Aix galericulata) in South Korea. We sequenced all eight genome segments of the virus, designated as A/Mandarin duck/Korea/K20-551-4/2020(H5N8), and conducted genetic characterization and comparative phylogenetic analysis to track its origin. Genome sequencing and phylogenetic analysis show that the hemagglutinin gene belongs to H5 clade 2.3.4.4 subgroup B. All genes share high levels of nucleotide identity with H5N8 HPAI viruses identified from Europe during early 2020. Enhanced active surveillance in wild and domestic birds is needed to monitor the introduction and spread of HPAI via wild birds and to inform the design of improved prevention and control strategies.In October 2020, a highly pathogenic avian influenza (HPAI) subtype H5N8 virus was identified from a fecal sample of a wild mandarin duck ( Anatidae family, are known as a natural reservoir of avian influenza virus (AIV) [Highly pathogenic avian influenza (HPAI) subtype H5Nx viruses have been causing substantial losses to the poultry industry and public health concerns since the detection of A/Goose/Guangdong/1/1996(H5N1) (Gs/GD) from domestic poultry in southern China. The descendent Gs/GD-lineage H5 viruses have evolved into 10 genetically independent hemagglutinin (HA) clades (0–9) and subclades [us (AIV) . Migratous (AIV) ,4,5,6,7.Since the first identification of HPAI H5N5 virus in China in 2008, Gs/Gd-lineage clade 2.3.4 HPAI H5Nx viruses have evolved into subclades 2.3.4.1–2.3.4.4 . The H5 During May–June 2016, novel reassortant clade 2.3.4.4 subgroup B H5N8 viruses containing internal genes of Eurasian low-pathogenic avian influenza (LPAI) viruses were identified in wild birds in Uvs Lake, Russia and Qinghai Lake, China ,13. ThesAix galericulata) collected in October 2020 during active wild bird surveillance for AIV in South Korea. We sequenced all eight genome segments of the virus, designated as A/Mandarin duck/Korea/K20-551-4/2020(H5N8), and conducted genetic characterization and comparative phylogenetic analysis to track its origin. The clade 2.3.4.4 subgroup B H5Nx viruses caused multiple outbreaks in South Korea in winter 2016–17 and 2017–18, which were most likely introduced by wild waterfowl ,22,23. TWe collected a total of 150 fresh fecal samples in wild bird habitat near Cheongmi Stream (GPS 37°07′09.0″ N 127°24′01.0″ E) in Anseong, South Korea, for active surveillance purposes. The fecal samples were stored at 4–8 ℃ and analyzed within 12 h.Fecal samples were placed in phosphate-buffered saline (PBS) containing 400 mg/mL gentamicin and thoroughly homogenized by vortexing for 1 min. The supernatant of samples were filtered using a 0.45 µm Minisart Syringe Filter  and inoculated into 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs. After 72 h of incubation at 37 ℃, the allantoic fluids were harvested and tested for hemagglutinin activity using 10% chicken red blood cells. RNA was extracted from the hemagglutinin-activity-positive allantoic fluid using the RNeasy Mini Kit  according to the manufacturer’s instruction and screened for the matrix (M) gene of avian influenza virus using real-time reverse transcription-PCR (rRT-PCR) as previously described . Reverse transcription was conducted using SuperScript IV First-Strand Synthesis System  and the eight gene segments were amplified as previously described using TaKaRa Ex Taq  according to the manufacturer’s instruction . The PCRhttps://platform.gisaid.org/) database and the NCBI’s Influenza Virus Resource at GenBank (https://www.ncbi.nlm.nih.gov/genomes/FLU) . The maximum-likelihood (ML) phylogenetic tree for each genome segment was constructed using RAxML v8 [The sequences of the highly pathogenic and low-pathogenic avian influenza viruses were retrieved from the GISAID Epiflu , hereafter referred to as 551-4/2020 virus, was identified as an HPAI virus on the basis of multiple basic amino acids at the HA proteolytic cleavage site (PLREKRRKR/G). The GISAID database BLAST searches showed that all eight gene segments of the 551-4/2020 virus shared high nucleotide identity (>99.05%) with HPAI H5N8 viruses identified from poultry farms and wild birds in Europe in early 2020 . In the (ML) phylogenies, all the eight gene segments of 551-4/2020 virus were closely clustered together with the clade 2.3.4.4 subgroup B subtype H5N8 viruses detected from poultry and wild birds in Europe during early 2020 . All genAnatidae birds including mallard (Anas platyrhynchos), spot-billed duck (Anas poecilorhyncha), and common teal (Anas crecca) and also the previous outbreak site of clade 2.3.4.4 subgroup B H5N6 in winter 2017–18 [South Korea is located in the East Asian–Australasian flyway and serves as a wintering and stopover site for wild migratory birds ,28,29. W 2017–18 . During Mandarin ducks are native to the Far and Southeast of Asia including China, North and South Korea, Japan, and Eastern Russia along the East Asian–Australasian flyway. Some mandarin ducks are resident in South Korea, and others are passage migrants that occasionally overwinter in Korea and move to and from southern Japan . The ideThe timing and location of the 551-4/2020 virus isolation, lack of identification of related clade 2.3.4.4 subgroup B H5Nx virus in South Korea in recent two and half years, and phylogenetic analysis and BLAST search results collectively suggest that migratory wild birds might have introduced the 551-4/2020 virus into South Korea during the fall migration of wild birds. However, the detailed transmission route of this virus could not be determined because of the lack of wild bird surveillance data during spring and summer 2020. It is most likely that the subgroup B H5Nx viruses belonging to clade 2.3.4.4 affected Europe during early 2020 and were then maintained in wild birds before being introduced into South Korea in fall 2020. Previous studies showed that numerous wild waterfowl species can be infected by clade 2.3.4.4 viruses without detectable clinical signs ,38, and Considering the continued emergence of novel reassortant viruses and global dissemination of the clade 2.3.4.4 HPAI H5Nx viruses, enhanced active surveillance in wild and domestic birds is required to monitor the introduction, spread, and reassortment of HPAI and to inform the design of improved prevention and control strategies.


