Fiber type composition of epaxial muscles is geared toward facilitating rapid spinal extension in the leaper Galago senegalensis.

OBJECTIVES: We hypothesized that the vertical leaper Galago senegalensis will have epaxial extensor muscles with a fast fiber phenotype to facilitate rapid spinal extension during leaping in comparison to the slow-moving quadruped Nycticebus coucang. To test this, we determined the percentage of fiber cross-sectional area (%CSA) devoted to Type 2 fibers in epaxial muscles of G. senegalensis compared to those of N. coucang. MATERIALS AND METHODS: Immunohistochemistry was used to identify Type 1, Type 2, and hybrid fibers in iliocostalis, longissimus, and multifidus muscles of G. senegalensis (n = 3) and N. coucang (n = 3). Serial muscle sections were used to estimate and compare proportions, cross-sectional areas (CSAs), and %CSAs of Type 1, Type 2, and hybrid fibers between species. RESULTS: Epaxial muscles of G. senegalensis were comprised predominantly of Type 2 fibers with large CSAs (%CSA range ≈ 83-94%; range of mean CSA = 1,218-1,586 µm2 ). N. coucang epaxial muscles were comprised predominantly Type 1 fibers with large CSAs (%CSA range ≈ 69-77%; range of mean CSA = 983-1,220 µm2 ). DISCUSSION: The predominance of Type 2 fibers in G. senegalensis epaxial muscles facilitates rapid muscle excursion and spinal extension during leaping, and is consistent with their relatively long muscle fibers. The predominance of Type 1 fibers in N. coucang epaxial muscles may aid in maintaining stable postures during bridging and cantilevering behaviors characteristic of slow-climbing. These histochemical characteristics highlight the major divergent locomotor repertoires of G. senegalensis and N. coucang.

Nuclear basic fibroblast growth factor regulates triple-negative breast cancer chemo-resistance.

INTRODUCTION: Chemotherapy remains the only available treatment for triple-negative (TN) breast cancer, and most patients exhibit an incomplete pathologic response. Half of patients exhibiting an incomplete pathologic response die within five years of treatment due to chemo-resistant, recurrent tumor growth. Defining molecules responsible for TN breast cancer chemo-resistance is crucial for developing effective combination therapies blocking tumor recurrence. Historically, chemo-resistance studies have relied on long-term chemotherapy selection models that drive genetic mutations conferring cell survival. Other models suggest that tumors are heterogeneous, being composed of both chemo-sensitive and chemo-resistant tumor cell populations. We previously described a short-term chemotherapy treatment model that enriches for chemo-residual TN tumor cells. In the current work, we use this enrichment strategy to identify a novel determinant of TN breast cancer chemotherapy resistance [a nuclear isoform of basic fibroblast growth factor (bFGF)]. METHODS: Studies are conducted using our in vitro model of chemotherapy resistance. Short-term chemotherapy treatment enriches for a chemo-residual TN subpopulation that over time resumes proliferation. By western blotting and real-time polymerase chain reaction, we show that this chemotherapy-enriched tumor cell subpopulation expresses nuclear bFGF. The importance of bFGF for survival of these chemo-residual cells is interrogated using short hairpin knockdown strategies. DNA repair capability is assessed by comet assay. Immunohistochemistry (IHC) is used to determine nuclear bFGF expression in TN breast cancer cases pre- and post- neoadjuvant chemotherapy. RESULTS: TN tumor cells surviving short-term chemotherapy treatment express increased nuclear bFGF. bFGF knockdown reduces the number of chemo-residual TN tumor cells. Adding back a nuclear bFGF construct to bFGF knockdown cells restores their chemo-resistance. Nuclear bFGF-mediated chemo-resistance is associated with increased DNA-dependent protein kinase (DNA-PK) expression and accelerated DNA repair. In fifty-six percent of matched TN breast cancer cases, percent nuclear bFGF-positive tumor cells either increases or remains the same post- neoadjuvant chemotherapy treatment (compared to pre-treatment). These data indicate that in a subset of TN breast cancers, chemotherapy enriches for nuclear bFGF-expressing tumor cells. CONCLUSION: These studies identify nuclear bFGF as a protein in a subset of TN breast cancers that likely contributes to drug resistance following standard chemotherapy treatment.

Immunohistochemical markers in fibrohistiocytic lesions: factor XIIIa, CD34, S-100 and p75.

