uterine cross sections were taken from the horn ipsilateral to the CL, fixed in 10% NBF, sectioned at 5 µm, and stained for transporters. For each image, the areas of fetal membrane (FM; chorioallantois), endometrium (ENDO), superficial glands (SG), deep glands (DG), and myometrium (MYO) were analyzed separately for relative intensity of fluorescence as an indicator of transporter abundance. Analysis of FM was only conducted on days 34 and 50. In ENDO, SLC7A5 was greater (P < 0.01) in CON compared with RES heifers. In SG, SLC1A1 was greater (P = 0.02) in day 16 RES compared with day 16 CON and days 34 and 50 RES. In DG, SLC1A1 was greater (P = 0.02) on day 16 compared with 50 of gestation. In MYO, SLC1A1 was greater (P = 0.02) in day 50 CON compared with day 16 CON and day 50 RES. Additionally, in MYO SLC38A2 was greater (P = 0.02) in day 16 RES compared with day 16 CON and day 34 RES. In FM, SLC7A5 tended (P = 0.08) to be greater in CON vs RES. Analysis of all uterine tissues on day 16 determined that expression of SLC1A1, SLC1A5, SL38A2, and SL38A7 differed across uterine tissue type (P < 0.01); however, only SLC7A5 tended (P = 0.10) to differ and be greater in CON compared with RES heifers. Analysis of all utero-placental tissues on days 34 and 50 determined that SLC1A1, SLC7A5, SLC38A2, and SLC38A7 were greater (P ≤ 0.03) in CON compared with RES heifers. Furthermore, abundance of all transporters investigated on days 34 and 50 differed across utero-placental tissue types (P < 0.01). These data support our hypothesis that maternal nutrition and day of gestation influence the abundance of neutral amino acid transporters in utero-placental tissues from days 16 to 50 of gestation. Additionally, these data combined with previously published works help further elucidate nutrient supply and demands of the maternal and fetal system during early gestation in beef heifers.

Laser-capture microdissection of murine lung for differential cellular RNA analysis.

The lung tissue contains a heterogeneous milieu of bronchioles, epithelial, airway smooth muscle (ASM), alveolar, and immune cell types. Healthy bronchiole comprises epithelial cells surrounded by ASM cells and helps in normal respiration. In contrast, airway remodeling, or plasticity, increases surrounding of bronchial epithelium during inflammation, especially in asthmatic condition. Given the profound functional difference between ASM, epithelial, and other cell types in the lung, it is imperative to separate and isolate different cell types of lungs for genomics, proteomics, and molecular analysis, which will improve the diagnostic and therapeutic approach to treat cell-specific lung disorders. Laser capture microdissection (LCM) is the technique generally used for the isolation of specific cell populations under direct visual inspection, which plays a crucial role to evaluate cell-specific effect in clinical and preclinical setup. However, maintenance of tissue RNA quality and integrity in LCM studies are very challenging tasks. It is obvious to believe that the major factor affecting the RNA quality is tissue-fixation method. The prime focus of this study was to address the RNA quality factors within the lung tissue using the different solvent system to fix tissue sample to obtain high-quality RNA. Paraformaldehyde and Carnoy's solutions were used for fixing the lung tissue and compared RNA integrity in LCM captured lung tissue samples. To further confirm the quality of RNA, we measured cellular marker genes in collected lung tissue samples from control and mixed allergen (MA)-induced asthmatic mouse model using qRT-PCR technique. RNA integrity number showed a significantly better quality of RNA in lung tissue samples fixed with Carnoy's solution compared to paraformaldehyde solution. Isolated RNA from MA-induced asthmatic murine lung epithelium, smooth muscle, and granulomatous foci using LCM showed a significant increase in remodeling gene expression compared to control which confirm the quality and integrity of isolated RNA. Overall, the study concludes tissue fixation solvent can alter the quality of RNA in the lung and the outcome of the results.

