Effects of the neonatal intensive care environment on circadian health and development of preterm infants.

The circadian system in mammals ensures adaptation to the light-dark cycle on Earth and imposes 24-h rhythmicity on metabolic, physiological and behavioral processes. The central circadian pacemaker is located in the brain and is entrained by environmental signals called Zeitgebers. From here, neural, humoral and systemic signals drive rhythms in peripheral clocks in nearly every mammalian tissue. During pregnancy, disruption of the complex interplay between the mother's rhythmic signals and the fetal developing circadian system can lead to long-term health consequences in the offspring. When an infant is born very preterm, it loses the temporal signals received from the mother prematurely and becomes totally dependent on 24/7 care in the Neonatal Intensive Care Unit (NICU), where day/night rhythmicity is usually blurred. In this literature review, we provide an overview of the fetal and neonatal development of the circadian system, and short-term consequences of disruption of this process as occurs in the NICU environment. Moreover, we provide a theoretical and molecular framework of how this disruption could lead to later-life disease. Finally, we discuss studies that aim to improve health outcomes after preterm birth by studying the effects of enhancing rhythmicity in light and noise exposure.

Seminal plasma metabolomics profiles following long (4-7 days) and short (2 h) sexual abstinence periods.

OBJECTIVE: Metabolomic profiling of seminal plasma has been suggested as a possible approach for a fast and non-invasive male infertility evaluation diagnosis. However, metabolomics profiles in normozoospermic men have not been thoroughly investigated, and the influence of ejaculation-abstinence has not been described. To provide interim reference values and find associations between the metabolomics profiles of human seminal plasma and length of ejaculation-abstinence period in normozoospermic men. STUDY DESIGN: Semen samples collected after long (4-7 days) and short abstinence (2 h) from 31 normozoospermic males were assessed for routine quality parameters before the seminal plasma was separated by centrifugation. Metabolomics profiles of the seminal plasma were then determined using untargeted Nuclear Magnetic Resonance Spectroscopy. RESULTS: In total, 30 metabolites were identified. Pyruvate showed a higher concentration, while fructose, acetate, choline, methanol, N-acetylglucosamine, O-acetylcarnitine, uridine, and sn-glycero-3-phosphocoline showed lower concentrations in samples collected after short abstinence (vs. long). All metabolites showed lower absolute amounts (volume × concentration) following shorter abstinence. However, the lower sperm concentration in samples collected after short abstinence resulted in higher absolute amounts of pyruvate and taurine per spermatozoa: pyruvate 1.92 (1.12-3.87) vs. 1.29 (0.83-2.62) (P < 0.001) and taurine 0.58 (0.36-0.92) vs. 0.43 (0.28-0.95) (P < 0.05) ng/106 spermatozoa. Simultaneously, there was a higher percentage of progressively motile spermatozoa in samples collected after the short abstinence. CONCLUSION: The generally lower concentrations of seminal metabolites after short abstinence periods may be related to the shorter time available for secretion and collection of these metabolites by the accessory glands and the epididymides. The concomitant lower number of spermatozoa in the second ejaculate resulted in increased absolute amounts of pyruvate and taurine per spermatozoa, accompanied by increased spermatozoa motility in these samples. The simultaneous increase in percentages of motile spermatozoa and absolute amounts of pyruvate and taurine per spermatozoa after shorter abstinence might indicate that these two metabolites play a more critical role in sperm motility, which should be further investigated in future studies.

Effects of beta-1,3-glucan (AletaTM) on vaccination response in broiler chickens.

This 42-day study evaluated the effects of dietary supplementation with ß-1,3-glucan (Aleta™) on the vaccination response to Newcastle disease virus (NDV), avian infectious bronchitis virus (IBV), and infectious bursal disease (IBD) in a non-challenged environment. This trial included 600 chicks (all vaccinated with IBD at the hatchery) which were assigned to 1 of 3 treatments: vaccination (NDV, IBV), no vaccination, or vaccination combined with feed supplemented with Aleta (100 g/MT of feed). The vaccination with Aleta treatment group showed a trend for improved FCR that was not statistically significant. Control birds that were not vaccinated for IBV had significantly lower IBV titers on day 21 compared to birds that were vaccinated (both with and without Aleta). Surprisingly, there was significant separation among treatment groups for NDV titer levels, especially on day 21, where birds vaccinated and supplemented with Aleta had significantly higher titer levels compared to vaccination alone or no vaccination at all. Critically, only 14% of the birds receiving the vaccine plus Aleta had titer levels below the critical titer threshold for immunity compared to 28% of the birds receiving the vaccine alone and 40% of the unvaccinated birds. This suggests that Aleta supplementation may help to improve the vaccination response by birds, especially for NDV.