BACKGROUND: The distinction between dermatofibroma (DF), dermatofibrosarcoma protuberans (DFSP), and other benign and malignant cutaneous spindle cell lesions frequently requires immunohistochemical staining. CD34 and factor XIIIa are the most commonly used immunostains; however, they may exhibit aberrant expression and introduce the potential for misdiagnosis. There is some data supporting that p75 and S100A6 may be additional helpful immunohistochemical markers. METHODS: We undertook a large case series examining the use of CD34 and factor XIIIa as well as p75 and S100A6 in DF, cellular DF, DFSP, indeterminate fibrohistiocytic lesion, and scar. RESULTS: As expected, CD34 stained DFSP, although it was usually negative in DF. Factor XIIIa was generally positive in DF and negative in DFSP. There were exceptions in both cases of DF and DFSP. S100A6 was routinely negative in all entities studied. P75 was negative in all cases except DFSP, approximately half of which showed weak and/or patchy positivity. CONCLUSIONS: We conclude that to date, CD34 and factor XIIIa remain the most reliable immunohistochemical markers for DF and DFSP.

FoxP3 and indoleamine 2,3-dioxygenase immunoreactivity in sentinel nodes from melanoma patients.

OBJECTIVE: 1) Assess FoxP3/indoleamine 2,3-dioxygenase immunoreactivity in head and neck melanoma sentinel lymph nodes and 2) correlate FoxP3/indoleamine 2,3-dioxygenase with sentinel lymph node metastasis and clinical recurrence. STUDY DESIGN: Retrospective cohort study. METHODS: Patients with sentinel lymph node biopsy for head and neck melanoma between 2004 and 2011 were identified. FoxP3/indoleamine 2,3-dioxygenase prevalence and intensity were determined from the nodes. Poor outcome was defined as local, regional or distant recurrence. The overall immunoreactivity score was correlated with clinical recurrence and sentinel lymph node metastasis using the chi-square test for trend. RESULTS: Fifty-six sentinel lymph nodes were reviewed, with 47 negative and 9 positive for melanoma. Patients with poor outcomes had a statistically significant trend for higher immunoreactivity scores (p=0.03). Positive nodes compared to negative nodes also had a statistically significant trend for higher immunoreactivity scores (p=0.03). Among the negative nodes, there was a statistically significant trend for a poor outcome with higher immunoreactivity scores (p=0.02). CONCLUSION: FoxP3/indoleamine 2,3-dioxygenase immunoreactivity correlates with sentinel lymph node positivity and poor outcome. Even in negative nodes, higher immunoreactivity correlated with poor outcome. Therefore higher immunoreactivity may portend a worse prognosis even without metastasis in the sentinel lymph node. This could identify a subset of patients that may benefit from future trials and treatment for melanoma through Treg and IDO suppression.

Tea polyphenols increase the antioxidant status of laying hens fed diets with different levels of ageing corn.

This study was conducted to evaluate the effects of ageing corn levels (stored for 4 years) with or without the supplementation of tea polyphenols (TPP) on the performance, egg quality and antioxidant status of laying hens. A total of 288 Lohmann commercial laying hens (63-week-old) were used under a 2 × 4 factorial arrangement with 4 levels of dietary ageing corn (0%, 25%, 50%, or 100%) and 2 levels of TPP (0 and 600 mg/kg) for 8 wk. Dietary ageing corn linearly decreased (P < 0.05) the egg production, serum total antioxidant capacity (T-AOC), liver glutathione peroxidase (GSH-Px) of laying hens, yolk index, yolk colour, 1,1-diphenyl-2-picrylhydrazyl (DPPH) value and the reducing power value of egg yolk, but it linearly increased (P < 0.05) the feed conversion rate, ovary malondialdehyde (MDA) content of laying hens, and the protein carbonyl content of egg yolk. Tea polyphenol supplementation increased (P < 0.05) the serum T-AOC, serum superoxide dismutase (SOD), liver SOD, liver GSH-Px, ovary SOD, GSH-Px, the expression of antioxidant-related genes of laying hens, albumen height, Haugh unit, DPPH value and the majority free amino acids of egg yolk, but it decreased (P < 0.05) the serum MDA content of laying hens, MDA and protein carbonyl of egg yolk. In conclusion, the ageing corn significantly reduced the performance, egg quality, antioxidant status and egg antioxidant capacity of laying hens, while TPP supplementation partially counteracted the adverse effects, especially antioxidant status and egg antioxidant capacity of laying hens.

