A Fluted Point from the Uptar Site, Northeastern Siberia

Lanceolate bifacial points, including one fluted specimen, have been collected from beneath an early Holocene tephra at the Uptar site, northeastern Siberia. Thus, the technology associated with the well-known Paleoindian tradition was not confined to the Americas. The Uptar collection does not compare readily with other Beringian complexes and demonstrates that there is greater diversity in the archaeological record of northeastern Siberia than traditional colonization models imply.     

Using 3D animations to teach intracellular signal transduction mechanisms: taking the arrows out of cells

Traditional methods of teaching intracellular biological processes and pathways use figures or flowcharts with the names of molecules linked with arrows. Many veterinary students, presented with such material, simply memorize the names or chemical structures of the molecules and are then likely to forget the material once the examination is completed. To address this problem, the authors designed, created, and field-tested new teaching media that incorporate realistic three-dimensional (3D) animations depicting the dynamic changes that occur in intracellular molecules during cellular activation. Testing found that veterinary students taught using traditional teaching media (e.g., lectures, handouts, textbooks) are proficient in memorizing the names and order of intracellular molecules but unable to appreciate the interactions between these elements or their spatial relationships within cells. In contrast, more than 90% of veterinary students taught using 3D animations not only recall the facts about the intracellular elements but also develop accurate mental images of the interactions among these molecules and their spatial relationships. These findings strongly suggest that the comprehension of complex biological processes by veterinary students can be enhanced by the use of dynamic 3D depictions of these processes in the classroom.     

Concentrations of serum amyloid A and lipopolysaccharide-binding protein in horses with colic

OBJECTIVE: To determine concentrations of 2 acute-phase proteins (serum amyloid A [SAA] and lipopolysaccharide-binding protein [LBP]) in serum samples obtained from horses with colic and identify relationships among these acute-phase proteins and clinical data. ANIMALS: 765 horses with naturally developing gastrointestinal tract diseases characterized by colic (ie, clinical signs indicative of abdominal pain) and 79 healthy control horses; all horses were examined at 2 university teaching hospitals. PROCEDURE: Serum concentrations of SAA and LBP were determined by immunoturbidometric and dot-blot assays, respectively. RESULTS: SAA and LBP concentrations were determined for 718 and 765 horses with colic, respectively. Concentrations of SAA were significantly higher in nonsurvivors than in survivors, and horses with enteritis or colitis and conditions characterized by chronic inflammation (eg, abdominal abscesses, peritonitis, or rectal tears) had SAA concentrations significantly greater than those for horses with other conditions. Serum concentrations of LBP did not correlate with outcome, disease process, or portion of the gastrointestinal tract affected. CONCLUSIONS AND CLINICAL RELEVANCE: Circulating concentrations of SAA were significantly higher at admission in horses with colic attributable to conditions having a primary inflammatory cause (eg, enteritis, colitis, peritonitis, or abdominal abscesses) and were higher in horses that failed to survive the episode of colic, compared with concentrations in horses that survived. Serum concentrations of LBP did not correlate with survival. Analysis of these findings suggests that evaluation of SAA concentrations may be of use in identifying horses with colic attributable to diseases that have inflammation as a primary component of pathogenesis.     

Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3h in PBG (PBS containing 0.5% BSA and 5mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01microg/ml or greater, and by zymosan concentrations of 0.1microg/ml or greater. ROS production in response to incubation with PMA was significantly increased starting at 10(-7)M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10(-7)M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3h in RPMI with LPS (0.1microg/ml), zymosan (10 microg/ml), PGN (10 microg/ml), and PMA (10(-7)M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.     