Principal coordinate analysis carried out using 21 STR allele frequency data mapped the Akans with Nigerian subpopulation groups , but separated from Thais of Thailand, Chechen of Jordan and Tijuana of Mexico.Short tandem repeat (STR) loci are widely used as genetic marker for ancestral and forensic analyses. The latter application includes for paternity testing and DNA profiling of samples collected from scenes of crime and suspects. This survey provides the first dataset for 21 STR loci across the Akan population in Ghana by genotyping of 109 unrelated healthy individuals using Investigator 24plex kit. None of the STR loci screened deviated from Hardy-Weinberg equilibrium after applying Bonferroni correction. Overall, 224 unique alleles were observed with allele frequencies ranging from 0.005 to 0.518. The combined match probability, combined power of exclusion and combined power discrimination were 1 in 4.07 × 10 Specifications TableValue of the data•In the Sub-Saharan region of Africa, population data for these 21 STR loci are only available for Nigerian subpopulations. The 21 STR dataset for the Akans of Ghana reported in this article is thus the first from a different country.•Our 21 locus STR dataset provides an important source of information to estimate their statistical value as DNA evidence  for this population group•Data from this survey also supports the general value of STR loci for population studies across the region; allele frequencies of the 21 STR loci can be used to examine the past history of population events in Akans including gene flow, natural selection and migration patterns.•The population genetic data and forensic statistics reported for the Akans can be used as a reference standard in future studies of other sub-population groups in Ghana; Ewe, Mole-Dagbon, Ga-Dangbe and Guang.1p = 0.05/21, at 95% significance level), no deviation from Hardy-Weinberg equilibrium (HWE)were observed. The highest power of discrimination (PD) and polymorphic information content (PIC) were 0.982 and 0.917 (respectively), recorded for the SE33 locus. Locus TH01 showed the lowest PIC (0.628) and PD (0.824) values. The combined matching probability (CMP), combined probability of exclusion (CPE) and combined discriminating power (CPD) were 1 in 4.07 × 10−25, 0.999999999 and 1, respectively.The allelic scores for the 21 STR loci examined across 109 unrelated Akan individuals are shown in Supplementary Table 1. Their allele frequency data and forensic parameters are shown in Principal coordinate (PCO) mapping performed using the allele frequency data across all 21 STR loci in the Akans and 7 other previously reported STR datasets (Supplementary Table 2) is shown in 22.1revd. 2019) and the Human Ethics Committee of University Sains Malaysia (USMKK), Health Campus, Kelantan, Malaysia (permit no: USM/JEPeM/16050188).This research was reviewed and approved by the Institutional Review Board of Nugochi Memorial Institute of Medical Research (NMIMR), University of Ghana . Total genomic DNA was quantified using Investigator Quantiplex Hyres kit according to manufacturer's recommendation . The Investigator 24plex QS amplification kits  was utilized to amplify the sex-determining marker Amelogenin, 1 Y-STR locus, 2 quality sensors and 21 autosomal STR loci namely CSF1PO, D10S1248, D12S391, D13S317, D16S539, D18S51, TPOX, D19S433, D1S1656, D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S818, D7S820, FGA, D8S1179, SE33, TH01 and vWA on the GeneAmp PCR System 9700 thermal cycler  in total of 12.5 ul reaction volume. PCR products were separated by multi-capillary electrophoresis in 3500XL Genetic Analyzer . The STR alleles were systematically called using the GeneMapper IDx v4.1 software . Allele designations were determined by comparison of the sample fragments with those of allelic ladders provided in the kit.2.4p > 0.00238 after Bonferroni correction . Allele frequencies for the 21 STR loci, PD, power of exclusion (PE), MP, typical paternity index (TPI), PIC and Ho values were computed using the Powerstats software version 1.2 The HWE and the expected heterozygosity (He) values were calculated using Arlequin v3.5.2.2 ,2. The sThe authors declare that they have no known competing interests or personal relationships that could have appeared to influence the work reported in this paper.


Staphylococcus epidermidis RNase J paralogs, RNase J1 and RNase J2. RNase J1 is a homodimer with exonuclease activity aided by two metal cofactors at the active site. RNase J2, on the other hand, has endonuclease activity and one metal ion at the active site and is predominantly a monomer. We note that the expression levels of these enzymes vary across Staphylococcal strains. Together, these observations suggest that multiple interacting RNase J paralogs could provide a strategy for functional improvisation utilizing differences in intracellular concentration, quaternary structure, and distinct active site architecture despite overall structural similarity.RNase J enzymes are metallohydrolases that are involved in RNA maturation and RNA recycling, govern gene expression in bacteria, and catalyze both exonuclease and endonuclease activity. The catalytic activity of RNase J is regulated by multiple mechanisms which include oligomerization, conformational changes to aid substrate recognition, and the metal cofactor at the active site. However, little is known of how RNase J paralogs differ in expression and activity. Here we describe structural and biochemical features of two  Escherichia coli, for example, RNase E, polynucleotide phosphorylase (PNPase), RhlB, and enolase are degradosome components with RNase E forming the scaffold for this multienzyme assembly  (rnj1) and double (rnj1 and rnj2) depletion mutants in B. subtilis strains were found to have substantial differences in the expression levels of multiple genes, suggesting that RNase J1 plays an extensive role in modulating gene expression , and the ΔrnjB strain (deletion of RNase J2) was attenuated in its ability to form biofilms  in a similar manner  activity of RNase J is also influenced by the quaternary structure of this enzyme. An illustration of this feature is nt dimer . Mutationt dimer . A surprhe dimer . The quahe dimer . Finallytivities . The knotivities .Staphylococcus epidermidis. The focus was to evaluate a structural or biochemical rationale for multiple RNase J paralogs in some Firmicutes, such as Staphylococci. In particular, we sought to understand the structural role of RNase J2 that has been ascribed to this paralog in the context of RNA degradation  bound at the active site. Furthermore, Ca2+ is the most preferred metal cofactor for RNase J2 activity in vitro. We also note that the expression levels of the two RNase J paralogs vary across Staphylococcal strains. Together, these observations suggest that multiple RNase J paralogs provide an evolutionary route for functional optimization within the conserved RNase J scaffold.Here we describe studies on two RNase J paralogs: RNase J1 and RNase J2 from radation . A compaB. subtilis RNase J paralogs interact, recombinant S. epidermidis RNase J1 and RNase J2 were coexpressed and purified  and RNase J2  was evaluated by