The Effects of Broiler Breeder Dietary Vitamin E and Egg Storage Time on the Quality of Eggs and Newly Hatched Chicks.

This study was conducted to investigate the effects of broiler breeder dietary vitamin E and egg storage time on the egg characteristics, hatchability, and antioxidant status of the egg yolks and newly hatched chicks. A total of 512 71-week-old Ross 308 breeder hens were fed the same basic diets containing 6 or 100 mg/kg vitamin E for 12 weeks. During this time, a total of 1532, 1464, and 1316 eggs were independently collected at weeks 8, 10, and 12, respectively, and subsequently stored for 0 or 14 d before hatching. The outcomes from three trials showed that prolonged egg storage time (14 vs. 0 d) negatively affected (p < 0.05) the egg characteristics, hatchability traits, and the yolk total antioxidant capacity (T-AOC) (p < 0.05). Chicks derived from the stored eggs exhibited higher malonaldehyde (MDA) and T-AOC in the serum and yolk sac (p < 0.05). Broiler breeder dietary vitamin E (100 vs. 6 mg/kg) increased (p < 0.05) the hatchability and the antioxidant status of the yolks as indicated by a higher α-tocopherol content and T-AOC and lower MDA level (p < 0.05). The supplementation of vitamin E also remarkably increased (p < 0.05) the total superoxide dismutase (T-SOD) activity (yolk sac, weeks 8 and 12) and T-AOC (serum, weeks 8, 10, and 12; yolk sac, weeks 8 and 12) and decreased (p < 0.05) the MDA content of chicks (yolk sac, week 10; serum, week 12). Interactions (p < 0.05) were found between the broiler breeder dietary vitamin E and egg storage time on the hatchability and antioxidant status of chick tissues. Broiler breeder dietary vitamin E (100 vs. 6 mg/kg) increased (p < 0.05) the hatchability and the T-AOC in the serum and liver of chicks, and decreased (p < 0.05) the early embryonic mortality and the MDA content in the yolk sacs of chicks derived from eggs stored for 14 d but not for 0 d. In conclusion, prolonged egg storage time (14 vs. 0 d) increased the embryonic mortality, decreased the hatchability, and impaired the antioxidant status of egg yolks and newly hatched chicks, while the addition of broiler breeder dietary vitamin E (100 vs. 6 mg/kg) could partly relieve these adverse impacts induced by long-term egg storage.

Effects of Dietary Iron Concentration on Manganese Utilization in Broilers Fed with Manganese-Lysine Chelate-Supplemented Diet.

Dietary iron (Fe) influences manganese (Mn) utilization in chickens fed with inorganic Mn-supplemented diet. This study aimed to determine if dietary Fe levels affect Mn utilization in broilers fed with organic Mn-supplemented diet. Nine hundred 8-day-old broilers were randomly assigned to 1 of 6 treatments in a 3 (Fe level) × 2 (Mn source) factorial arrangement after feeding Mn- and Fe-unsupplemented diets for 7 days. The broilers were fed the basal diets (approximately 28 mg Mn/kg and 60 mg Fe/kg) supplemented with 0, 80, or 160 mg/kg Fe (L-Fe, M-Fe, or H-Fe), and 100 mg/kg Mn from Mn sulfate (MnSO4) or manganese-lysine chelate (MnLys) for 35 days. The H-Fe diet decreased (P < 0.05) body weight gain and feed intake as compared with L-Fe and M-Fe diets regardless of dietary Mn sources. Dietary Fe levels did not influence (P > 0.10) serum Mn concentration in MnLys-treated broilers, but serum Mn concentration decreased (P < 0.05) with dietary Fe increasing in MnSO4-treated broilers. The Mn concentration in the duodenum and tibia decreased (P < 0.05) with increasing dietary Fe levels regardless of dietary Mn sources, and MnLys increased (P < 0.04) these indices as compared with MnSO4. Dietary Fe levels did not significantly influence (P > 0.11) Mn concentration and activity and mRNA abundance of manganese-containing superoxide dismutase (MnSOD) in the heart of MnLys-treaded broilers, but the H-Fe diet decreased (P < 0.05) these indices in MnSO4-treated broilers as compared with M-Fe and L-Fe diets. The L-Fe diet increased (P < 0.001) duodenal divalent metal transporter 1 mRNA abundance when compared with the M-Fe and H-Fe diets on day 42, regardless of dietary Mn sources. The M-Fe and H-Fe diets decreased (P < 0.001) duodenal ferroportin 1 (FPN1) mRNA level when compared with the L-Fe diet in MnSO4-treated broilers, while dietary Fe levels did not significantly influence (P > 0.40) duodenal FPN1 mRNA abundance in MnLys-treated broilers. These results indicated dietary Fe levels decreased Mn utilization in MnSO4-treated broilers, but did not influence Mn utilization in MnLys-treated broilers evaluated by Mn concentrations in the serum and heart, and the activity and mRNA expression of heart MnSOD.