Evaluation of the effects of feeding dried algae containing beta-1,3-glucan on broilers challenged with Eimeria.

This study determined the effect of a dried algae product containing beta-1,3-glucan on broiler performance and immunity during an Eimeria challenge. Heterotrophically grown Euglena gracilis, which contained ∼55% beta-1,3-glucan, was dried and milled for inclusion into a non-medicated starter diet. Two experiments were conducted to evaluate dietary treatments containing 0, 50, 100, 150, and 200 g/ton dried algae. In both experiments, male broilers were orally challenged on day 14 with a coccidial inoculum consisting of E. acervulina, E. maxima, and E. tenella. Fecal matter was collected 120-144 hours post-exposure to determine relative amounts of oocyte shedding and birds were sacrificed on day 20 for lesion scoring. Broiler performance was assessed on a weekly basis. In the first experiment, birds receiving dried algae at 50 and 200 g/ton showed a significant improvement in FCR compared to the infected control during the challenge period (days 14-20). In the second experiment, the dried algae treatment had no significant effect on FCR, but lesion scores were significantly reduced in the groups receiving 50, 150, and 200 g/ton dried algae relative to the infected control. In both experiments, the dried algae treatment did not significantly impact mortality or oocyte shedding. In the second experiment, staining of intestinal samples with fluorescently tagged antibodies demonstrated that dried algae at 100 g/ton increased the number of intestinal macrophages compared to the infected control. A significant and dose-dependent increase in intestinal MHC-II+ expression was also observed for birds fed dried algae, with an 8-fold increase observed in the 200 g/ton group relative to the infected control. Similarly, increased total immune cell density (measured by the mean fluorescence intensity of CD45+ cells) was also observed at 150 and 200 g/ton. Overall, these data suggest that dried algae rich in beta-1,3-glucan can help improve gut immunity and host protection, thereby reducing morbidity associated with coccidiosis.

Cut-off values for normal sperm morphology and toxicology for automated analysis of rat sperm morphology and morphometry.

We used automated sperm morphology analysis to investigate rat sperm morphometry and morphology in Sprague-Dawley and Wistar rats in three research centers to develop normal baseline values for sperm morphometry and to quantify the percentage of morphologically normal sperm in healthy rats. The participating centers were IRSN in Paris, France (Sprague-Dawley rats), University of the Western Cape, South Africa (Wistar rats) and Stellenbosch University (Wistar rats), South Africa. All three centers used identical sperm isolation techniques from the cauda epididymis, the same staining protocols, identical computer-aided sperm morphometry analysis (CASMA) software and microscopes with similar optics. With CASMA, fully automated analysis of the different parts of stained sperm, e.g., head, acrosome, mid-piece, can be performed, many sperm morphometric features can be measured accurately and eventually normal sperm morphology can be defined. We found that it is possible to distinguish sperm morphometric characteristics of Sprague-Dawley and Wistar rats. We also developed cut-off values for evaluating the percentage normal sperm in these two rat strains using the automatic analysis mode. Normal sperm morphology varied between 67 and 74% by contrast with previous findings of > 90%.

Early- and late-onset preeclampsia and the DNA methylation of circadian clock and clock-controlled genes in placental and newborn tissues.

