Heparanase deglycanation of syndecan-1 is required for binding of the epithelial-restricted prosecretory mitogen lacritin

Cell surface heparan sulfate (HS) proteoglycans are carbohydrate-rich regulators of cell migratory, mitogenic, secretory, and inflammatory activity that bind and present soluble heparin-binding growth factors (e.g., fibroblast growth factor, Wnt, Hh, transforming growth factor β, amphiregulin, and hepatocyte growth factor) to their respective signaling receptors. We demonstrate that the deglycanated core protein of syndecan-1 (SDC1) and not HS chains nor SDC2 or -4, appears to target the epithelial selective prosecretory mitogen lacritin. An important and novel step in this mechanism is that binding necessitates prior partial or complete removal of HS chains by endogenous heparanase. This limits lacritin activity to sites where heparanase appears to predominate, such as sites of exocrine cell migration, secretion, renewal, and inflammation. Binding is mutually specified by lacritin's C-terminal mitogenic domain and SDC1's N terminus. Heparanase modification of the latter transforms a widely expressed HS proteoglycan into a highly selective surface-binding protein. This novel example of cell specification through extracellular modification of an HS proteoglycan has broad implications in development, homeostasis, and disease

Detection of bovine inflammatory cytokines IL-1β, IL-6, and TNF-α with a multiplex electrochemiluminescent assay platform

Commercially available bovine-specific assays are limited in number, and multiplex assays for this species are rare. Our objective was to develop a multiplex assay for the bovine inflammatory cytokines IL-1β, IL-6, and TNFα using the Meso Scale Discovery U-PLEX platform. Do-It-Yourself ELISA kits that contained polyclonal antibodies, both unlabeled and biotinylated, and the specific recombinant bovine cytokine standard, were purchased for each of these three cytokines. The biotinylated antibodies were coupled to linkers that bind to specific locations within each well of the U-PLEX plate. Unique linkers were used for each of the cytokines. The unlabeled antibodies were conjugated with electrochemiluminescent labels to serve as detection antibodies. Each cytokine assay was optimized individually prior to performing an optimization on the multiplex assay containing reagents for all three cytokines. To calculate cytokine concentrations, standard curves were developed using the recombinant cytokines and were run concurrently on each plate. Standard curves for IL-1β and TNF-α were run at concentrations ranging from 0 to 50,000 pg/mL, and for IL-6 from 0 to 10,000 pg/mL. The average lowest level of detection concentration measured by the standard curves were 5.3 pg/mL, 0.92 pg/mL, and 22.34 pg/mL for IL-1β, IL-6, and TNF-α respectively, as determined by data from seven plates containing bovine plasma samples from a combination of healthy and diseased cattle. The U-PLEX platform was a viable means to develop custom analyte- and species-specific multiplex assays using privately developed or purchased sets of commercially available reagents

Large genomic differences between Moraxella bovoculi isolates acquired from the eyes of cattle with infectious bovine keratoconjunctivitis versus the deep nasopharynx of asymptomatic cattle

Citation: Dickey, A. M., Loy, J. D., Bono, J. L., Smith, T. P. L., Apley, M. D., Lubbers, B. V., . . . Clawson, M. L. (2016). Large genomic differences between Moraxella bovoculi isolates acquired from the eyes of cattle with infectious bovine keratoconjunctivitis versus the deep nasopharynx of asymptomatic cattle. Veterinary Research, 47, 11. doi:10.1186/s13567-016-0316-2Moraxella bovoculi is a recently described bacterium that is associated with infectious bovine keratoconjunctivitis (IBK) or "pinkeye" in cattle. In this study, closed circularized genomes were generated for seven M. bovoculi isolates: three that originated from the eyes of clinical IBK bovine cases and four from the deep nasopharynx of asymptomatic cattle. Isolates that originated from the eyes of IBK cases profoundly differed from those that originated from the nasopharynx of asymptomatic cattle in genome structure, gene content and polymorphism diversity and consequently placed into two distinct phylogenetic groups. These results suggest that there are genetically distinct strains of M. bovoculi that may not associate with IBK

Tissue Transglutaminase Is a Negative Regulator of Monomeric Lacritin Bioactivity