size exclusion chromatography (SEC). We note that RNase J1 is a dimer in solution whereas RNase J2 is seen to be a mostly monomeric enzyme with a minor dimeric species . Subsequpurified . We note its own . The lowangement , 20.Figuin situ by a protease contaminant that evaded the multiple protein purification steps. The diffraction data and refinement statistics for crystals of both paralogs are compiled in B. subtilis RNase J1 as a search model   could be crystallized. Although RNase J2 could be readily crystallized, analysis of the protein sample from a single crystal by MALDI-TOF MS suggested that a polypeptide  alone had crystallized and not the full-length RNase J2 that was used in the crystallization trials. It appears likely that the flexible C-terminal domain was proteolytically cleaved ctively) .Table 1DS. epidermidis RNase J1 and RNase J2 is similar to previously determined structures which include B. subtilis RNase J1 and other RNase J homologs from T. thermophilus, S. coelicolor, D. radiodurans, and M. psychrophilus (S. epidermidis RNase J2 (2096 Å3) when compared with S. epidermidis RNase J1 (3108 Å3) or B. subtilis RNase J1 (2649 Å3)  . For thi2649 Å3) .S. coelicolor RNase J/RNA complex (PDB ID: 5A0T)  are similar to other characterized RNase J enzymes  specifically act on 5′ mono-phosphorylated substrates, whereas others are more liberal in substrate recognition , a putative RNA substrate was modeled to evaluate potential differences in substrate recognition. The RNA template used was the 5′ mono-phosphorylated 5-nucleotide from the D: 5A0T) . From th enzymes   is similar to other characterized RNase J homologs, most other RNase J enzymes have two Zn2+ ions (2+) at the active site  have two metal ions at the active site. This observation formed the basis for a two metal ion–dependent catalytic mechanism . Assays to monitor endo- and exoRNase activity were performed using protocols described earlier with few modifications (B). In the case of RNase J1 (0.6 µm concentration), no product corresponding to endoRNase activity was seen in this reaction condition. When the activity assays for RNase J2 were performed at the same concentration as that for RNase J1, no product corresponding to either exo- or endoRNase activity was seen. This observation is similar to that reported for B. subtilis and E. faecalis RNase J2, which suggests that this paralog has substantially lower enzymatic activity compared with RNase J1 (m). At this concentration of RNase J2, we note both endoRNase activity and exonuclease activity .The structural information on RNase J1 and RNase J2 aided the design of experiments to evaluate the catalytic activity of these paralogs and mutant enzymes A. Assaysications . In thesications B. In theRNase J1 , 18. Them RNase J1 and 0.25 nm of the 20-mer RNA substrate) as that of the full-length RNase J1. We note that RNase J1ΔCTD is monomeric (B) and also has endoRNase activity (presence of bands between 20-mer RNA and mono-ribonucleotides) in addition to exoRNase activity (B). Together, the activity assays suggest that whereas full-length RNase J1 functions primarily as an exoRNase, RNase J1ΔCTD has both endoRNase and exoRNase activity.To evaluate whether the C-terminal deletion construct employed in the structural analysis of RNase J1 is functionally active, enzyme assays were performed with the same protein preparation used in the crystallization trials . These assays were performed with the same concentration of the enzyme and substrate . In the case of RNase J2, the H80A,E166A double mutant showed substantially impaired catalytic activity (E). To enable a comparison with enzyme assays performed on S. aureus RNase J1 (98% identity with S. epidermidis RNase J1), enzyme activity was evaluated using a 27-mer RNA substrate reported earlier  arising from a contaminant in enzyme preparations, activity assays were performed with active site mutants of RNase J1 and RNase J2. In the case of RNase J1, the D78A mutation led to an inactive enzyme D. In theactivity E. To ena earlier . DNase a earlier . The resRNase J2 . These eD). The finding that the RNase J1 H368A mutant has substantially lower catalytic activity than the WT enzyme is particularly significant because this is consistent with the hypothesis that His-368, located in the loop connecting the β-lactamase and β-CASP domains, plays an important role in catalysis .The crystal structure of RNase J1 suggested that residues His-364, Ser-366, and His-368 could be potentially involved in phosphate recognition . WhereasFig. 4S. epidermidis RNase J1 was evaluated in the presence of different metal ions   and competing metal ions (2 mm) were added to the reaction mixture  . Presencnd Zn2+) . We noten et al. , in the  mixture . These e2+, followed by Mg2+ and Mn2+, whereas Co2+ and Zn2+ cofactors elicited negligible catalytic activity (2+ was kept constant (1 mm) and different metal ions  were added (2 mm) to the reaction mixture  and other metal ions  were added at a higher concentration (2 mm)  . The repn (2 mm) . It is wB. subtilis, the genes encoding the two RNase J paralogs (rnjA and rnjB) lie in different operons. However, the expression of rnjA (encoding RNase J1) was not found to be at levels significantly different from rnjB (encoding RNase J2). This finding suggested that RNase J1 does not exist alone to a significant extent. This inference is consistent with the observation that the B. subtilis paralogs RNase J1 and RNase J2 interact with 1:1 stoichiometry (rnj1 and rnj2) also belong to different operons in Staphylococci, it was important to quantify the expression levels of these genes. One caveat of this inference is that expression level of the genes translates to corresponding changes in the intracellular level of these proteins. To evaluate expression levels of rnj1 and rnj2, we quantified the transcript levels of both rnj1 and rnj2 at two time points (log and stationary phase) in eight representative Staphylococcal strains (A). These strains were chosen to represent different lineages, nosocomial and community backgrounds . For example, the expression levels of rnj1 at the exponential phase of growth in the LVP2 strain are higher when compared with other clinical S. aureus strains, including COL (A). The LVP2 strain belongs to the ST772 lineage (S. epidermidis rnj2 are higher when compared with rnj1.In the case of hiometry . Because strains A. These kgrounds . Alongsiine tool  28). On. OnB. su strains A. For exding COL A. The LV lineage . We also2+ ions at the active site  seeks to reorient from an irregular pentacoordinated arrangement to the preferred octahedral geometry, the enzyme-substrate complex is destabilized to drive catalysis , enzyme assays suggest that RNase J2 prefers Ca2+ for its catalytic activity in vitro. A related observation is that mutating residues that coordinate the bound metal ion in RNase J2 (His-80 and Glu-166) influence enzyme activity (E). It thus appears likely that the metal cofactor plays a more structural role in this enzyme as opposed to direct involvement in the catalytic mechanism. It however remains to be determined whether RNase J2 is bound to Mn2+ or Ca2+in vivo. In this context, a phylogenetic analysis of