The placenta is important in providing a healthy environment for the fetus and plays a central role in the pathophysiology of preeclampsia (PE). Fetal and placental developments are influenced by epigenetic programming. There is some evidence that PE is controlled to an altered circadian homeostasis. In a nested case-control study embedded in the Rotterdam Periconceptional Cohort, we obtained placental tissue, umbilical cord leukocytes (UCL), and human umbilical venous endothelial cells of 13 early-onset PE, 16 late-onset PE and 83 controls comprising 36 uncomplicated and 47 complicated pregnancies, i.e. 27 fetal growth restricted and 20 spontaneous preterm birth. To investigate the associations between PE and the epigenetics of circadian clock and clock-controlled genes in placental and newborn tissues, genome-wide DNA methylation analysis was performed using the Illumina HumanMethylation450K BeadChip and a candidate-gene approach using ANCOVA was applied on 939 CpGs of 39 circadian clock and clock-controlled genes. DNA methylation significantly differed in early-onset PE compared with spontaneous preterm birth at 6 CpGs in placental tissue (3.73E-5 ≤ p ≤ 0.016) and at 21 CpGs in UCL (1.09E-5≤ p ≤ 0.024). In early-onset PE compared with fetal growth restriction 2 CpGs in placental tissue (p < 0.05) and 8 CpGs in uncomplicated controls (4.78E-5≤ p ≤ 0.049) were significantly different. Moreover, significantly different DNA methylation in early-onset PE compared with uncomplicated controls was shown at 6 CpGs in placental tissue (1.36E-4≤ p ≤ 0.045) and 11 CpGs in uncomplicated controls (2.52E-6≤ p ≤ 0.009). No significant associations were shown with late-onset PE between study groups or tissues. The most differentially methylated CpGs showed hypomethylation in placental tissue and hypermethylation in uncomplicated controls. In conclusion, DNA methylation of circadian clock and clock-controlled genes demonstrated most differences in UCL of early-onset PE compared with spontaneous preterm birth. Implications of the tissue-specific variations in epigenetic programming for circadian performance and long-term health need further investigation.

Improved sperm kinematics in semen samples collected after 2 h versus 4-7 days of ejaculation abstinence.

STUDY QUESTION: Does a short abstinence period of only 2 h yield spermatozoa with better motility characteristics than samples collected after 4-7 days? SUMMARY ANSWER: Despite lower semen volume, sperm concentration, total sperm counts and total motile counts, higher percentages of motile spermatozoa with higher velocity and progressiveness were detected in samples obtained after 2 h. WHAT IS KNOWN ALREADY: Most studies that have assessed the effect of abstinence periods on sperm motility parameters in men with a sperm concentration below 15 million/ml have detected a higher percentage of motile spermatozoa in samples obtained after short abstinence periods. Studies of men with sperm concentrations above 15 million/ml have reported significantly decreased motile sperm counts after 24 h of abstinence compared with longer abstinence periods. STUDY DESIGN, SIZE, DURATION: This study had a controlled repeated-measures design based on semen samples from 43 male partners, in couples attending for IVF treatment, who had a sperm concentration above 15 million/ml. Data were collected between June 2014 and December 2015 in the Fertility Unit of Aalborg University Hospital (Aalborg, Denmark). PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants provided a semen sample after 4-7 days of abstinence followed by another sample after only 2 h. For both ejaculates, sperm concentration, total sperm counts, motility groups and detailed kinematic parameters were assessed and compared by using the Sperm Class Analyzer (SCA) computer-aided sperm analysis system before and after density gradient selection. The laboratory's local manual method (Makler chamber) was used for comparison. MAIN RESULTS AND THE ROLE OF CHANCE: The second raw ejaculate demonstrated lower semen volume (P < 0.0001), sperm concentration (P = 0.003) and sperm counts in all motility sub-groups (P < 0.001) but higher percentages of spermatozoa with higher velocity (P < 0.01), progressiveness (P < 0.001) and hyperactivation (P < 0.001), compared with the first raw ejaculate. LIMITATIONS, REASONS FOR CAUTION: The first ejaculate in this study was also used for the IVF/ICSI treatments and therefore only patients with a semen volume ≥2 ml and concentration ≥15 million/ml were included. Further validation in large prospective randomized controlled trials, more purposely directed at normozoospermic males with partners having problems conceiving when there appears to be no female factor, is needed to confirm the potential advantage of using a second semen sample in improving fertilization and pregnancy rates in assisted reproduction. WIDER IMPLICATIONS OF THE FINDINGS: Despite the significantly lower semen volume, sperm concentration and total sperm counts in all motility sub-groups, the significantly higher percentage of spermatozoa with better motility characteristics (velocity, progressiveness and hyperactivation) in the second ejaculate, may provide and allow for a simpler and more effective selection of higher quality spermatozoa. This could prove to be an advantage for ART procedures such as intracytoplasmic sperm injection where a large number of spermatozoa is not needed. It can also be speculated that pooling two consecutive ejaculates obtained after 4-7 days and after 2 h, could be an advantage for intrauterine insemination where a large number of motile spermatozoa are needed. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by internal grants from the Department of Health Science and Technology, Faculty of Medicine, Aalborg University (Aalborg, Denmark). The SCA® was provided by a grant from 'Ferring Pharmaceuticals' to Aalborg University Hospital (H.I.N). G.V.D.H. is an external senior scientific consultant to Microptic S/L (Barcelona, Spain). H.A. has provided scientific input and presentations for Microptic S/L (Barcelona, Spain) on several occasions. All other authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Autoantibodies to box A of high mobility group box 1 in systemic lupus erythematosus.