Oxidized Oils and Oxidized Proteins Induce Apoptosis in Granulosa Cells by Increasing Oxidative Stress in Ovaries of Laying Hens.

The storage and preparation of corn for animal feed inevitably lead to lipid and protein peroxidation. Granulosa cells play an important role in follicular development in the ovaries, and hen laying productivity is likely to be dependent on follicle health and number. We hypothesized that oxidized oil and protein induce apoptosis via oxidative stress in laying hen granulosa cells. A sample of 360 38-week-old Lohmann commercial laying hens was used in a 2 × 2 factorial design for 8 weeks. Dietary treatments included dietary oil (fresh corn oil (FO) or oxidized corn oil (OO)) and corn gluten meal (fresh corn gluten meal (FP) or oxidized corn gluten meal (OP)). Productivity, ovarian histology, granulosa cell apoptosis, and indicators of oxidative stress were evaluated in all groups. Both dietary OO and OP decreased egg production and the average daily feed intake (ADFI) of laying hens. Flow cytometry, TUNEL, and real-time PCR revealed that both dietary OO and OP induced granulosa cell apoptosis in prehierarchical and hierarchical follicles. Furthermore, dietary OO and OP caused oxidative stress in prehierarchical and hierarchical follicles, as indicated by the downregulation of antioxidant-related-gene expression. Moreover, forkhead box O1 (FoxO1), extracellular regulated protein kinase (ERK), and c-Jun NH2 kinase (JNK) are involved in potential apoptosis regulation pathways in the granulosa cells of laying hens fed OO and OP, as indicated by the upregulation of FoxO1 expression and downregulation of ERK/JNK expression. These results indicate that OO and OP induce granulosa cell apoptosis via oxidative stress, and the combined use of OO and OP aggravates the adverse effects of oxidative stress in laying hens.

Effects of Dietary Iron on Manganese Utilization in Broilers Fed with Corn-Soybean Meal Diet.

To investigate the effects of dietary iron (Fe) levels on manganese (Mn) utilization, 900 8-day-old broilers were randomly assigned to 1 of 6 treatments in a 3 (Fe level) × 2 (Mn level) factorial arrangement after feeding Mn- and Fe-unsupplemented diet for 7 days. The broilers were then fed with basal corn-soybean meal diets (approximately 28 mg Mn/kg and 60 mg Fe/kg) added with 0, 80, or 160 mg/kg Fe (L-Fe, M-Fe, or H-Fe), and 0 or 100 mg/kg Mn for 35 days. Body weight gain was lower for H-Fe broilers than that for L-Fe and M-Fe broilers. On day 42, H-Fe broilers had lower serum Mn concentration as compared with L-Fe and M-Fe broilers, and tibia Mn concentration decreased as dietary Fe increased. In Mn-supplemented broilers, liver Mn was lower in L-Fe and H-Fe treatments than that in M-Fe treatment. H-Fe treatment decreased Mn concentration and manganese-containing superoxide dismutase (MnSOD) activity in the heart when compared with L-Fe and M-Fe treatments. Dietary Fe did not significantly influence Mn concentrations in the liver and heart, and heart MnSOD activity in Mn-unsupplemented broilers. In the duodenum, L-Fe treatment decreased divalent metal transporter 1 (DMT1) mRNA abundance when compared with M-Fe and H-Fe treatments, and ferroportin 1 (FPN1) mRNA level was higher in M-Fe treatment than that in L-Fe and H-Fe treatments. These results suggested H-Fe diet decreased Mn status in broilers evaluated by Mn concentrations in serum and heart, and heart MnSOD activity. Dietary Fe influenced Mn absorption possibly through effects on duodenal DMT1 and FPN1 expression.