RNase J paralogs also provided an interesting insight (B). RNase J2 paralogs appear to be phylogenetically distinct; whether other RNase J2 paralogs have a similar active site architecture characterized by one metal cofactor remains to be determined. Together, the structural and biochemical data on S. epidermidis RNase J1 and RNase J2 reveal that (i) oligomerization is an important determinant in the switch between exo- and endoRNase activity; (ii) the metal cofactor and stoichiometry determine the reaction mechanism; this difference is hardwired in RNase J paralogs; (iii) RNase J1 is phylogenetically closer to ancestral RNase J than RNase J2; and (iv) the expression levels of these paralogs vary across different Staphylococcal strains.Two prominent differences between the structures of the two RNase J paralogs are the active site architecture and the stoichiometry of the metal cofactor at the active site. The structures reveal that the RNase J1 active site is closer to other characterized RNase J enzymes or even eukaryotic MRE-11 with two Mnive site . Indeed,nificant . In the atalysis . In thisomplexes . Structuactivity E. It thu insight B. RNase S. epidermidis RNase J paralogs also provide a rationale to understand observations made on the B. subtilis RNase J1 and RNase J2 enzymes. Similarity in RNA substrate recognition, for example, can be rationalized from the observation that residues in the monophosphate interaction site are conserved in both RNase J1 and RNase J2  is relevant. The C-terminal segment that governs oligomeric assembly is thus an important determinant of the switch between exo- and endoRNase activity.The bacterial RNase J–specific C-terminal domain appears to be an important part of the evolutionary diversification. In the case of Staphylococcal RNase J paralogs is also intriguing in the light of some recent findings: RNase J2 has been suggested to potentially serve as a stabilizer of RNase J1 in S. aureus  when compared with the other strains that were examined (A).The functional and evolutionary rationale for the . aureus . The typ and RNA . Indeed, and RNA . In this and RNA ; Fig. S1 and RNA . Albeit examined A.S. epidermidis RNase J paralogs suggest functional optimization utilizing differences in active site architecture and preferred quaternary arrangement. Whereas RNase J1 is closer to the canonical RNase J , RNase J2 is distinct as a monomeric enzyme with a single Mn2+ ion at the active site. Because the genes encoding these two enzymes are not part of the same operon, changes in the expression level of these paralogs could, in effect, form a regulatory mechanism. RNase J paralogs in Staphylococci thus represent an example of a multi-layered regulatory mechanism enabling functional diversity, ranging from phage immunity, RNA maturation, RNA recycling, and changes in gene expression.Put together, the E. coli BL21 Star (DE3) strain (Novagen). A single transformant colony was inoculated in Luria Bertani broth containing 50 µg/ml ampicillin. E. coli cultures were induced with 0.5 mm isopropyl β-d-1-thiogalactopyranoside at an A600nm of 0.5. The cells were further grown for 12 h at 18 °C before harvesting by centrifugation.The genes encoding RNase J1 (amino acids 1–560), the C-terminal domain deletion construct of RNase J1 , and RNase J2 were cloned in Multiple Cloning Site (MCS) I of the pETDuet-1 expression vector between the BamHI and HindIII restriction sites . The recm Tris-HCl, pH 8.0, 250 mm NaCl, 0.5 mm MnCl2, 2 mm β-mercaptoethanol (β-ME), and 10% glycerol) in the presence of 2 mm PMSF. After centrifugation, the supernatant was incubated with nickel-nitrilotriacetic acid resin. A high salt wash (1.5 m NaCl in buffer A) with ∼10 column volumes helped in the removal of bound nucleic acid contaminants. The recombinant protein bound to the resin was then eluted stepwise with imidazole concentrations ranging from 250–500 mm in buffer A. The eluted proteins were further subjected to size exclusion chromatography using a Hi-prep 16/60 Sephacryl S-200 column  in buffer B phosphine, and 10% glycerol).Proteins were purified using immobilized metal affinity chromatography. Briefly, pellets were lysed in buffer A (25 mrnj2 cloned in the MCS-I of the pETDuet-1 expression vector was used as a backbone to insert rnj1 in MCS II between the MfeI and XhoI restriction sites (E. coli BL21 Star (DE3) strain . A single transformed colony was inoculated in Luria Bertani broth containing 50 µg/ml ampicillin. E. coli cultures were induced with 0.5 mm isopropyl β-d-1-thiogalactopyranoside at an A600nm of 0.5. The cells were further grown for 12 h at 18 °C before harvesting by centrifugation.The plasmid containing on sites . In thism Tris, pH 8.0, 250 mm NaCl, 2 mm β-ME, 10% glycerol, and 2 mm PMSF. After centrifugation, the supernatant was incubated with nickel-nitrilotriacetic acid resin. The recombinant protein bound to the resin was then eluted stepwise with imidazole concentrations ranging from 100–400 mm. The eluted protein was digested with 20 µg/ml trypsin (Sigma-Aldrich) and the fragments were analyzed by MS .The coexpressed proteins were copurified by immobilized metal affinity chromatography. Briefly, the cells were lysed in lysis buffer containing 50 mm Tris-HCl, pH 8.0, containing 250 mm NaCl, 10% glycerol, 4 mm β-ME, 0.5 mm tris(2-carboxyethyl)phosphine, and 5 mm MgCl2. The same size exclusion column and identical buffer conditions were maintained for SEC-MALS experiments performed on the two RNase J paralogs.Analytical size exclusion chromatography was used to analyze the oligomeric state of the purified recombinant enzymes. In these experiments, 250 μg of freshly purified RNase J1, RNase J1ΔCTD, and RNase J2 were injected separately into a Superdex200 column  equilibrated in 50 mm Tris-HCl, pH 8.0, 250 mm NaCl, 2 mm β-mercaptoethanol, and 10% glycerol). Crystallization trials were performed using both microbatch and hanging-drop methods. Although crystalline precipitates were obtained in a variety of conditions, the best diffracting crystals of RNase J1 were obtained using the microbatch method in a condition containing 10 mm citric acid, 70 mm bis-tris propane, pH 9.3, 20% w/v PEG 3350, and 0.1 m spermine tetrahydrochloride. A typical 2-µl drop contained protein:crystallization condition in a 1:1 ratio. The protein crystals were cryoprotected as-is without soaking in any metal ion–containing buffer. All crystallization experiments were performed at 20 °C. Diffraction data were collected at the ID30A-1 beamline of the European Synchrotron Radiation Facility, Grenoble, France. The diffraction data were integrated using iMosflm and scaled using SCALA (B. subtilis RNase J (PDB: 3ZQ4) as a search model. The model corresponding to RNase J1 was further built using Coot and refined using REFMAC and Phenix  of the truncated RNase J1 enzyme (comprising residues 1–450). Sparse-matrix sampling using commercial crystallization screens (Hampton Research and Jena Biosciences) was performed with freshly purified and concentrated protein samples . To improve crystal quality, additives (Hampton Research) were screened in the ratio of 2:2:0.4 (condition:protein:additive) alongside seeding (both micro-seeding and streak