Autoantibodies to nuclear structures are a hallmark of systemic lupus erythematosus (SLE), including autoantibodies to nuclear protein high mobility group box 1 (HMGB1). HMGB1 consists of three separate domains: box A, box B and an acidic tail. Recombinant box A acts as a competitive antagonist for HMGB1 and might be an interesting treatment option in SLE. However, antibodies to box A might interfere. Therefore, levels of anti-box A were examined in SLE patients in association with disease activity and clinical parameters. Serum anti-box A was measured in 86 SLE patients and 44 age- and sex-matched healthy controls (HC). Serum samples of 28 patients with primary Sjögren's syndrome and 32 patients with rheumatoid arthritis were included as disease controls. Anti-HMGB1 and anti-box B levels were also measured by enzyme-linked immunosorbent assay during quiescent disease [SLE Disease Activity Index (SLEDAI) ≤ 4, n = 47] and active disease (SLEDAI ≥ 5, n = 39). Anti-box A levels in active SLE patients were higher compared to quiescent patients, and were increased significantly compared to HC and disease controls. Anti-box A levels correlated positively with SLEDAI and anti-dsDNA levels and negatively with complement C3 levels. Increased levels of anti-box A antibodies were present in the majority of patients with nephritic (73%) and non-nephritic exacerbations (71%). Antibodies to the box A domain of HMGB1 might be an interesting new biomarker, as these had a high specificity for SLE and were associated with disease activity. Longitudinal studies should be performed to evaluate whether these antibodies perform better in predicting an exacerbation, especially non-nephritic exacerbations.

A zebrafish xenograft model for studying human cancer stem cells in distant metastasis and therapy response.

Lethal and incurable bone metastasis is one of the main causes of death in multiple types of cancer. A small subpopulation of cancer stem/progenitor-like cells (CSCs), also known as tumor-initiating cells from heterogenetic cancer is considered to mediate bone metastasis. Although over the past decades numerous studies have been performed in different types of cancer, it is still difficult to track small numbers of CSCs during the onset of metastasis. With use of noninvasive high-resolution imaging, transparent zebrafish embryos can be employed to dynamically visualize cancer progression and reciprocal interaction with stroma in a living organism. Recently we established a zebrafish CSC-xenograft model to visually and functionally analyze the role of CSCs and their interactions with the microenvironment at the onset of metastasis. Given the highly conserved human and zebrafish genome, transplanted human cancer cells are able to respond to zebrafish cytokines, modulate the zebrafish microenvironment, and take advantage of the zebrafish stroma during cancer progression. This chapter delineates the zebrafish CSC-xenograft model as a useful tool for both CSC biological study and anticancer drug screening.

Standing sedation with medetomidine and butorphanol in captive African elephants (Loxodonta africana).

Doses for standing sedation allowing for various procedures in otherwise inaccessible, untrained captive African elephant bulls are presented. Thirty-three standing sedations were performed in 12 males aged 8-30 years (one to four sedations per animal). Each bull received a combination of 0.009 ± 0.002 mg/kg medetomidine and 0.03 ± 0.007 mg/kg butorphanol. Full sedation was reached on average 25.5 min after injection. The addition of hyaluronidase (1000-2000 IU) significantly reduced time to full sedation to 16.5 min (paired t test, P = 0.024). Reversal was induced with intramuscular atipamezole 0.008 (±0.002) and naltrexone 0.035 (±0.015) mg/kg. Recovery took on average 7 min (3-18 min). The medetomidine/butorphanol combination provided safe standing sedation for smaller procedures.