Maternal nutrition and stage of early pregnancy in beef heifers: impacts on hexose and AA concentrations in maternal and fetal fluids1.

We examined the hypothesis that maternal nutrition and day of gestation would affect the concentrations of AAs and hexoses in bovine utero-placental fluids and maternal serum from days 16 to 50 of gestation. Forty-nine cross-bred Angus heifers were bred via artificial insemination and fed a control diet (CON = 100% of requirements for growth) or a restricted diet (RES = 60% of CON) and ovariohysterectomized on days 16, 34, or 50 of gestation; nonpregnant controls were not bred and ovariohysterectomized on day 16 of the synchronized estrous cycle. The resulting design was a completely randomized design with a 2 × 3 factorial + 1 arrangement of treatments. Maternal serum, histotroph, allantoic fluid, and amniotic fluid were collected at time of ovariohysterectomy. Samples were then analyzed for concentrations of AAs and intermediary metabolites: alanine (Ala), arginine, asparagine (Asn), aspartate (Asp), citrulline, cysteine, glutamine, glutamate (Glu), glycine (Gly), histidine, isoleucine, leucine (Leu), lysine, methionine (Met), ornithine, phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan, tyrosine (Tyr), and valine (Val). The concentrations of Gly, Ser, and Thr in maternal serum were greater (P ≤ 0.05) in CON compared with RES. Furthermore, day of gestation affected (P ≤ 0.05) concentrations of Asn, Glu, Phe, Thr, and Tyr in maternal serum. Status of maternal nutrition affected the Asp concentration of histotroph where RES was greater (P = 0.02) than CON. In histotroph, Ala, Leu, Met, and Val concentrations were greater (P ≤ 0.05) on day 50 compared with day 16. Additionally, Glu and Pro concentrations in histotroph were greater (P < 0.01) on days 34 and 50 compared with day 16. A day × treatment interaction was observed for the concentration of Val in allantoic fluid where day 34 CON was greater (P = 0.05) than all other days and nutritional treatments. In addition, the concentration of Gln in amniotic fluid experienced a day × treatment interaction where day 34 RES was greater (P ≤ 0.05) than day 34 CON, which was greater (P ≤ 0.05) than day 50 CON and RES. These data support our hypothesis that day of gestation and maternal nutrition affect the concentrations of various neutral and acidic AA in beef heifer utero-placental fluids and maternal serum from days 16 to 50 of gestation.

Placental development during early pregnancy in sheep: nuclear estrogen and progesterone receptor mRNA expression in the utero-placental compartments.

Sex steroid hormones are major regulators of uterine and placental growth and functions, as well as many other biological processes. To examine the mRNA expression of nuclear estrogen (ESR1 and 2) and progesterone (PGRAB and B) receptors in different compartments of the uterus and placenta, tissues were collected in experiment 1 on days 16, 20, and 28 after natural mating (NAT) and on day 10 after estrus (nonpregnant controls [NP]); and in experiment 2 on day 22 of NAT, and pregnancies established after transfer of embryos generated through mating of FSH-treated ewes (NAT-ET), in vitro fertilization (IVF), or in vitro activation (parthenotes). In experiment 1, ESR1 expression in endometrial stroma (ES), endometrial glands (EGs), and myometrial blood vessels (MBVs), ESR2 in endometrial blood vessels (EBV), PGRAB in ES, and PGRB in ES, EG, and MBV was greater in pregnant than NP ewes depending on the day of pregnancy. The day of pregnancy affected the expression of ESR1 in MBV, ESR2 in EBV and MBV, and PGRAB in ES. In experiment 2, ESR1, PGRAB, and PGRB in EG, but not in other compartments, was greater in NAT-ET than NAT, and PGRB was greater for NAT-ET than IVF. These data demonstrate that ESR and PGR expression differ in pregnant versus NP ewes in selected compartments and was affected by pregnancy stage or embryo origin in selected utero-placental compartments. Thus, sex steroid hormone mRNA expression is differentially regulated in a spatiotemporal manner in the uterus and placenta and is affected by the application of assisted reproductive technology in sheep.

