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1 Global Veterinary Summit-August 31-2 September 2015.

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1 1 Global Veterinary Summit-August 31-2 September 2015

2 Global Veterinary Summit-A11ugust 31-2 September 2015 2 Overview and related workOur research Characterization ovine SERPINA1 cDNA and gene Differential expression of sheep SERPINA1 gene during lactation ConclusionFuture work

3 Global Veterinary Summit-A11ugust 31-2 September 2015 3 SERPINA1 Fig. 1 3D- structure of SerpinA AAT REACTIVE CENTER LOOP HINGE REGION BREACH REGION SHUTTER DOMAIN GATE REGION Expression: liver, muscle, mammary gland, macrophages and neutrophil Biological processes: blood coaugulation, apoptosis, reproduction, fibrinolysis and inflamamtory responses Primary fucntion: inactivation of neutrophil elastase

4 Global Veterinary Summit-A11ugust 31-2 September 2015 4 AJRI 2001, 45: 266-272 ISRN Immunology 2012, Article ID 354365 DIABETES, VOL. 56, MAY 2007

5 By Lindberg et al 1982 Global Veterinary Summit-A11ugust 31-2 September 2015 5  Protection of infant liver by inactivation of endogenous proteases  Regulaiton of proteolitic activity in mammary gland  Inactivation of macrophages proteases  Increase of survival of other milk proteins via partial inhibition of proteolisys

6 Global Veterinary Summit-A11ugust 31-2 September 2015 6 AAT: bioactivity in human milkAAT: protect other milk protein from proteolysis

7 Global Veterinary Summit-A11ugust 31-2 September 2015 7

8 8

9 9 218.519341171.8

10 Global Veterinary Summit-A11ugust 31-2 September 2015 10 Overview and related workOur research Characterization ovine SERPINA1 cDNA and gene Differential expression of sheep SERPINA1 gene during lactationSecond experiment ConclusionFuture work

11 Global Veterinary Summit-A11ugust 31-2 September 2015 11

12 Global Veterinary Summit-A11ugust 31-2 September 2015 12 RNA extraction from milk somatic cells 4 SARDA 3 GENTILE DI PUGLIA 3 COMISANA RNA extraction from 11 tissues 1 SARDA 1 GENTILE DI PUGLIA Gene sequencing 4 SARDA 3 GENTILE DI PUGLIA 3 COMISANA

13 Global Veterinary Summit-A11ugust 31-2 September 2015 13 This transcript codes for a protein of 416aa This transcript codes for a protein of 230aa This transcript codes for a protein of 112aa

14 Global Veterinary Summit-A11ugust 31-2 September 2015 14

15 Global Veterinary Summit-A11ugust 31-2 September 2015 15 Ampliconeprimers N° primer Lunghezza bpSequenziamento Ex1-Ex5cDNAFWD-cDNARWD1-29000no 5’UTR5’UTRFWD-5’UTRRWD3-42009si Ex1-Intr1- Ex2 cDNAFWD-I IntronRWD1-55000No completato Ex2-Intr2- Ex3 II IntronFWD-II IntronRWD6-71495si Ex3-Intr3- Ex4 III IntronFWD-III IntronRWD8-91278si Ex4-Intr4- Ex5 IV IntronFWD-cDNARWD10-21132si 3’UTR3’UTRFWD-3’UTRRWD11-122082si I esone II esone IIII esone IV esone V 3’UTR 5’UTR - 1 3 4 681011 12

16 Global Veterinary Summit-A11ugust 31-2 September 2015 16 Region n° synonimousnon synonimous 5’UTR24-- I exon440 II exon1349 II intron11-- III exon303 III intron2-- IV exon624 IV intron10-- V exon927 3’UTR15-- TOTAL971223

17 Global Veterinary Summit-A11ugust 31-2 September 2015 17  Isolation of 3 different transcripts  Long transcript codes for a complete AAT  Alternative splicing is not influenced by SNPs identified in intron regions  Upstream region to II exon could influence the trasncription of SERPINA1 cDNA  The ovine SERPINA1 gene exon and intron organization is similar to human and bovine  12 of 23 non synonimous aa change could affect AAT function  None aa change is identified in RCL region important for inhibitory action

18 Global Veterinary Summit-A11ugust 31-2 September 2015 18 Overview and related workOur research Characterization ovine SERPINA1 cDNA and gene Differential expression of sheep SERPINA1 gene during lactation ConclusionFuture work

19 Global Veterinary Summit-A11ugust 31-2 September 2015 19 SARDA GENTILE DI PUGLIA Is there a differential expression in milk of SERPINA1 gene during lactation?