The nucleotide excision repair protein XPC is essential for bulky DNA adducts to promote interleukin-6 expression via the activation of p38-SAPK.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants, and many are potent carcinogens. Benzo[a]pyrene (B[a]P), one of the best-studied PAHs, is metabolized ultimately to the genotoxin anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE). BPDE triggers stress responses linked to gene expression, cell death and survival. So far, the underlying mechanisms that initiate these signal transduction cascades are unknown. Here we show that BPDE-induced DNA damage is recognized by DNA damage sensor proteins to induce activation of the stress-activated protein kinase (SAPK) p38. Surprisingly, the classical DNA damage response, which involves the kinases ATM and ATR, is not involved in p38-SAPK activation by BPDE. Moreover, the induction of p38-SAPK phosphorylation also occurs in the absence of DNA strand breaks. Instead, increased phosphorylation of p38-SAPK requires the nucleotide excision repair (NER) and DNA damage sensor proteins XPC and mHR23B. Interestingly, other genotoxins such as cisplatin (CDDP), hydrogen peroxide and ultraviolet radiation also enhance XPC-dependent p38-SAPK phosphorylation. In contrast, anti-benzo[c]phenanthrene-3,4-dihydrodiol-1,2-epoxide, the DNA adducts of which are not properly recognized by NER, does not trigger p38-SAPK activation. As a downstream consequence, expression and secretion of the pro-inflammatory cytokine interleukin-6 is induced by BPDE and CDDP in vitro and by CDDP in the murine lung, and depends on XPC. In conclusion, we describe a novel pathway in which DNA damage recognition by NER proteins specifically leads to activation of p38-SAPK to promote inflammatory gene expression.

miR-25 Modulates Invasiveness and Dissemination of Human Prostate Cancer Cells via Regulation of αv- and α6-Integrin Expression.

Altered microRNA (miRNA; miR) expression is associated with tumor formation and progression of various solid cancers. A major challenge in miRNA expression profiling of bulk tumors is represented by the heterogeneity of the subpopulations of cells that constitute the organ, as well as the tumor tissue. Here, we analyzed the expression of miRNAs in a subpopulation of epithelial stem/progenitor-like cells in human prostate cancer [prostate cancer stem cell (PCSC)] and compared their expression profile to more differentiated cancer cells. In both cell lines and clinical prostate cancer specimens, we identified that miR-25 expression in PCSCs was low/absent and steadily increased during their differentiation into cells with a luminal epithelial phenotype. Functional studies revealed that overexpression of miR-25 in prostate cancer cell lines and selected subpopulation of highly metastatic and tumorigenic cells (ALDH(high)) strongly affected the invasive cytoskeleton, causing reduced migration in vitro and metastasis via attenuation of extravasation in vivo. Here, we show, for the first time, that miR-25 can act as a tumor suppressor in highly metastatic PCSCs by direct functional interaction with the 3'-untranslated regions of proinvasive αv- and α6-integrins. Taken together, our observations suggest that miR-25 is a key regulator of invasiveness in human prostate cancer through its direct interactions with αv- and α6-integrin expression.

Metallic taste in cancer patients treated with chemotherapy.

BACKGROUND: Metallic taste is a taste alteration frequently reported by cancer patients treated with chemotherapy. Attention to this side effect of chemotherapy is limited. This review addresses the definition, assessment methods, prevalence, duration, etiology, and management strategies of metallic taste in chemotherapy treated cancer patients. METHODS: Literature search for metallic taste and chemotherapy was performed in PubMed up to September 2014, resulting in 184 articles of which 13 articles fulfilled the inclusion criteria: English publications addressing metallic taste in cancer patients treated with FDA-approved chemotherapy. An additional search in Google Scholar, in related articles of both search engines, and subsequent in the reference lists, resulted in 13 additional articles included in this review. Cancer patient forums were visited to explore management strategies. FINDINGS: Prevalence of metallic taste ranged from 9.7% to 78% among patients with various cancers, chemotherapy treatments, and treatment phases. No studies have been performed to investigate the influence of metallic taste on dietary intake, body weight, and quality of life. Several management strategies can be recommended for cancer patients: using plastic utensils, eating cold or frozen foods, adding strong herbs, spices, sweetener or acid to foods, eating sweet and sour foods, using 'miracle fruit' supplements, and rinsing with chelating agents. INTERPRETATION: Although metallic taste is a frequent side effect of chemotherapy and a much discussed topic on cancer patient forums, literature regarding metallic taste among chemotherapy treated cancer patients is scarce. More awareness for this side effect can improve the support for these patients.

Rituximab impairs immunoglobulin (Ig)M and IgG (subclass) responses after influenza vaccination in rheumatoid arthritis patients.