seeding). Diffracting crystals were obtained after addition of 40% formamide and 1 m lithium chloride. RNase J2 crystals diffracted beyond 2.6 Å with 10 min of exposure at the home source (Rigaku FRE X-ray generator and R-axis VI detector). As in the case of RNase J1, the diffraction data were integrated using iMosflm and scaled using SCALA (B. subtilis RNase J (PDB: 3ZQ4) as a search model. The model was rebuilt using Coot and refined using REFMAC and Phenix  was selected as the seed point, and the cavity profile was generated with 12 Å of radius around this seed point. Similarly, the cavity shape profile was generated taking the equivalent atom position with respect to the ND1 of His-76 in other RNase J homologs.The active sites of RNase J1 and RNase J2 were visualized using a protocol implemented in HOLLOW . To ensuS. coelicolor RNase J (PDB ID: 5A0T). Structures of RNase J1 and RNase J2 were superposed on the S. coelicolor homolog to obtain an initial model with a putative RNA substrate  and a 27-mer single-stranded RNA (5′-UCU-UUACGGUGCUAUUUUGUUUUGUUC-3′) (32P (γATP). After radiolabeling using polynucleotide kinase (New England Biolabs), the radiolabeled oligos were purified by ethanol precipitation . The RNase activity levels of RNase J1 and RNase J2 was compared at different concentrations of RNase J1  and RNase J2 . For ease in comparison, we evaluated RNase activity at a fixed time point (30 min). In an effort to determine the most preferred metal ion for RNase J1 and RNase J2 catalytic activity, assays were performed in the presence of different divalent cations . Apo enzymes were prepared by incubating freshly purified enzyme with a chelator (0.001 m EDTA) followed by extensive dialysis. ICP-MS analysis was used to evaluate the presence of the bound metal ion. In these metal ion competition experiments, the concentration of one metal ion was maintained at a constant level (1 mm) and the potential competing metal ion  was added to the reaction mixture.Enzyme assays for RNase J1, RNase J2, and the C-terminal deletion constructs were performed in a buffer containing 50 mGUUC-3′) . DNase apitation . The actStaphylococcal strains used in this study are listed in S. epidermidis (ATCC 12228), S. aureus COL , and characterized S. aureus strains belonging to a different sequence type from those in our collection , 200 µg of Proteinase K (Qiagen), and 100 µg of lysostaphin (Sigma-Aldrich). No changes were made to the protocol suggested by the manufacturer. cDNA was prepared using QuantiTect® Reverse Transcription kit (Qiagen) according to the manufacturer's instructions. Real-time PCR was carried out for samples from different strains  to quantitate the levels of rnj1 and rnj2 using SYBR green super mix on a Bio-Rad iQ5TM thermo cycler. 16S rRNA was used as internal control in these experiments  using RNeasy mini kit (Qiagen). Initially, the cells were lysed in 30 meriments . The priThe data produced by this study are available in this article and in the 6K6S and 6K6W) have been deposited in the Protein Data Bank.The atomic coordinates and structure factors (


Entamoeba histolytica, the protozoan parasite responsible for amoebic dysentery and liver abscess in humans. E. histolytica encodes two isotypes of eIF5A and two isotypes of enzymes, deoxyhypusine synthase (DHS), responsible for their PTM. Both of the two eIF5A isotypes are functional, whereas only one DHS , is catalytically active. The DHS activity increased ~2000-fold when EhDHS1 was co-expressed with EhDHS2 in Escherichia coli, suggesting that the formation of a heteromeric complex is needed for full enzymatic activity. Both EhDHS1 and 2 genes were required for in vitro growth of E. histolytica trophozoites, indicated by small antisense RNA-mediated gene silencing. In trophozoites, only eIF5A2, but not eIF5A1, gene was actively transcribed. Gene silencing of eIF5A2 caused compensatory induction of expression of eIF5A1 gene, suggesting interchangeable role of the two eIF5A isotypes and also reinforcing the importance of eIF5As for parasite proliferation and survival. Furthermore, using a sibling species, Entamoeba invadens, we found that eIF5A1 gene was upregulated during excystation, while eIF5A2 was downregulated, suggesting that eIF5A1 gene plays an important role during differentiation. Taken together, these results have underscored the essentiality of eIF5A and DHS, for proliferation and potentially in the differentiation of this parasite, and suggest that the hypusination associated pathway represents a novel rational target for drug development against amebiasis.The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein and is essential in all eukaryotes. However, the specific roles of eIF5A in translation and in other biological processes remain elusive. In the present study, we described the role of eIF5A, its posttranslational modifications (PTM), and the biosynthetic pathway needed for the PTM in  E. histolytica. We found by small antisense RNA-mediated gene silencing that EhDHS1/2 and EheIF5A2 are essential for growth of E. histolytica trophozoites. We further found that only one of two isotypes of eIF5A, EheIF5A2, was constitutively expressed in the trophozoite stage and silencing of EheIF5A2 gene caused overexpression of the other eIF5A isotype (EheIF5A1) to partially rescue the growth defect in this parasite. Furthermore, we found that transcription of eIF5A1 gene was stage-specifically upregulated during excystation in E. invadens. Taken together, we have demonstrated for the first time that the two eIF5As play important and distinct roles in Entamoeba biology. This study has also provided an answer to a long-standing conundrum on the biological importance of polyamines: spermidine is needed for eIF5A hypusination, which is essential for protein translation in Entamoeba. Our work will provide an insight into the physiological significance of eIF5A and its post-translational modifications in other pathogenic eukaryotes and potentially lead to the formulation of control measures against parasitic diseases.Eukaryotic initiation factor 5A is a ubiquitous protein that is essential for cell proliferation. We examined the maturation, regulation, and function of eIF5A in  Polyamines are low-molecular-weight nitrogenous bases that are essential for the regulation of cell growth and development . Due to E. histolytica is a unicellular parasitic protozoan responsible for human amebiasis. The World Health Organization estimates that approximately 50 million people worldwide suffer from invasive amebic infections, resulting in 40 to 100 thousand deaths annually  containing 10–50 mM of imidazole. Bound proteins were eluted with buffer A containing 100–300 mM imidazole to obtain recombinant EhDHS1, EhDHS2, a combination of EhDHS1 and 2, EhDHS1_K295F, EhDHS2_F302K, EheIF5A1, and EheIF5A2. After the integrity and the purity of recombinants protein were confirmed with 12% SDS-PAGE analysis, followed by Coomassie Brilliant Blue staining, they were extensively dialyzed twice against the 300-fold volume of 50 mM Tris-HCl, 150 mM NaCl, pH 8.0 containing 10% glycerol (v/v) and Complete Mini protease inhibitor cocktail  for 18 hr at 4°C. The dialyzed proteins were