PD-L1 Assay Concordance in Metastatic Renal Cell Carcinoma and Metastatic Urothelial Carcinoma.

BACKGROUND: Immune checkpoint inhibitors are now standard of care for many patients with metastatic renal cell carcinoma (mRCC) and metastatic urothelial carcinoma (mUC). Given real-world limitations in programmed death-ligand 1 (PD-L1) testing, concordance studies between PD-L1 assays are needed. We undertook comparisons of Dako 28-8 and Ventana SP142 assays in mRCC and Dako 22C3 and Ventana SP263 assays in mUC. PATIENTS AND METHODS: Thirty-two patients with mRCC and 18 patients with mUC who had received immune checkpoint inhibitor therapy were identified. Formalin-fixed paraffin-embedded tumor samples for patients with mRCC were evaluated with Dako 28-8 and Ventana SP142 PD-L1 immunohistochemistry assays. For patients with mUC, formalin-fixed paraffin-embedded tumor samples were evaluated with Dako 22C3 and Ventana SP263 PD-L1 immunohistochemistry assays. RESULTS: The majority (29/32; 91%) of mRCC cases were concordant between assays. The majority (17/18; 94%) of mUC cases were also concordant between assays. CONCLUSIONS: There was strong concordance between PD-L1 assays chosen for comparison in both mRCC and mUC, with similar performance characteristics. One limitation is the small number of cases in this study; larger comparison studies are needed for this biomarker in mRCC and mUC.

Uptake of Manganese from the Manganese-Lysine Complex in Primary Chicken Intestinal Epithelial Cells.

Organic manganese (Mn) sources can replace inorganic Mn as dietary Mn supplements in poultry. To compare the uptake of Mn from the Mn-lysine complex (MnLys) and MnSO4, we first established the primary chicken intestinal epithelial cells (IECs) model and used it to determine Mn uptake. The MnLys increased the uptake of Mn compared to MnSO4. The uptake of Mn decreased in the IECs with Fe addition in the medium regardless of the Mn sources. The MnLys decreased the Mn2+ efflux transporter ferroportin 1 (FPN1) mRNA level but did not influence the Mn2+ influx transporter divalent metal transporter 1 (DMT1) mRNA expression when compared to MnSO4. The results above indicated that the increase of Mn accumulation for MnLys at least partly was due to the decrease of Mn efflux by reduced FPN1 expression. The addition of N-ethylmaleimide, an L-lysine transport system y+ inhibitor, decreased the uptake of Mn from MnLys but did not affect the uptake of Mn from MnSO4. The cycloheximide, as an L-lysine transport system b0,+ activator, increased the uptake of Mn from MnLys, whereas they did not influence the uptake of Mn from MnSO4. The MnLys increased the system y+ members cationic amino acid transporter (CAT) 1 and CAT2, and system b0,+ components rBAT and b0,+AT mRNA expression when compared to MnSO4. These results suggested that the uptake of MnLys complex might be transported by CAT1/2 and system b0,+, which was different from the ionized Mn2+ uptake pathway. In conclusion, the uptake of Mn from MnLys complex not only might be uptake through the ionized Mn2+ pathway, but also appeared to be transported through the CAT1/2 and system b0,+ in primary chicken IECs.

High Dietary Iron Differentially Influences the Iron Distribution in the Livers and the Spleens of Laying Hens After Salmonella Typhimurium Infection.

Salmonella and the host battle for iron (Fe), due to its importance for fundamental cellular processes. To investigate Fe redistribution of Salmonella-infected hens and the effects of high dietary Fe on it, Salmonella-free hens were randomly assigned to 1 of 4 treatments in 2 (two dietary Fe level) × 2 (Salmonella-inoculation or -noninoculation) factorial assignment. After feeding a basal diet supplemented with 60 (adequate, control) or 300 mg Fe/kg (high-Fe) for 4 weeks, 59-week-old Salmonella-free hens were orally inoculated with 5 × 107 colony-forming units of Salmonella Typhimurium (infection) or PBS (vehicle). Blood, spleen, and liver samples (n = 8) were collected at 14 days post-inoculation to determine Fe concentration and Fe transporters expression. Salmonella infection decreased (P < 0.05) hematocrit, serum Fe concentration, and splenic Fe concentration regardless of high-Fe or control hens, whereas increased (P < 0.05) Fe centration in the livers of high-Fe-treated hens. High dietary Fe increased hematocrit and serum Fe concentration, but did not affect (P = 0.11) splenic Fe concentration in Salmonella-infected hens. Salmonella infection did not influence (P = 0.31) liver Fe centration in control hens, but increased (P = 0.04) it in high-Fe-treated hens. High dietary Fe decreased (P < 0.01) the mRNA abundance of divalent metal transporter 1 and transferrin receptor, but increased (P < 0.02) ferroportin-1 (FPN1) mRNA and protein in the spleens and the livers regardless of Salmonella-infected or vehicle hens. Salmonella infection increased (P < 0.02) FPN1 mRNA and protein expression in the spleens, but did not influence its expression in the livers. These results suggested Salmonella infection and high dietary Fe differently influence the Fe distribution in the spleen and the liver of Salmonella-infected hens.