20 Global Veterinary Summit-A11ugust 31-2 September 2015 20 RAZZASTADIO DELLA LATTAZIONE 60 GIORNI90 GIORNI120 GIORNI SARDA1413 GENTILE DI PUGLIA12104 1. RNA extraction from a total of 71 milk samples 60, 90, 120 d RNeasy Lipid Tissue Mini kit QIAGEN 2.RNA quality check: Agilent 2100 and Bioanalyzer High quality vs degraded RNA 3. RT-PCR and qPCR

21 Global Veterinary Summit-A11ugust 31-2 September 2015 21 Tab.1 Primer sequences, concentration, T.a., amplicon lenght GenePrimers nM T.aAmplicon cDNA/DNA SERPINA15’AGGGATCACCGAGGAAACAG3’ 5’GTTGCTCATTCACGTGGAAG3 300 200 58112/900 ATP5B5’TTTGGACTCCACGTCTCGCATC3’ 5’TCCTGGAGGGATTTGTAGTCCTG3’ 200 61108/NO EIF2B25’CCGTTCCCATTATGCTCAACTCCAG3 5’TCCGTGTCCCTTCCAGTTCCAC3’ 200 100 6181/NO SDHA5’ACGATTACTCCAAGCCCATCCAG3’ 5’AACGTAGGAGAGCGTGTGCTTC3’ 200 100 6180/80 POLR2A5’ AATGGAAGCATGTCAATGAGGACTCTC3’ 5’ CACAGGCAGCACAGTGACGATC3’ 100 61164/164 UXT5’TGTGGCCCTTGGATATGGTT3’ 5’GGTTGTCGCTGAGCTCTGTG3’ 300 58101/NO RPS95’CCTCGACCAAGAGCTGAAG3’ 5’CCTCCAGACCTCACGTTTGTTC3’ 200 5854/250 MRPS155’GCAGCTTATGAGCAAGGTCGT3’ 5’GCTCATCAGCAGATAGCGCTT3’ 300 58151/2100 a. Evaluation of the optimal set of reference genes b. SERPINA expression analyses

22 Global Veterinary Summit-A11ugust 31-2 September 2015 22 Determination of HK more stable 0.67 0.90 1.05 SPOL MRSP15 SEIF2UXT323QATPQSDHRPS9 V2/3 V3/4 V4/5 V5/6 V6/7 0.00 0.150 0.235 Determination of the optimal number of HK

23 RazzaGentileSarda N2640 µ ± SE1.64 ± 0.041.69 ± 0.05 Giorni6090120 N262317 µ ± SE1.65 ± 0.071.70 ± 0.041.64 ± 0.07 RazzaGentileSardaGentileSardaGentileSarda GiorniN121410134 60µ ± SE1.67 ± 0.081.63 ± 0.07 90µ ± SE1.60 ± 0.071.80 ± 0.10 120µ ± SE1.66 ± 0.041.63 ± 0.06 Global Veterinary Summit-A11ugust 31-2 September 2015 23 GentileSarda Results 1 We did not find any difference in expression levels of SERPINA1 between two breed 1206090 Results 2 We did not find any difference in expression levels Analysis of data by a repeated measure analysis In GLM procedure of SAS software

24  Our findings confirm the results obtained in pig, goat and cow  In other research in mammals a higher concentration of AAT was observed in the first lactation days or in mastitic mammary gland after calving  We hyphothesize that any SERPINA1 differential expression was observed because the first sampling was far away lambing (60 days) Global Veterinary Summit-A11ugust 31-2 September 2015 24

25 Global Veterinary Summit-A11ugust 31-2 September 2015 25 Immune response Could we consider SERPINA1 a biomarker for infiammation? Which is expression of SERPINA1 during peripartum? Does SERPINA1 Regulate expression of other immune genes as CD62L and IL8 Is there a differential expression of SERPINA1 in different blood cell

26 Global Veterinary Summit-A11ugust 31-2 September 2015 26 DESIGN and REALIZATION of experiments Dr.ssa Alessandra Crisà Dr. Francesco Napolitano Dr.ssa Bianca Moiloli Dr.ssa Elisa Mostarda Sig. Francesco Grandoni Funding This study was supported by the research programme “Improvement of Italian livestock through the use of innovative biotechnologies: functional genomics, transcriptomics and proteomics (GENZOOT)” funded by the Italian Ministry of Agriculture. Care and management of animal CRA-ZOE staff

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