stored at –80°C with 30% glycerol in small aliquots until further use. Protein concentrations were spectrophotometrically determined by the Bradford method using bovine serum albumin as a standard as previously described , H. sapiens (P49366) and E. invadens . The catalytic lysine residue is shown in red background, whereas NAD+ and spermidine binding sites are shown in green and yellow background, respectively. The conserved residues are marked by asterisks (*) while similar amino acids are shown either with periods (.) or colons (:). Sequence alignment was performed using ClustalW.Alignment of DHS protein sequences from (TIF)Click here for additional data file.S2 FigAccession numbers of these sequences are as follows: EHI_186480 (XP_651531), EHI_151810 (XP_657397), EHI_151540 (XP_657374), and EHI_177460 (XP_655916). The conserved residues are marked by asterisks (*) while similar amino acids are shown either with periods (.) or colons (:). The conserved lysine residue, which was supposed to be hypusinated, is highlighted in red. Sequence alignment was performed using ClustalW.(TIF)Click here for additional data file.S3 Fig(A) Purification of recombinant EhDHS1, EhDHS2, EheIF5A1, and EheIF5A2. Protein samples at each step of purification were subjected to 15% SDS-PAGE under reducing conditions, and then stained with Coomassie Brilliant Blue R250. (B) Purification of the recombinant EhDHS mutants.(TIF)Click here for additional data file.S4 FigApproximately 40 μg of total lysates were electrophoresed on a SDS-PAGE gel under reducing conditions and subjected to immunoblot analysis using anti-HA and anti-hypusine antibodies.(TIF)Click here for additional data file.S5 Fig+, 3μg of co-expressed recombinant EhDHS1 and 2 , and the mixtures were subjected to SDS-PAGE and immunoblot analyses using anti-hypusine, anti-His, and anti-S tag antibodies.Recombinant EheIF5A1 or EheIF5A2 was incubated with 1 mM spermidine, 0.5 mM NAD(TIF)Click here for additional data file.S6 FigApproximately 40 μg of total lysate from the transformant expressing HA-EheIF5A1 and HA-EheIF5A2 and HA was electrophoresed on a SDS-PAGE gel under reducing conditions and subjected to immunoblot analysis using anti-EheIF5A1, anti-EheIF5A2 and anti-HA antibodies.(TIF)Click here for additional data file.S7 Fig(A) Percent amino acid identity among E. histolytica and E. invadens eIF5A isoforms by ClustalW multiple sequence alignment score. GenBank accession numbers: EheIF5A1 (XP_657374), EheIF5A2 (XP_651531), EieIF5A1 (XP_004255257), EieIF5A2 (XP_004258351), EieIF5A3 (XP_004260381). (B) Amino acid sequence alignment of E. histolytica, E. invadens and Homo sapiens eIF5A isoforms. Accession numbers of these sequences are as follows: EheIF5A1 (XP_657374); EheIF5A2 (XP_651531); EieIF5A1 (XP_004255257), EieIF5A2 (XP_004258351), EieIF5A3 (XP_004260381), HseIF5A1 (P63241); and HseIF5A2 (AAG23176). The conserved residues are marked by asterisks (*) while similar amino acids are shown either with periods (.) or colons (:). The conserved lysine residue, which was supposed to be hypusinated, is highlighted in red. Sequence alignment was performed using ClustalW.(TIF)Click here for additional data file.S8 FigMaximum likelihood tree inferred by MEGA X program with JTT matrix-based model is shown. 132 unambiguously aligned positions from 39 sequences were used for the analysis. Branch lengths are proportional to estimated numbers of substitutions. Bootstrap proportion (BP) values (shown in percentage) are shown on the internal branches. Species names and accession numbers of the sequences are also indicated.(TIF)Click here for additional data file.S9 FigApproximately 30 μg of total lysates was electrophoresed on a 15% SDS-PAGE gel under reducing conditions and subjected to immunoblot analysis using anti-hypusine, anti-EheIF5A1 and anti-EheIF5A2 antibodies.(TIF)Click here for additional data file.S10 Fig(A) Kinetics of encystation. The percentages of the amoebae resistant to 0.05% sarkosyl during encystation. (B) The steady-state levels of transcripts of EieIF5A1, EieI5FA2, EieIF5A1, and EiActin genes measured by semi-quantitative RT-PCR during encystation. cDNA from different time points during encystation was subjected to 30 cycles of PCR using specific primers for the EheIF5A1, EheI5FA2, EheIF5A3 and Ehactin genes. Ehactin gene served as a control. PCR analysis of samples without reverse transcription was also used to exclude the possibility of genomic DNA contamination.(TIF)Click here for additional data file.S11 Fig(A) Representative immunofluorescence assay (IFA) micrographs of HA-eIF5A1 and HA-eIF5A2 expressed in E. histolytica trophozoites, double stained with anti-HA antibody (green) and anti-CS1 antiserum (red) respectively. EhCS1 (Cysteine synthase 1) served as a cytosolic control. Scale bar, 10 μm. (B) eIF5A2gs (only eIF5A1 expressed) and control .(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Since their discovery in the United States in 1963, outbreaks of infection with equine influenza virus (H3N8) have been associated with serious respiratory disease in horses worldwide. Genomic analysis suggests that equine H3 viruses are of an avian lineage, likely originating in wild birds. Equine-like internal genes have been identified in avian influenza viruses isolated from wild birds in the Southern Cone of South America. However, an equine-like H3 hemagglutinin has not been identified. We isolated 6 distinct H3 viruses from wild birds in Chile that have hemagglutinin, nucleoprotein, nonstructural protein 1, and polymerase acidic genes with high nucleotide homology to the 1963 H3N8 equine influenza virus lineage. Despite the nucleotide similarity, viruses from Chile were antigenically more closely related to avian viruses and transmitted effectively in chickens, suggesting adaptation to the avian host. These studies provide the initial demonstration that equine-like H3 hemagglutinin continues to circulate in a wild bird reservoir. Aquatic birds are the reservoir of influenza A viruses and responsible for the evolution and long-distance spread of the virus  and stored in 1-mL universal transport media tubes . They were then transported at 4°C to the Faculty of Veterinary Science of the University of Chile  and stored at −80°C until analysis.Fresh feces from birds were collected from the environment during 2013–2017 at different wetlands across the central region of Chile. These samples were collected by using sterile flocked swabs  on a Kingfisher Flex Magnetic Particle Processor . Quantitative reverse transcription PCR was performed on a CFX96 Real-Time PCR System with the 4x TaqMan Fast Virus Master Mix (ThermoFisher Scientific) and primers and probes specific for the influenza A matrix gene  embryonated chicken eggs as described  as described (https://www.illumina.com) as described (http://webapp.cabgrid.res.in/biocomp/CLCBio/clc-bio.html). The H3 sequences from Chile used in this study have been deposited into GenBank .To identify the host species, genetic barcoding was performed by using primers that amplified the cytochrome oxidase I gene as described (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was performed to ensure that sequences from Chile not have