PEG-b-poly (carbonate)-derived nanocarrier platform with pH-responsive properties for pancreatic cancer combination therapy.

A pH-responsive nanoparticle platform, based on PEG-b-poly (carbonate) block copolymers have been proposed that can respond to low pH as found in many cancer micro- and intracellular environment, including that in pancreatic cancer. The hydrophobic domain, i.e., the poly (carbonate) segment has been substituted with tertiary amine side chains, such as N, N'-dibutylethylenediamine (pKa = 4.0, DB) and 2-pyrrolidin-1-yl-ethyl-amine (pka = 5.4, Py) to generate two different sets of block copolymers namely PEG-DB and PEG-PY systems. These side-chain appended amines promote disassembly of nanoparticles and activation of drug release in response to pH conditions mimicking extra- (pH 6.9-6.5) and intracellular compartments (5.5-4.5, from early endosome to lysosome) of cancer tissues respectively. A frontline chemotherapy used for pancreatic cancer, i.e., gemcitabine (GEM) and a Hedgehog inhibitor (GDC 0449) has been used as the model combination to evaluate the encapsulation and pH-dependent release efficiency of these block copolymers. We found that, depending on the tertiary amine side chains appended to the polycarbonate segment, these block copolymers self-assemble to form nanoparticles with the size range of 100-150 nm (with a critical association concentration value in the order of 10-6 M). We also demonstrated an approach where GEM and GDC 0449-encapsulated PEG-DB and PEG-PY nanoparticles, responsive to two different pH conditions, when mixed at a 1:1 vol ratio, yielded a pH-dependent co-release of the encapsulated contents. We envision that such release behaviour can be exploited to gain spatiotemporal control over drug accumulation in pathological compartments with different pH status. The mixture of pH-responsive nanoparticles was found to suppress pancreatic cancer cell proliferation when loaded with anticancer agents in vitro. Cell-proliferation assay showed that both variants of PEG-b-polycarbonate block copolymers were inherently non-toxic. We have also immobilized iRGD peptide on intracellularly activable PEG-DB systems to augment cellular uptake. These targeted nanoparticles were found to promote selective internalization of particles in pancreatic cancer cells and tumor tissue.

Tissue-Penetrating, Hypoxia-Responsive Echogenic Polymersomes For Drug Delivery To Solid Tumors.

Hypoxia in solid tumors facilitates the progression of the disease, develops resistance to chemo and radiotherapy, and contributes to relapse. Due to the lack of tumor penetration, most of the reported drug carriers are unable to reach the hypoxic niches of the solid tumors. We have developed tissue-penetrating, hypoxia-responsive echogenic polymersomes to deliver anticancer drugs to solid tumors. The polymersomes are composed of a hypoxia-responsive azobenzene conjugated and a tissue penetrating peptide functionalized polylactic acid-polyethylene glycol polymer. The drug-encapsulated, hypoxia-responsive polymersomes substantially decreased the viability of pancreatic cancer cells in spheroidal cultures. Under normoxic conditions, polymersomes were echogenic at diagnostic ultrasound frequencies but lose the echogenicity under hypoxia. In-vivo imaging studies with xenograft mouse model further confirmed the ability of the polymersomes to target, penetrate, and deliver the encapsulated contents in hypoxic pancreatic tumor tissues.

Placental development during early pregnancy in sheep: Progesterone and estrogen receptor protein expression.