Effect of fetal bovine serum and heat-inactivated fetal bovine serum on microbial cell wall-induced expression of procoagulant activity by equine and canine mononuclear cells in vitro

OBJECTIVE: To determine the effect of fetal bovine serum (FBS) and heat-inactivated FBS (HI-FBS) on lipopolysaccharide (LPS)- and zymosan-induced procoagulant activity of equine and canine mononuclear cells. SAMPLE POPULATION: Mononuclear cells from 18 horses and 3 dogs. PROCEDURES: Cells were incubated with various concentrations of FBS, HI-FBS, LPS, zymosan, polymyxin B, and anti-LPS-binding protein monoclonal antibody or combinations of these constituents. A 1 stage recalcification assay was used to determine procoagulant activity. RESULTS: Addition of FBS to media significantly increased procoagulant activity; equine and canine cells were stimulated by 1% and 10% FBS, respectively. Coincubation of cells with FBS and polymyxin B did not reduce this effect, suggesting that the response was not attributable to LPS contamination. Addition of HI-FBS to media did not stimulate procoagulant activity of equine or canine cells, and the sensitivity of the equine cells to LPS was significantly increased by HI-FBS. This increased LPS sensitivity was reduced 40% with monoclonal antibody directed against human recombinant LPS-binding protein. Increasing concentrations of HIFBS significantly increased LPS- and zymosan-induced procoagulant activity of canine cells. CONCLUSION AND CLINICAL RELEVANCE: Procoagulant activity production in equine and canine mononuclear cells was significantly increased by addition of FBS, whereas heat inactivation of FBS eliminated this effect. Heat inactivation did not eliminate the function of serum proteins involved in enhancement of LPS and zymosan-induced procoagulant activity. Results suggest that HI-FBS can be used as a source of serum proteins that increase the sensitivity of mononuclear cells to bacterial and yeast cell wall components.     

Altrenogest Treatment Associated with a Farrowing Induction Protocol to Avoid Early Parturition in Sows

This study investigated the effect of altrenogest treatment on the farrowing development of sows, and birth weight (BW) and piglet survival until the third day of life. Three control groups were used: (i) sows that farrowed spontaneously before 114 day of gestation (CONT <114); (ii) sows that spontaneously farrowed at ≥114 day of gestation (CONT ≥114); (iii) sows that farrowed at ≥114 day with cloprostenol treatment (CONTCLOPR). Other sows were treated with altrenogest (Regumate^®) for 3 days (days 111, 112 and 113 of gestation): one group gave birth spontaneously (ALT) and the other group received altrenogest until day 113 and cloprostenol on day 114 (ALTCLOPR). There were no differences (p > 0.05) in farrowing duration, BW, coefficient of variation (CV) of BW, stillborn piglets, mummified foetuses, percentage of light piglets and survival until Day 3 between sows with and without cloprostenol treatment, in both control (CONT ≥114 vs CONTCLOPR) and altrenogest‐treated sows (ALT vs ALTCLOPR). Further comparisons were performed taking into account three groups: sows with early delivery (CONT <114 – farrowing before 114 days of gestation; n = 56), sows with longer gestation (CONT ≥114 – with and without cloprostenol treatment sows; n = 103) and ALT sows (with and without cloprostenol treatment; n = 105). Gestation length of CONT ≥114 and ALT sows was similar (p > 0.05), but higher than in CONT <114 sows. There were no differences (p > 0.05) between groups in farrowing duration, CV of BW, and percentages of stillborn piglets and mummified foetuses. Sows of CONT <114 group had a larger litter size and a lower BW than sows of the other two groups (p < 0.05). Sows of CONT <114 group had a higher percentage of lighter piglets and a lower piglet survival rate (p < 0.05) than ALT sows. In conclusion, altrenogest treatment proved to be an efficient method to avoid early parturition in 3–5 parity sows resulting in heavier piglets at birth.     