Effect of High Dietary Manganese on the Immune Responses of Broilers Following Oral Salmonella typhimurium Inoculation.

Manganese (Mn) is an essential nutrient for both host and pathogen. Recent studies have demonstrated the nutritional immunity of Mn against Salmonella infection in mammals. To investigate the effect of high dietary Mn on immune responses of broilers following Salmonella challenge, 144 1-day-old male broilers were fed a basal diet (containing 20.04 mg Mn/kg) plus an additional 40 (the control group) or 400 mg Mn/kg (the H-Mn group) for 7 days. The 72 broilers in each group were then orally inoculated with 5 × 107 CFUs of Salmonella typhimurium (ATCC#14028) or phosphate-buffered saline. Peripheral blood, spleens, cecal tonsils, and bursa of Fabricius were collected from Salmonella-inoculated and Salmonella-noninoculated broilers (n = 6) at 2 days post inoculation (2 DPI) and 7 days post inoculation (7 DPI). Peripheral blood lymphocyte subpopulations were determined by flow cytometry. The messenger RNA (mRNA) abundance of genes was determined by quantitative real-time polymerase chain reaction. Salmonella counts were higher (P < 0.05) in the H-Mn group than that in the control group at 2 DPI in the cecal contents of Salmonella-inoculated broilers. High dietary Mn increased CD3+CD4+ and CD3+CD8+ percentages in the peripheral blood of Salmonella-inoculated broilers at 2 DPI. Salmonella inoculation increased interleukin (IL)-6 mRNA expression in spleens and bursa of Fabricius at 2 DPI and increased IL-1ß and IL-6 mRNA expression in cecal tonsils at 7 DPI in the H-Mn group. These changes were not observed in the control group. High dietary Mn increased interferon-γ (IFN-γ) in spleens and decreased IFN-γ and IL-12 mRNA expression in cecal tonsils of Salmonella-inoculated broilers at 2 DPI. High dietary Mn decreased IL-17 mRNA expression in the bursa of Fabricius at 7 DPI, but increased this expression in cecal tonsils at 2 and 7 DPI in Salmonella-inoculated broilers. These results suggested that dietary Mn level affected T helper (Th) 1-cytokine reaction in spleens and cecal tonsils, and Th17-mediated immunity in cecal tonsils and the bursa of Fabricius of broilers when challenged with Salmonella.

Long-term effect of dietary overload lithium on the glucose metabolism in broiler chickens.

Lithium, like insulin, activates glycogen synthase and stimulates glucose transport in rat adipocytes. To investigate the effect of dietary overload lithium on glucose metabolism in broiler chickens, one-day-old chicks were fed a basal diet supplemented with 0 (control) or 100mg lithium/kg (overload lithium) for 35days. Compared to controls, glucose disappearance rates were lower (p=0.035) 15-120min after glucose gavage, and blood glucose concentrations were lower (p=0.038) 30min after insulin injection in overload lithium broilers. Overload lithium decreased (p<0.05) glycogen and glucose-6-phosphate concentrations in liver, but increased (p<0.05) their concentrations in pectoralis major. Overload lithium increased (p<0.05) mRNA expression of glucose transporter (GLUT) 3 and GLUT9 in liver, and GLUT1, GLUT3, GLUT8, and GLUT9 in pectoralis major, but decreased (p<0.05) cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in liver and mitochondrial PEPCK in pectoralis major. These results suggest that dietary overload lithium decreases glucose tolerance and gluconeogenesis, but increases insulin sensitivity and glucose transport in broiler chickens.

Dietary overload lithium decreases the adipogenesis in abdominal adipose tissue of broiler chickens.