The aim of this study was to evaluate the pattern of protein expression of the steroid receptor isoforms of nuclear progesterone receptors (PGR) A and B, and estrogen receptors (ESR1 and 2) in utero-placental compartments during early pregnancy. Utero-placental tissues were collected from days 14-30 (n = 4 ewes/day), and uterine tissues were collected from non-pregnant ewes on day 10 after estrus (n = 4). Cross sections of formalin-fixed and paraffin embedded tissues were immunofluorescently stained to detect PGRAB, PGRB, ESR1 and ESR2, followed by image generation of entire cross-sections of uterine and utero-placental tissues, confocal imaging of individual uterine and utero-placental compartments, and image and statistical analyses. PGRAB, PGRB, ESR1 and ESR2 were detected in several compartments of uterine and utero-placental tissues. Quantitative image analysis of staining intensity demonstrated that compared to non-pregnant controls 1) expression of PGRAB and PGRB was less in luminal epithelium and endometrial glands from day 14-16 till 30; 2) PGRAB expression tended to be greater in endometrial and myometrial blood vessels on days 28 and/or 30; 3) PGRB expression in myometrum was lower on days 16 and 28; 4) ESR1 in endometrial stroma was lower in all days of pregnancy; 5) ESR2 expression was similar in all compartments and not affected by pregnancy stage; and 6) in FM, expression of steroid receptors was similar. Thus, we have demonstrated spatial and temporal expression of nuclear PGR and ESR isoforms in utero-placental compartments during early pregnancy.

The effects of nutrient restriction on mRNA expression of endogenous retroviruses, interferon-tau, and pregnancy-specific protein-B during the establishment of pregnancy in beef heifers.

We hypothesize that syncytin-Rum1, bovine endogenous retrovirus-K1 (BERV-K1), pregnancy-specific protein-B (PSP-B), and interferon-τ (IFN-τ) will be influenced by maternal nutrient restriction and be differentially expressed during key stages (day 16, 34, and 50) of the establishment of gestation when fed to meet industry standards. Commercial crossbred heifers (n = 49) were maintained on a total mixed ration and supplemented with dried distillers grains with solubles. All heifers were subjected to 5-d CO-Synch + CIDR estrus synchronization protocol. Non-pregnant, non-bred control (NP-NB) heifers (n = 6) were ovariohysterectomized on day 16, and the remaining heifers were AI to a single Angus sire (day of breeding = day 0). On the day of breeding, heifers were randomly assigned to dietary treatments. One half were assigned to control treatment (CON) targeted to gain 0.45 kg/d, and the remaining half were assigned to restricted treatment (RES), which received 60% of control diets. Heifers were subjected to ovariohysterectomy on day 16, 34, or 50 of gestation. Utero-placental tissues were obtained from the uterine horn ipsilateral (P) and contralateral (NP) to the corpus luteum and separated into maternal caruncle (CAR), maternal endometrium, inter-caruncle, (ICAR), and fetal membrane (FM). There were no interactions between stage of gestation and nutritional treatment for syncytin-Rum1 or PSP-B (P > 0.22). Expression of BERV-K1 was influenced by a treatment × stage of gestation interaction (P = 0.03) in NP-CAR. On day 50, heifers fed the CON diet had greater BERV-K1 expression compared with CON heifers on day 16 and 34 and RES heifers at all sampling time points. There was a treatment × stage of gestation interaction (P < 0.01) for IFN-τ in FM tissue. On 16 d, mRNA expression of IFN-τ was greater (P < 0.01) compared with day 34 and 50 for both CON and RES heifers, but RES FM had greater (P < 0.01) IFN-τ expression compared with CON FM. In P-CAR, PSP-B expression increased (P < 0.01) by 18 000-fold on day 50 compared with NP-NB heifers. In P-ICAR, expression of syncytin-Rum1 in P-ICAR was greater (P = 0.01) on day 16 with a 14.14-fold increase compared with relative expression on day 34 and 50; whereas, PSP-B was increased (P < 0.01) on day 34 and 50 compared with day 16. In conclusion, 40% nutrient restriction had limited influence on mRNA of ERVs, PSP-B, and IFN-τ but stage of gestation differences reinforced the importance of these genes during the establishment of pregnancy.

Peptide-targeted, stimuli-responsive polymersomes for delivering a cancer stemness inhibitor to cancer stem cell microtumors.