Adenosine A2A receptor agonists inhibit lipopolysaccharide-induced production of tumor necrosis factor-alpha by equine monocytes

Adenosine is an endogenous nucleoside that regulates many physiological processes by activating one or more adenosine receptor subtypes, namely A1, A2A, A2B and A3. The results of previous studies indicate that adenosine analogues inhibit lipopolysaccharide (LPS)-induced production of reactive oxygen species (ROS) by equine neutrophils primarily through activation of A2A receptors. Because peripheral blood monocytes produce cytokines that are responsible for many of the deleterious effects of LPS, the current study was performed to evaluate the effects of an array of novel adenosine receptor agonists on LPS-induced production of tumor necrosis factor-alpha (TNF-alpha), and to assess the selectively of these agonists for equine adenosine A2A over the A1 receptor. Radioligand binding studies performed with equine tissues expressing adenosine A1 and A2A receptor subtypes yielded a rank order of affinity for the equine A2A receptor of ATL307>ATL309 approximately ATL310 approximately ATL313>ATL202 approximately ATL361 approximately ATL376>ATL372>CGS21680>NECA. Co-incubation of equine peripheral blood monocytes with LPS and these agonists resulted in inhibition of TNF-alpha production with a rank order of potency that strongly correlated with their binding affinities for equine adenosine A2A receptors. Results of experiments performed with one of the adenosine receptor agonists (ATL313) and selective adenosine receptor antagonists confirmed that inhibition of LPS-induced production of TNF-alpha occurred via stimulation of A2A receptors. Although incubation of monocytes with IB-MECA, a compound purported to act as an adenosine A3 receptor agonist, reduced LPS-induced TNF-alpha production, this effect of IB-MECA was inhibited by the A2A selective antagonist ZM241385 but not by the A3 receptor antagonist MRS1220. These results indicate that the adenosine receptor subtype responsible for regulation of LPS-induced cytokine production by equine monocytes is the A2A receptor. To address the signal transduction mechanism responsible for the anti-inflammatory effects of ATL313 in equine monocytes, production of cAMP was compared in the presence and absence of either the adenosine A2A receptor antagonist ZM241385 or the adenosine A2B receptor antagonist MRS1706. In the absence of the antagonists, ATL313 increased production of cAMP; ZM241385 inhibited this effect of ATL313, whereas MRS1706 did not. Furthermore, incubation of monocytes with either the stable analogue of cAMP, dibutyryl cAMP, or forskolin, an activator of adenylyl cyclase, also inhibited LPS-induced production of TNF-alpha production by equine monocytes. Collectively, the results of the current study indicate that adenosine analogues inhibit LPS-induced production of TNF-alpha by equine monocytes primarily via activation of adenosine A2A receptors and do so in a cAMP-dependent manner. The results of this study indicate that stable adenosine analogues that are selective for adenosine A2A receptors may be suitable for development as anti-inflammatory drugs in horses.     

Can Microfiltered Seminal Plasma Preserve the Morphofunctional Characteristics of Porcine Spermatozoa in the Absence of Antibiotics? A Preliminary Study

Artificial insemination is extensively performed in pig farms in Europe, the United States and Canada. Antibiotics are typically added to the inseminating dose to limit bacterial growth during liquid phase storage at 16°C, as bacterial contamination is unavoidable. The World Organization for Animal Health (OIE) and the Food and Agriculture Organization (FAO) take action to control and reduce antibiotic use in animals as more bacteria are becoming resistant to antimicrobials. To avoid the use of antibiotics, we prepared inseminating doses using microfiltered seminal plasma (SP). Microfiltration is a common technology used to reduce bacterial contamination but may retain seminal substances, influencing sperm quality during storage. Therefore, the aim of this study was to evaluate the morphofunctional parameters of spermatozoa during storage at 16°C in doses prepared with or without microfiltered SP, with or without the addition of antibiotics, in a Latin square design. Artificial insemination doses with microfiltered SP and without antibiotic addition preserved spermatozoa viability, mitochondrial membrane potential, acrosome integrity and objective motility, with absolute values equal or even better than those observed in conventional doses. In conclusion, although the results could be considered preliminary due to the small sample size, this study suggests that microfiltration of SP can be a simple method, feasible on farms, to replace antibiotic use in extended doses stored in the liquid phase at 16°C for up to 7 days.