To investigate the toxic effects of dietary overload lithium on the adipogenesis in adipose tissue of chicken and the role of hypothalamic neuropeptide Y (NPY) in this process, one-day-old male chicks were fed with the basal diet added with 0 (control) or 100mg lithium/kg diet from lithium chloride (overload lithium) for 35days. Abdominal adipose tissue and hypothalamus were collected at day 6, 14, and 35. As a percentage of body weight, abdominal fat decreased (p<0.001) at day 6, 14, and 35, and feed intake and body weight gain decreased during day 7-14, and day 15-35 in overload lithium treated broilers as compared to control. Adipocyte diameter and DNA content in abdominal adipose tissue were significantly lower in overload-lithium treatment than control at day 35, although no significant differences were observed at day 6 and 14. Dietary overload lithium decreased (p<0.01) transcriptional expression of preadipocyte proliferation makers ki-67 (KI67), microtubule-associated protein homolog (TPX2), and topoisomerase 2-alpha (TOP2A), and preadipocyte differentiation transcriptional factors peroxisome proliferator-activated receptor-γ (PPARγ), and CCAAT/enhancer binding protein (C/EBP) α mRNA abundance in abdominal adipose tissue. In hypothalamus, dietary overload lithium influenced (p<0.001) NPY, and NPY receptor (NPYR) 6 mRNA abundance at day 6 and 14, but not at day 35. In conclusion, dietary overload lithium decreased the adipogenesis in abdominal adipose tissue of chicken, which was accompanied by depressing transcriptional expression of adipogenesis-associated factors. Hypothalamic NPY had a potential role in the adipogenesis in abdominal adipose tissue of broilers with a short-term overload lithium treatment.

Prothrombin and beta 2-glycoprotein I frequently contribute to antiphospholipid antibody interactions with phospholipids and the generation of abnormal waveform profiles in coagulation assays.

Transmittance waveforms are generated during clot formation on photo-optical coagulation analyzers. We previously showed that 61.5% of patients with antiphospholipid antibodies (APLA) exhibited a negative deflection in the pre-coagulation phase of the prothrombin time (PT slope 1). The current studies investigated the 'molecular basis' of this abnormal parameter. We found that the negative PT slope 1 is IgG-mediated and is not dependent on the presence of fibrinogen or thrombin activity. We also found that IgG from most of the patients required a specific thromboplastin and the presence of prothrombin or beta(2)-glycoprotein I beta(2) GPI) to produce an abnormal IgG wave-form assay. In addition, the abnormal IgG waveform required cofactor binding to phospholipids when beta(2) GPI was the cofactor, and annexin V could partially block this interaction. In conclusion, these results showed that the interactions of IgG with phospholipids via beta(2) GPI or prothrombin constitute the core mechanisms of the abnormal waveforms.

Anti-beta(2)-glycoprotein I antibody-mediated inhibition of activated protein C requires binding of beta(2)-glycoprotein I to phospholipids.

To clarify the role(s) of anti-beta(2) GPI antibodies on thrombosis in anti-phospholipid antibody syndromes (APS), the effect of IgG from three patients on activated protein C (APC) was investigated using phospholipid vesicles and purified proteins. Two of the total IgG inhibited APC activity in the presence of beta(2)GPI, whereas the third IgG did not. In addition, one IgG inhibited APC activity without beta(2)GPI. Anti-beta(2)GPI IgG from the two inhibitory IgG preparations inhibited APC activity only in the presence of beta(2)GPI. Inhibition was suppressed partially by excess APC and almost completely by excess phospholipid vesicles. Cleaved beta(2)GPI, a non-phospholipid-binding form, did not support inhibitory activity, even though the anti-beta(2)GPI IgG bound to the cleaved molecule. This study confirms that anti-beta(2)GPI antibodies from APS patients inhibit APC activity, and demonstrates the requirement of phospholipid binding of beta(2)GPI for expression of the inhibitory activity of these antibodies.

Nuclear Snail1 and nuclear ZEB1 protein expression in invasive and intraductal human breast carcinomas.