Rituximab (RTX) treatment in rheumatoid arthritis (RA) patients severely hampers humoral response after influenza vaccination as determined by haemagglutination inhibition assay (HI). It is not known whether HI reflects both immunoglobulin (Ig)M and IgG (subclass) influenza response, and whether IgM antibodies contribute to the low rate of influenza infection seen in RA patients. Twenty RA patients on methotrexate (MTX), 23 on RTX and 28 healthy controls (HC) received trivalent influenza subunit vaccination. Before and 28 days after vaccination, H1N1- and H3N2-specific antibodies were measured by HI and by IgM and IgG (subclass) enzyme-linked immunosorbent assay (ELISA). B cell activating factor (BAFF) levels were determined in serum samples before vaccination. Vaccination induced a significant increase of IgM and IgG (IgG1 and IgG3) antibodies against both strains in the HC and MTX groups (all P < 0·01), but not in the RTX group. HI correlated significantly in all cases with IgG (IgG1) but not with IgM. In RTX late patients (RTX treatment 6-10 months before vaccination), IgG (IgG1 and IgG3) response to vaccination was restored, but not IgM response. BAFF levels were significantly increased in RA-RTX patients and correlated with total IgG levels. Haemagglutination inhibition assay, used as gold standard, detects primarily IgG (IgG1) responses. IgM- and IgG influenza-specific antibodies increase after vaccination in HC and RA patients except in patients on RTX treatment. BAFF levels are increased in both early and late RTX-treated patients, but do not correlate with an influenza-specific antibody response.

To like or not to like: neural substrates of subjective flavor preferences.

Flavor preferences vary; what one enjoys may be disgusting to another. Previous research has indicated several brain regions associated with flavor preferences. However, by using different stimuli or different internal states to obtain differences in liking, results of these studies may be confounded. Therefore, we used one target stimulus (grapefruit juice) and fMRI to compare brain activation patterns between participants that either liked (n=16) or disliked (n=18) this stimulus. Our first aim was to investigate whether differential neural activation exists that accounts for the difference in subjective flavor preference for the target stimulus. Secondly, multivariate analysis was used to investigate whether differences in subjective liking for the target revealed similar activation patterns as differences in general liking for a sweet and bitter solution. A direct comparison of likers and dislikers of the target stimulus revealed only small differences in activations in orbitofrontal cortex (OFC) and dorsal anterior cingulate cortex (dACC). However, when using multivariate analysis, a broader activation pattern (including OFC, dACC, pregenual anterior cingulate, anterior insula and ventral striatum) was identified that discriminated likers from dislikers with an 88% success rate. Interestingly though, little overlap was found between this pattern and the pattern that discriminates liking for the sweet and bitter solutions and lesser voxels contributed to the former compared with the latter. These differences between patterns discerning innate versus learned preferences may suggest that different mechanisms are at work and highlight the importance of elucidating the neural processes of how subjective preferences are learned and acquired.

Accurate automated quantitative imaging of tortoise erythrocytes using the NIS image analysis system.

The standard method for assessing blood cell characteristics using an ocular micrometer is time-consuming and limited. We used the Nikon NIS Elements imaging software and May- Grünwald-Giemsa staining to determine whether automated image analysis is suitable for rapid and accurate quantitative morphometry of erythrocytes. Blood was collected during four seasons from 126 geometric tortoises and the blood smears were evaluated for cell (C) and nuclear (N) characteristics of the erythrocytes. We measured area, length (L), width (W), perimeter, elongation and pixelation intensity, and calculated L/W and N/C areas. Erythrocyte size differed among cohorts; females, the larger sex, had smaller erythrocytes than either males or juveniles. Males had more elongated erythrocytes than females and erythrocytes of adults were more elongated than those of juveniles. Erythrocyte size and shape influence the efficiency of gas exchange owing to surface area to volume ratios, which are greater for small, elongated cells than for large, round cells. The high N/C ratio and low pixelation intensities of males and juveniles indicate that they may have had more immature erythrocytes in their circulation than females. The use of pixelation intensity to indicate the presence of immature erythrocytes was validated by seasonal differences that corresponded to the biology of the tortoises. Pixelation intensity was lowest in winter. We found that automated image analysis is a rapid and reliable method for determining cell size and shape, and it offers the potential for distinguishing among developmental stages that differ in staining intensity. The method should be useful for rapid health assessments, particularly of threatened species, and for comparative studies among different vertebrates.

Isolated pancreatic tuberculosis: a case report and radiological comparison with cystic pancreatic lesions.