PURPOSE. Molar accounting of bioactive fluids can expose new regulatory mechanisms in the growing proteomic focus on epithelial biology. Essential for the viability of the surface epithelium of the eye and for normal vision is the thin, but protein-rich, tear film in which the small tear glycoprotein lacritin appears to play a prominent prosecretory, cytoprotective, and mitogenic role. Although optimal bioactive levels in cell culture are 1 to 10 nM over a biphasic dose optimum, ELISA suggests a sustained tear lacritin concentration in the midmicromolar range in healthy adults. Here we identify a reconciling mechanism. METHODS. Monoclonal anti-lacritin 1F5 antibody was generated, and applied together with a new anti-C-terminal polyclonal antibody to tear and tissue Western blotting. In vitro tissue transglutaminase (Tgm2) cross-linking was monitored and characterized by mass spectrometry. RESULTS. Blotting for lacritin in human tears or saliva surprisingly detected immunoreactive material with a higher molecular weight and prominence equal or exceeding the~23 to 25 kDa band of monomeric glycosylated lacritin. Exogenous Tgm2 initiated lacritin cross-linking within 1 minute and was complete by 90 minutes-even with as little as 0.1 nM lacritin, and involved the donors lysine 82 and 85 and the acceptor glutamine 106 in the syndecan-1 binding domain. Lacritin spiked into lacritin-depleted tears formed multimers, in keeping with~0.6 lM TGM2 in tears. Cross-linking was absent when Tgm2 was inactive, and cross-linked lacritin, unlike recombinant monomer, bound syndecan-1 poorly. Enhanced TGM2 expression correlates with reduced cell viability, caspase activation, TNF receptor clustering, 7 and mitochondrial dysfunction 8 associated with hyperosmolar stress in dry eye. 14 Could TGM2 in tears regulate ocular surface biology? Lacritin is a 12.3 kDa tear prosecretory mitogen 15 with glutamine and lysine residues suitable for TGM2 catalyzed cross-linking. Lacritin promotes corneal epithelial cell survival (Zimmerman K, et al. IOVS 2012;53:ARVO E-Abstract 4231) and proliferation

Comparing imaging, acoustics, and radar to monitor Leach’s storm-petrel colonies

Seabirds are integral components of marine ecosystems and, with many populations globally threatened, there is a critical need for effective and scalable seabird monitoring strategies. Many seabird species nest in burrows, which can make traditional monitoring methods costly, infeasible, or damaging to nesting habitats. Traditional burrow occupancy surveys, where possible, can occur infrequently and therefore lead to an incomplete understanding of population trends. For example, in Oregon, during the last three decades there have been large changes in the abundance of Leach’s storm-petrels (Hydrobates leucorhoa), which included drastic declines at some colonies. Unfortunately, traditional monitoring failed to capture the timing and magnitude of change, limiting managers’ ability to determine causes of the decline and curtailing management options. New, easily repeatable methods of quantifying relative abundance are needed. For this study, we tested three methods of remote monitoring: passive acoustic monitoring, time-lapse cameras, and radar. Abundance indices derived from acoustics and imagery: call rates, acoustic energy, and counts were significantly related to traditional estimates of burrow occupancy of Leach’s storm-petrels. Due to sampling limitations, we were unable to compare radar to burrow occupancy. Image counts were significantly correlated with all other indices, including radar, while indices derived from acoustics and radar were not correlated. Acoustic data likely reflect different aspects of the population and hold the potential for the further development of indices to disentangle phenology, attendance of breeding birds, and reproductive success. We found that image counts are comparable with standard methods (e.g., radar) in producing annual abundance indices. We recommend that managers consider a sampling scheme that incorporates both acoustics and imaging, but for sites inaccessible to humans, radar remains the sole option. Implementation of acoustic and camera based monitoring programs will provide much needed information for a vulnerable group of seabirds

Growth of the C4 dicot Flaveria bidentis: photosynthetic acclimation to low light through shifts in leaf anatomy and biochemistry

In C4 plants, acclimation to growth at low irradiance by means of anatomical and biochemical changes to leaf tissue is considered to be limited by the need for a close interaction and coordination between bundle sheath and mesophyll cells. Here differences in relative growth rate (RGR), gas exchange, carbon isotope discrimination, photosynthetic enzyme activity, and leaf anatomy in the C4 dicot Flaveria bidentis grown at a low (LI; 150 μmol quanta m2 s−1) and medium (MI; 500 μmol quanta m2 s−1) irradiance and with a 12 h photoperiod over 36 d were examined. RGRs measured using a 3D non-destructive imaging technique were consistently higher in MI plants. Rates of CO2 assimilation per leaf area measured at 1500 μmmol quanta m2 s−1 were higher for MI than LI plants but did not differ on a mass basis. LI plants had lower Rubisco and phosphoenolpyruvate carboxylase activities and chlorophyll content on a leaf area basis. Bundle sheath leakiness of CO2 (ϕ) calculated from real-time carbon isotope discrimination was similar for MI and LI plants at high irradiance. ϕ increased at lower irradiances, but more so in MI plants, reflecting acclimation to low growth irradiance. Leaf thickness and vein density were greater in MI plants, and mesophyll surface area exposed to intercellular airspace (Sm) and bundle sheath surface area per unit leaf area (Sb) measured from leaf cross-sections were also both significantly greater in MI compared with LI leaves. Both mesophyll and bundle sheath conductance to CO2 diffusion were greater in MI compared with LI plants. Despite being a C4 species, F. bidentis is very plastic with respect to growth irradiance