Snail1 and ZEB1 are transcriptional repressors that drive tumor initiation and metastasis in animal models. Snail1 and ZEB1 are frequently coexpressed in tumor cell lines, suggesting that these factors may cooperate to promote tumor progression. However, coexpression of these transcriptional repressors in primary human cancer specimens has not been investigated. Previous studies assessed expression in primary breast cancers of Snail1 messenger RNA, which does not reflect Snail1 activity because Snail1 is subject to posttranslational modifications that inhibit its nuclear localization/activity. In the current study, using breast tumor cell lines of known Snail1 and ZEB1 expression status, we developed immunohistochemistry protocols for detecting nuclear Snail1 and nuclear ZEB1 proteins. Using these protocols, we assessed nuclear Snail1 and nuclear ZEB1 expressions in primary human breast cancers of varying subtypes (n = 78). Nuclear Snail1 and estrogen receptor α expressions were inversely associated in primary breast cancers, and nuclear Snail1 was expressed in approximately 80% of triple-negative breast cancers (lacking estrogen receptor α, progesterone receptor, and human epidermal growth factor receptor 2 overexpression). In contrast, nuclear ZEB1 was expressed at a significantly lower frequency in these breast cancers. Notably, nuclear Snail1 protein was detected in 45% of ductal carcinoma in situ specimens (n = 29), raising the important possibility that nuclear Snail1 expression in early stage breast lesions may predict future development of invasive breast cancer. Collectively, our studies demonstrate frequent expression of nuclear Snail1, but not nuclear ZEB1, in invasive, triple-negative breast cancers as well as in intraductal carcinomas.

Protein microarray analysis of mammary epithelial cells from obese and nonobese women at high risk for breast cancer: feasibility data.

BACKGROUND: Obesity is a well-established risk factor for cancer, accounting for up to 20% of cancer deaths in women. Studies of women with breast cancer have shown obesity to be associated with an increased risk of dying from breast cancer and increased risk of developing distant metastasis. While previous studies have focused on differences in circulating hormone levels as a cause for increased breast cancer incidence in postmenopausal women, few studies have focused on potential differences in the protein expression patterns of mammary epithelial cells obtained from obese versus nonobese women. METHODS: Protein expression was assessed by reverse-phase protein microarray in mammary epithelial cells from 31 random periareolar fine needle aspirations performed on 26 high-risk women. RESULTS: In this pilot and exploratory study, vimentin (unadjusted P=0.028) expression was significantly different between obese and nonobese women. CONCLUSIONS: Vimentin is integral both to adipocyte structure and function and to the epithelial-to-mesenchymal transition needed for cancer cell metastasis. Further research is needed to confirm this finding and determine the possible effects of the adipocyte microenvironment on the initiation and progression of breast cancer in high-risk women. IMPACT: Differential protein expression patterns obtained from a future expanded study may serve to elaborate the underlying pathology of breast cancer initiation and progression in obese women and identify potential biomarkers of response to preventative interventions such as dietary changes and exercise.

Antiphospholipid antibodies after surgical exposure to topical bovine thrombin.

Exposure to topical bovine thrombin during surgery frequently results in the development of antibodies to multiple protein and carbohydrate antigens. We investigated the frequency of increased levels of antibodies to cardiolipin and beta(2)-glycoprotein I (beta(2)-GPI) in two groups of patients, one exposed to bovine thrombin during cardiovascular surgery (n = 151) and a "control" group undergoing cardiovascular surgery but without exposure to bovine thrombin (n = 11). Anticardiolipin antibody levels were increased before surgery in 10 of the 151 patients exposed to topical thrombin (6.6%). Four to 8 weeks after surgery, 84 patients (55.6%) had increased anticardiolipin antibody levels (P <.0001). In the control group, an increased anticardiolipin antibody level was present in a single patient before and after surgery (9%). Increased levels of antibodies to bovine and human beta(2)-GPI were also observed after surgery in the patients exposed to topical thrombin (37.7% and 38.2%, respectively). Increased anticardiolipin levels correlated with higher levels of antibody to bovine, but not human, beta(2)-GPI. In addition, increased levels of anticardiolipin antibody were associated with higher levels of antibodies to bovine factor V and prothrombin, as well as human factor V. Antibody binding on an enzyme-linked immunosorbent assay conducted to detect anticardiolipin antibody was dependent on the presence of anionic phospholipid, indicating that binding was not linked to the fetal bovine serum in the blocking buffer alone. Seven of 8 patients with delayed thromboembolic complications had increased anticardiolipin IgG antibody levels after surgery, but this association was not statistically significant. Nevertheless, our findings support the recommendation that the clinical safety of these commonly used hemostatic agents should be reassessed.