Pancreatic tuberculosis is rare and can occur in the absence of evidence of tuberculosis elsewhere in the body. Here we review the radiological appearance of pancreatic tuberculosis and compare it with other cystic pancreatic lesions, including common lesions (pseudocysts, serous or mucinous cystadenomas, intraductal papillary mucinous neoplasm) and rare lesions such as solid pseudopapillary tumors, etc. Their typical localizations within the pancreas and their malignant potential are presented. Knowledge of these can assist radiologists and clinicians in selecting the best approach towards making the correct diagnosis.

Quantification and identification of sperm subpopulations using computer-aided sperm analysis and species-specific cut-off values for swimming speed.

Motility is an essential characteristic of all flagellated spermatozoa and assessment of this parameter is one criterion for most semen or sperm evaluations. Computer-aided sperm analysis (CASA) can be used to measure sperm motility more objectively and accurately than manual methods, provided that analysis techniques are standardized. Previous studies have shown that evaluation of sperm subpopulations is more important than analyzing the total motile sperm population alone. We developed a quantitative method to determine cut-off values for swimming speed to identify three sperm subpopulations. We used the Sperm Class Analyzer(®) (SCA) CASA system to assess the total percentage of motile spermatozoa in a sperm preparation as well as the percentages of rapid, medium and slow swimming spermatozoa for six mammalian species. Curvilinear velocity (VCL) cut-off values were adjusted manually for each species to include 80% rapid, 15% medium and 5% slow swimming spermatozoa. Our results indicate that the same VCL intervals cannot be used for all species to classify spermatozoa according to swimming speed. After VCL intervals were adjusted for each species, three unique sperm subpopulations could be identified. The effects of medical treatments on sperm motility become apparent in changes in the distribution of spermatozoa among the three swimming speed classes.

Improved semen collection method for wild felids: urethral catheterization yields high sperm quality in African lions (Panthera leo).

For wild and domestic felids, electroejaculation (EE) is the most common semen collection method. However, the equipment is expensive, there is a risk of urine contamination and animals usually show strong muscular contraction despite general anesthesia. Accordingly, we tested the feasibility of a different approach using urethral catheterization (UC) in seven African lions, previously described for domestic cats only. After general anesthesia with the α2-agonist medetomidine (which also stimulates semen release into the urethra) and ketamine, a transrectal ultrasound was performed to locate the prostate. A commercial dog urinary catheter (2.6 or 3.3 mm in diameter) was advanced approximately 30 cm into the urethra to allow semen collection into the lumen of the catheter by capillary forces. After retraction, sperm volumes between of 422.86 ± 296.07 µl yielded motility of 88.83 ± 13.27% (mean ± SD) with a mean sperm concentration of 1.94 × 10(9)/ml. Here we describe a simple, field friendly and effective method to attain highly concentrated semen samples with excellent motility in lions and potentially other wild felid species as an alternative to electroejaculation.

The BMP2/7 heterodimer inhibits the human breast cancer stem cell subpopulation and bone metastases formation.

Accumulating evidence suggests that a subpopulation of breast cancer cells, referred to as cancer stem cells (CSCs), have the ability to propagate a tumor and potentially seed new metastases. Furthermore, stimulation of an epithelial-to-mesenchymal transition by factors like transforming growth factor-ß (TGFß) is accompanied with the generation of breast CSCs. Previous observations indicated that bone morphogenetic protein-7 (BMP7) antagonizes the protumorigenic and prometastatic actions of TGFß, but whether BMP7 action is mechanistically linked to breast CSCs has remained elusive. Here, we have studied the effects of BMP7, BMP2 and a BMP2/7 heterodimer on the formation of human breast CSCs (ALDH(hi)/CD44(hi)/CD24(-/low)) and bone metastases formation in a preclinical model of intra-cardiac injection of MDA-MB-231 cells in athymic nude (Balb/c nu/nu) mice. The BMP2/7 heterodimer was the most efficient stimulator of BMP signaling and very effectively reduced TGFß-driven Smad signaling and cancer cell invasiveness. The tested BMPs-particularly the heterodimeric BMP2/7-strongly reduced the size of the ALDH(hi)/CD44(hi)/CD24(-/low) CSC subpopulation. In keeping with these in vitro observations, pretreatment of cancer cells with BMPs for 72 h prior to systemic inoculation of the cancer cells inhibited the formation of bone metastases. Collectively, our data support the notion that breast CSCs are involved in bone metastasis formation and describe heterodimeric BMP2/7 as a powerful TGFß antagonist with anti-metastatic potency.