\u3ci\u3eChoanotaenia atopa\u3c/i\u3e n. sp. (Cestoda: Dilepididae) from a Domestic Cat in Kansas

Choanotaenia atopa (Cestoda: Dilepididae) is described (host: domestic cat from the vicinity of Manhattan, Kansas); its natural host is presumed to be a rodent. Choanotaenia atopa is morphologically similar to cestodes of 6 species, all from rodents, formerly placed in the genus Rodentotaenia Matevosian, 1953, and subsequently removed to the genera Choanotaenia Railliet, 1896, or Monopylidium Fuhrmann, 1899. The systematic position of those cestodes is discussed; Monopylidium and Rodentotaenia are treated as synonyms of Choanotaenia. Choanotaenia atopa is distinguished by size and form of rostellar hooks, regularly alternating genital pores, and other characters in genital organs

Cytokine and Haptoglobin Profiles From Shipping Through Sickness and Recovery in Metaphylaxis- or Un-Treated Cattle

Fifty-six head of cattle, 28 animals with bovine respiratory disease complex (BRDC), and 28 healthy animals that were matched by treatment, sale barn of origin, day, and interactions among these variables, were identified from a population of 180 animals (60 each purchased at three sale barns located in Missouri, Tennessee, and Kentucky) enrolled in a study comparing animals receiving metaphylaxis to saline-treated controls. Cattle were transported to a feedlot in KS and assigned to treatment group. Blood samples were collected at Day 0 (at sale barn), Day 1, Day 9, and Day 28 (at KS feedlot), and transported to the US Meat Animal Research Center in Clay Center, NE where plasma was harvested and stored at −80°C until assayed for the cytokines IFN-γ, IL-1β, IL-6, and TNF-α, and the acute stress protein haptoglobin (HPT). Our objectives were to determine if cytokine and haptoglobin profiles differed between control and metaphylaxis treatment groups over time, and if profiles differed between animals presenting with BRDC and those that remained healthy. There was no difference between the treated animals and their non-treated counterparts for any of the analytes measured. Sale barn of origin tended to affect TNF-α concentration. Differences for all analytes changed over days, and on specific days was associated with state of origin and treatment. The Treatment by Day by Case interaction was significant for HPT. The analyte most associated with BRDC was HPT on D9, possibly indicating that many of the cattle were not exposed to respiratory pathogens prior to entering the feedlot

Use of recombinant bovine cytokines in pigs vaccinated and challenged with streptococcus suis

An experiment was conducted to determine the adjuvanticity of recombinant bovine interleukin-1β (rBoIL-1β) and recombinant bovine interleukin-2 (rBoIL-2) administered in conjunction with a single S. suis vaccination in pigs. Sixty, 4-wk-old pigs were allotted to 8 groups: 1) nonvaccinated controls; 2) vaccinated controls; 3) rBoIL-Iβ, 100 ng/kg; 4)rBoIL-lβ, 1000 ng/kg; 5) rBoIL-1β, 10,000 ng/kg; 6) rBoIL-2, 2.5 µg/kg; 7) rBoIL-2, 25 µg/kg; and 8) rBoIL-2, 250 µg/kg. All pigs (except group 1) were vaccinated on d 0 with a commercial S. suis vaccine (serotypes 1 and 2). At vaccination, pigs were injected intramuscularly with their respective cytokine treatments. Pigs received additional cytokine injections for 2 consecutive days. On d 21, all pigs were injected intravenously with 3.5 x 109 CFU of a log phase culture of S. suis (serotype 2). The highest dose of rBolL-1β exceeded the maximum tolerable dose for the cytokine; however, this dose of rBoIL-1β protected pigs from the S. suis challenge. In pigs receiving rBoIL-1β at 10,000 ng/kg, pathological lesions caused by S. suis were lowest when compared to other treatment groups. No mortality from S. suis challenge was observed in pigs that received the highest dose of rBoIL-1β. These data clearly show that rBoIL-1β (10,000 ng/kg), administered intramuscularly for 3 consecutive days at vaccination, is more effective than the S. suis vaccine alone in protecting pigs against a S. suis challenge