CK-2 of rainbow trout (Oncorhynchus mykiss) has two differentially regulated alleles that encode a functional chemokine

The final publication is available at Elsevier via https://doi.org/10.1016/j.vetimm.2018.02.003. © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/Rainbow trout chemokine 2 (CK-2) is currently the only known CC chemokine to have a mucin stalk. Further analysis of the mucin stalk region revealed a second, related CC chemokine sequence, denoted here as CK-2.1. This second sequence was determined to be an allele of CK-2 following genomic PCR analysis on several outbred individuals. Furthermore, in both in vivo and in vitro trials, CK-2 and CK-2.1 were both present, but appeared to have differential tissue expression in both control and PHA stimulated samples. Upon the development of a polyclonal antibody to rCK-2, CK-2 was only observed in the brain, liver and head kidney of PHA stimulated rainbow trout tissues. In comparison, when using the rainbow trout monocyte/macrophage-like cell line, RTS-11, CK-2 protein was observed in both control and PHA stimulated conditions. When studying the function of CK-2, a chemotaxis assay revealed that both peripheral blood leukocytes and RTS-11 cells migrated towards rCK-2 significantly at all concentrations studied when compared to truncated β2m. Interestingly, this migration was lowest at both the highest concentration and the lowest concentrations of CK-2. Thus, teleostean chemokine receptors may become desensitized when overstimulated as has been observed in mammalian models. The observed chemotactic function was indeed due to rCK-2 as cell migration was inhibited through pre-treatment of both the cells and the polyclonal antibody with rCK-2. As has been observed thus far with all other chemokines, CK-2 does appear to function through binding to a G-coupled protein receptor as chemotaxis could be inhibited through pre-treatment with pertussis toxin. Overall, the results of this study indicate that CK-2 is a functional chemokine that is encoded by two differentially expressed alleles in rainbow trout, CK-2 and CK-2.1.Natural Sciences and Engineering Research Council || 21752

Functional Characterization of Rainbow Trout (<em>Oncorhynchus mykiss</em>) Chemokine 2 (CK-2)

Chemokines are cytokines with chemoattractant ability, and comprise one of the major groups of molecules in immune system. These are small, secreted proteins cause the migration of leukocytes to the sites of injury. Over 40 mammalian chemokines have been identified to date, and they have been implicated in a number of immune mediated processes, including regulation of inflammation, antigen presentation, blood cell development, metastasis, viral infection and wound healing. In rainbow trout, there have been fewer chemokines reported and only one functional study has been published. Rainbow trout chemokine 2 (CK-2) is the only known CC chemokine with a mucin stalk, which has the potential for extensive O-glycosylation. However, no functional characterization has been performed on this molecule yet. CK-2 shares the presence of a mucin stalk with the mammalian chemokines, fractalkine (CX3CL1), lymphotactin (XCL1), and CXCL16. Another related trout CC chemokine sequence, CK-2. 1, has been discovered recently, which has 98% nucleotide sequence identity with CK-2. CK-2. 1 was believed to be a separate gene due to its apparent differential regulation in challenged rainbow trout. The question remained, however, whether or not CK-2. 1 was a separate gene or an allele of CK-2. The goal of this project was to further characterize both CK-2 and CK-2. 1. Through genomic PCR on several outbred individuals it was shown that CK-2. 1 is an allele of CK-2 but not a separate gene. Reverse transcriptase (RT) PCR analysis revealed an increased level of transcript both CK-2 and CK-2. 1 in response to phytohaemagglutinin (PHA) stimulation of head kidney leukocytes (HKL) and peripheral blood leukocytes (PBL) collected from fish with different allelic distributions. Similar results were also observed in the rainbow trout macrophage/monocyte cell line, RTS11. Moreover, an anti-CK-2 antiserum was developed in rabbits, which cross-reacted with CK-2. 1. This newly produced antibody was used to determine the protein expression levels in PHA stimulated rainbow trout tissues. RT-PCR was also performed on the same tissues in order to examine the transcript expression. Rainbow trout with both CK-2 and CK-2. 1 were used for this experiment. An overall decreasing pattern of transcript (both CK-2 and CK-2. 1) was observed in brain and HK over 24 hours, while protein was still detected at 24 hours post stimulation. However, in spleen the CK-2 transcript showed a slight upregulation at 4 hours post stimulation along with a very little or no CK-2. 1 expression, although no protein was detected in spleen. Liver showed a very low level of CK-2 and CK-2. 1 transcript at 8 hours post stimulation; while protein was again detected at 24 hours post stimulation. In addition, the sizes of the proteins found in different tissues were larger than expected (&le;30 kDa for CK-2 or &le;35 for CK-2. 1), perhaps due to the presence of extensive O-glycosylation at the mucin stalk of the protein. A chemotaxis assay was carried out, which is the definitive assay for chemokine activity. This assay showed migration of peripheral blood leukocytes across a membrane with 5µm pores toward CK-2 at an optimal concentration of 500ng/ml (17nm). Moreover, by pre-treating the recombinant chemokine with the polyclonal antisera, it was shown that the chemokine was actually causing the chemotactic activity. Pre-treatment of the cells with pertussis toxin, an inhibitor of G-protein signalling inhibited the migration of PBLs, established the fact that CK-2 caused chemotaxis by binding to a 7 transmembrane, G-coupled receptor just like all other known chemokines. Interestingly, CK-2 was also shown to attract RTS-11 cells. Overall, the above findings indicate that CK-2 is functionally a chemokine with two very different alleles in rainbow trout. It is probably heavily O-glycosylated and different tissues express different sizes of the protein. This is only the second functional study of a fish chemokine