Often cancer relapses after an initial response to chemotherapy because of the tumor's heterogeneity and the presence of progenitor stem cells, which can renew. To overcome drug resistance, metastasis, and relapse in cancer, a promising approach is the inhibition of cancer stemness. In this study, the expression of the neuropilin-1 receptor in both pancreatic and prostate cancer stem cells was identified and targeted with a stimuli-responsive, polymeric nanocarrier to deliver a stemness inhibitor (napabucasin) to cancer stem cells. Reduction-sensitive amphiphilic block copolymers PEG1900-S-S-PLA6000 and the N3-PEG1900-PLA6000 were synthesized. The tumor penetrating iRGD peptide-hexynoic acid conjugate was linked to the N3-PEG1900-PLA6000 polymer via a Cu2+ catalyzed "Click" reaction. Subsequently, this peptide-polymer conjugate was incorporated into polymersomes for tumor targeting and tissue penetration. We prepared polymersomes containing 85% PEG1900-S-S-PLA6000, 10% iRGD-polymer conjugate, and 5% DPPE-lissamine rhodamine dye. The iRGD targeted polymersomes encapsulating the cancer stemness inhibitor napabucasin were internalized in both prostate and pancreatic cancer stem cells. The napabucasin encapsulated polymersomes significantly (p < .05) reduced the viability of both prostate and pancreatic cancer stem cells and decreased the stemness protein expression notch-1 and nanog compared to the control and vesicles without any drug. The napabucasin encapsulated polymersome formulations have the potential to lead to a new direction in prostate and pancreatic cancer therapy by penetrating deeply into the tumors, releasing the encapsulated stemness inhibitor, and killing cancer stem cells.

Enterolactone alters FAK-Src signaling and suppresses migration and invasion of lung cancer cell lines.

BACKGROUND: Systemic toxicity of chemotherapeutic agents and the challenges associated with targeting metastatic tumors are limiting factors for current lung cancer therapeutic approaches. To address these issues, plant-derived bioactive components have been investigated for their anti-cancer properties because many of these agents are non-toxic to healthy tissues. Enterolactone (EL) is a flaxseed-derived mammalian lignan that has demonstrated anti-migratory properties for various cancers, but EL has not been investigated in the context of lung cancer, and its anticancer mechanisms are ill-defined. We hypothesized that EL could inhibit lung cancer cell motility by affecting the FAK-Src signaling pathway. METHODS: Non-toxic concentrations of EL were identified for A549 and H460 human lung cancer cells by conducting 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Dephenyltetrazolium Bromide (MTT) assays. The anti-migratory and anti-invasive potential of EL for lung cancer cell lines was determined by scratch wound healing and Matrigel® invasion assays. Changes in filamentous actin (F-actin) fiber density and length in EL-treated cells were determined using phalloidin-conjugated rhodamine dye and fluorescent microscopy. Vinculin expression in focal adhesions upon EL treatment was determined by immunocytochemistry. Gene and protein expression levels of FAK-Src signaling molecules in EL-treated lung cancer cells were determined using PCR arrays, qRT-PCR, and western blotting. RESULTS: Non-toxic concentrations of EL inhibited lung cancer cell migration and invasion in a concentration- and time-dependent manner. EL treatment reduced the density and number of F-actin fibers in lung cancer cell lines, and reduced the number and size of focal adhesions. EL decreased phosphorylation of FAK and its downstream targets, Src, paxillin, and decreased mRNA expression of cell motility-related genes, RhoA, Rac1, and Cdc42 in lung cancer cells. CONCLUSIONS: Our data suggest that EL suppresses lung cancer cell motility and invasion by altering FAK activity and subsequent activation of downstream proteins needed for focal adhesion formation and cytoskeletal rearrangement. Therefore, administration of EL may serve as a safe and complementary approach for inhibiting lung tumor cell motility, invasion, and metastasis.