higher homology to influenza A virus in other hosts. Representative sequences were selected by clustering similar sequences using CD-HIT-EST  Influenza Virus Database. BLAST analysis (https://evolution.genetics.washington.edu) and visualized by using the ggtree package (http://tree.bio.ed.ac.uk).Maximum-likelihood trees were constructed by using the DECIPHER and PHANGORN packages in R  containing 200 mmol/L GlutaMAX  and 10% fetal bovine serum . All viruses used were natural isolates.We propagated A/California/04/2009(H1N1) and H3Nx viruses A/equine/Miami/1/1963 (H3N8), A/equine/Uruguay/1/1963(H3N8), A/equine/New York/2016(H3N8), A/equine/New York/2016(H3N8), A/red-gartered coot/Chile/C16030/2016(H3N4), A/red-fronted coot/Chile/5/2013(H3N6), A/yellow-billed pintail/Chile/C2014/2015(H3N8), A/mallard/Oregon/449221–105/2006(H3N6), A/duck/Minnesota/34/1976(H3N8), A/mallard/Wisconsin/22/1974(H3N5), A/blue-winged teal/Wisconsin/279/1975(H3N8), A/green winged teal/Alaska/292/2011(H3N8), A/northern pintail/Alaska/496/2012(H3N8), and A/northern pintail/Alaska/870/2014(H3N8) in 10-day-old embryonated chicken eggs as described (https://denka-seiken.com) and serially diluted 2-fold in 25 μL of phosphate-buffered saline in duplicate in a 96 well v-bottom plate. Each homologous virus was adjusted to 4 hemagglutination units, and 25 μL were added to the serum dilutions and incubated for 30 min at 4°C. Finally, 50 μL of 0.5% chicken erythrocytes were added to each of the wells, and the plate was incubated at 4°C for 30 min, after which results of the assay were read.We tested 2 equine H3 viruses [A/equine/Uruguay/1/1963(H3N8) and A/equine/Chile/EQCL003/2018(H3N8)]; 3 wild bird viruses from Chile ; and 7 wild bird origin North American viruses  in a hemagglutination inhibition (HI) assay against a panel of 3 ferret-generated antiserum  according to World Health Organization guidelines  and immediately stored at −80°C for the determination of virus titers by 50% median tissue culture infectious dose assay in MDCK cells as described  24 hours later. All chickens were observed daily for clinical signs of illness, such as body weight loss, labored breathing , and diarrhea. To assess virus shedding, cloacal and tracheal swab specimens were collected at 3, 5, 7, 9, and 12 dpi. Swab specimens were stored in viral transport medium at −80°C until virus titration. Viral titers were established by using the method of Reed and Munch by performing 50% median tissue culture infectious dose assay analysis of the swab specimen inoculum with 50 μL of allantoic fluid and 50 μL of 0.5% chicken erythrocytes (The chicken experiment was performed as described (https://www.graphpad.com). Area under the curve analysis for measuring cumulative shedding was performed by using GraphPad Prism version 8. All animal experiments, procedures, and sampling activities were approved by the St. Jude Children’s Research Hospital Institutional Animal Care and Use Committee.Mean infectious titers were analyzed by 2-way analysis of variance using GraphPad Prism version 8  of the total HA diversity. Six of the H3 viruses, A/cinnamon teal/Chile/C19368/2016 (H3N8), A/red-fronted coot/Chile/5/2013 (H3N6), A/yellow-billed pintail/Chile/C2014/2015 (H3N8), A/red-gartered coot/Chile/C16030/2016 (H3N4), A/yellow-billed pintail/Chile/C30974/2017 (H3N8), and A/yellow-billed pintail/Chile/C34473/2017 (H3N8), were isolated from different regions of Chile , had higBayesian molecular clock analysis was used to estimate the tMRCA between the H3 AIVs from Chile and 1963 H3 EIV, and between avian H3 sequences not from Chile and EIV H3 , panel AThe nucleotide similarity with the EIV H3 HA prompted us to conduct a detailed phylogenetic analysis. Like HA, the H3 viruses from Chile had NS1  Figure 3Bayesian molecular clock analysis was performed for the NP and PA gene segments to estimate the age of tMRCA between the 1963 H3 EIV and sequences from Chile  Figure 8The NS1 segment had insufficient temporal signal for molecular clock analysis. Overall, our phylogenetic analysis suggests that many of the gene segments collected from wild birds in South America were distinct from those collected from wild birds in North America; the H3, PA, NP, and NS1 genes were more closely related to the 1963 H3N8 EIV, supporting the hypothesis that the 1963 H3N8 EIV originated from wild birds in South America. Our results are consistent with those of Rimondi et al., who reported that viruses from birds from Argentina also carry PA and NP sequences with homology to the EIV  when the eq/Miami/63 virus replicated to significantly higher titers compared with the other H3 viruses (p<0.05) , panel A6 50% egg infective dose/0.5 mL virus (n = 3 per group) and then placed in direct contact with virus-naive chickens (n = 9 per group) 24 hours later. Oropharyngeal and cloacal viral shedding was monitored for 12 days postinfection (dpi), and seroconversion was measured at 21 dpi. At 3 dpi, each group infected with H3 virus from Chile had at >1 donor bird showing oropharyngeal virus shedding. The group inoculated with A/red-footed Coot/Chile/5/2013(H3N6) (RF Coot) virus also showed shedding by the cloacal route at this time point. After 5 dpi, donors were shedding virus by mixed oropharyngeal and cloacal routes. After 7 dpi, only shedding by the cloacal route was detected in the group inoculated with A/red-gartered coot/Chile/C16030/2016 virus (RG Coot H3N4)  . AlthougSeroconversion, as measured by HI titers at 21 dpi, was similar across all experimental groups and titers ranged from 1:20 to 1:80. In contrast, the eq/Miami/63 or the equine/NY/2016 viruses did not infect or were transmitted in chickens. However, 1 eq/NY/2016 donor chicken did have HI titers (1:60) at 21 dpi, suggesting infection. Overall, these studies demonstrate that there are H3 IAVs circulating in wild birds in Chile that are antigenically unique from H3 EIV and other wild bird AIVs and are capable of infecting and transmitting in poultry.In these studies, we isolated several H3 influenza viruses from wild birds in Chile that contain HA, NP, PA, and NS segments that are genetically similar to the 1963 EIVs, suggesting that the direct descendants of the virus that originated the EIV pandemic continue to circulate in wild birds in Chile. Previous AIV surveillance studies in South America have identified internal genes that were putatively related to the ancestral virus that originated the 1963 equine pandemic (Although H3 is the most common subtype found in wild birds from North America (Global trade of thoroughbred horses from South America carrying the original 1963 H3N8 EIV, and not migratory birds, was responsible for the spread of these avian-origin gene segments (In summary, our data provide evidence that gene segments, including HA, that are the closest ancestor of the 1963 H3N8 EIV continue to circulate in wild bird reservoirs. We recommend increased surveillance to better clarify the role of this subtype in the context of genetic diversity of IAVs in South America, its epidemiology and ecology, and the risk that this new subtype represents to avian and mammalian hosts.Additional information on equine-like H3 avian influenza viruses in wild birds, Chile.