Achieving Sustainable Growth at Uber Freight

The freight industry creates 8% of the world’s greenhouse gas emissions through shipping. If companies measure precisely, they can begin the benchmarking process toward improvement. The Global Logistics Emission Council (GLEC) provides a current consolidated Framework for calculating carbon emissions for freight transportation. Uber Freight, a third party software platform based trucking logistics service provider, requested a process to calculate and reduce their carbon emissions. After creating a calculation and forecast for carbon emissions, we complete an in-depth analysis of key lanes and activities to target for improvement. For long-term reduction, we present a projection of emissions through 2050 based on current activities, along with a Science Based Target that Uber Freight can use to set a climate goal. We provide Uber Freight with a strategy and method for measuring, tracking, and reducing their overall company environmental footprint along with the tools to enhance the environmental footprints of their shipper and carrier partners. For future accuracy improvement, Uber Freight can collect data on carriers’ specific CO2e per tkm and equipment type to avoid using general factors.Uber Freigh

Trend and risk factors of fatal pregnancy termination: A long-term nationwide population-based cross-section survey in Bangladesh.

BackgroundPregnant women often experience the fatal outcome of their pregnancy both in developed and impoverished countries. Due to strong health systems and services, factual and historical data are available from developed countries. However, the prevalence trend and risk factors of a fatal termination of pregnancy in developing countries like Bangladesh are still lacking.ObjectiveThe objective of the current study was to determine the 20 years trend of prevalence and risk factors of fatal pregnancy termination from 1997 to 2018 in Bangladesh.MethodThis study utilised the publicly available seven consecutive cross-data on Bangladesh Demographic and Health Surveys data since 1997 following identical methods among women of reproductive age. Respondent was asked if they had had a fatal pregnancy termination ever. A Generalised Linear model with a log-Poisson link was used to estimate the relative risk of different predictors for four survey time points (1998, 2004, 2011, 2018).ResultsThe proportions of fatal pregnancy termination in urban and rural areas were 24% vs. 19% and 24% vs. 22% in 1997 and 2018, respectively. In multivariable analysis, maternal age 30 years and above and obesity were strongly associated in all survey time points. The richest wealth index had a weak association in 1997 but was strongly associated in 2011 and 2018. A significant modest association with secondary complete education level was only observed in 2018.ConclusionThe overall proportions of fatal pregnancy termination in Bangladesh remain nearly static; however, its risk factors differed across different survey time points

A comparative study of microleakage between giomer and ormocer restoration in class I cavity of first permanent premolar teeth in vivo

This study was performed to compare the marginal microleakage of ormocer restorative material with that of giomer in vivo. Forty Class I cavities were prepared in non-carious permanent premolar teeth from 10 patients. Twenty cavities were filled with giomer and the remaining 20 cavities were filled by ormocer restorative materials. After one month, teeth were extracted, immersed in rhodamine dye solution, and then longitudinally bisected to assess the degree of dye penetration by stereoscopy. Furthermore, the gap between the dental material and tooth tissue were observed by the scanning electron microscope. The results showed that no microleakage (score 0) was detected in 15 ormocer and 5 giomer restorations. The remaining restorations were associated with dye penetration which was due to gap formation as seen in stereoscopic and scanning electron microscopic observations. The differences between ormocer and giomer restorative materials in respect to dye penetration were statistically significant. It can be concluded that ormocer restorative material shows less microleakage than that of giomer

A comparative study of microleakage between giomer and ormocer restoration in class I cavity of first permanent premolar teeth in vivo

This study was performed to compare the marginal microleakage of ormocer restorative material with that of giomer in vivo. Forty Class I cavities were prepared in non-carious permanent premolar teeth from 10 patients. Twenty cavities were filled with giomer and the remaining 20 cavities were filled by ormocer restorative materials. After one month, teeth were extracted, immersed in rhodamine dye solution, and then longitudinally bisected to assess the degree of dye penetration by stereoscopy. Furthermore, the gap between the dental material and tooth tissue were observed by the scanning electron microscope. The results showed that no microleakage (score 0) was detected in 15 ormocer and 5 giomer restorations. The remaining restorations were associated with dye penetration which was due to gap formation as seen in stereoscopic and scanning electron microscopic observations. The differences between ormocer and giomer restorative materials in respect to dye penetration were statistically significant. It can be concluded that ormocer restorative material shows less microleakage than that of giomer