MEN1 germline mutations, is characterised by parathyroid, pancreatic and pituitary tumours. MEN1 mutations also cause familial isolated primary hyperparathyroidism (FIHP), a milder condition causing hyperparathyroidism only. Identical mutations can cause either MEN1 or FIHP in different families, thereby implicating a role for genetic modifiers in altering phenotypic expression of tumours. We therefore investigated the effects of genetic background and potential for genetic modifiers on tumour development in adult +/-Men1 mice, which develop tumours of the parathyroids, pancreatic islets, anterior pituitary, adrenal cortex and gonads, that had been backcrossed to generate C57BL/6 and 129S6/SvEv congenic strains. A total of 275 +/-Men1 mice, aged 5–26 months were macroscopically studied, and this revealed that genetic background significantly influenced the development of pituitary, adrenal and ovarian tumours, which occurred in mice over 12 months of age and more frequently in C57BL/6 females, 129S6/SvEv males and 129S6/SvEv females, respectively. Moreover, pituitary and adrenal tumours developed earlier, in C57BL/6 males and 129S6/SvEv females, respectively, and pancreatic and testicular tumours developed earlier in 129S6/SvEv males. Furthermore, glucagon-positive staining pancreatic tumours occurred more frequently in 129S6/SvEv +/-Men1 mice. Whole genome sequence analysis of 129S6/SvEv and C57BL/6 +/-Men1 mice revealed >54,000 different variants in >300 genes. These included, Coq7, Dmpk, Ccne2, Kras, Wnt2b, Il3ra and Tnfrsf10a, and qRT-PCR analysis revealed that Kras was significantly higher in pituitaries of male 129S6/SvEv mice. Thus, our results demonstrate that Kras and other genes could represent possible genetic modifiers of Men1.Multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant disorder caused by  MEN1 gene, which is located on chromosome 11q13 and encodes the ubiquitously expressed, predominantly nuclear scaffold tumour-suppressor protein, menin , a disorder characterised by the sole occurrence of parathyroid tumours  is an autosomal dominant disorder characterised by the occurrence of parathyroid, pancreatic islet and anterior pituitary tumours. In addition, some patients may also develop adrenal cortical tumours, carcinoids, facial angiofibromas, collagenomas and lipomas  . PatientPla2s) gene is a major modifier of the adenomatous polyposis coli (APC) gene, such that its absence is associated with increased numbers of intestinal polyps in APC mutant mice that are on a C57BL/6J background, which are null for Pla2s activity, when compared to the APC mutant mice on MA/MyJ or Mus castaneus (CAST) backgrounds that highly express Pla2s  (-/-Tgfb1), which have vascular defects similar to those in patients with hereditary haemorrhagic telangiectasia, bred on NIH/Ola and C57BL/6J/Ola backgrounds revealed the presence of a major codominant modifier gene for embryonic lethality on mouse proximal chromosome 7 (M1HSCCftr/M1HSCCftr), which usually died of intestinal obstruction similar to that observed in patients with cystic fibrosis, bred on C57BL/6J and BALB/cJ backgrounds, revealed a modifier on mouse chromosome 7 that was associated with prolonged survival, likely due to a partial rectification of the Na+ and Cl- transport abnormalities (-/-) which develop multiple tumours similar to those in patients with the Li-Fraumeni syndrome , bred on CE/J and 129/Sv backgrounds revealed the presence of a modifier for embryonic lethality on mouse chromosome 11  . Furthermosome 7 ; cystic malities ; and p53osome 11 .-/-Men1 mouse embryos  was applied, followed by counterstaining with 1% eosin and permanent mounting (Vector Laboratories mounting media), as previously described . For immhttp://pantherdb.org) (DNA was extracted from ear biopsies of C57BL/6J and 129S6/SvEv mice using the Blood Core II kit (Qiagen) and used to generate a library for whole genome sequencing (WGS) that ustilised the Illumina HiSeq platform at the Oxford Genomics Centre , and single nucleotide variants called, as previously described . The genrdb.org) .Pituitary, ovary and testes tissues were harvested and placed immediately into RNAlater solution (Life Technologies). Pancreatic islets were picked from pancreatic tissue digested following direct intra pancreatic injection of 2 mL of 0.2 mg/mL Liberase (Roche) and placed immediately into RNAlater solution . Total RMouse pituitaries were lysed in NP40 lysis buffer and prepared in 4× Laemmli loading dye, as previously described . Samples+/-Men1 mice with tumours, including tumour subtypes, was compared between two different genetic backgrounds by performing a Fisher’s exact test, with the threshold for statistical significance set at P < 0.05. A Bonferroni correction was applied to account for multiple tests. To evaluate the trend of tumour development, a Chi squared test for trends was applied. For all other analyses one-way ANOVA or t-tests were used, as previously described  on 129S6/SvEv (n = 1166) and C57BL/6 (n = 1192) genetic background strains and backcross generations 10–18 were produced. There were no significant differences in the live births or survival to age 18–21 months between the +/-Men1 or +/+Men1 mice or between the background strains  vs +/-Men1 ; and survival: +/+Men1  vs +/-Men1  (Supplementary Tables 1 and 2)). From these, a total of 275 +/-Men1 mice from 129S6/SvEv (n = 137) and C57BL/6 (n = 138) genetic background strains, aged 5–26 months, were macroscopically assessed for the prevalence of endocrine tumours in the pancreas, pituitary, adrenals and gonads. This age range was chosen as our previous study indicated that mice develop a significant number of tumours after 12 months of age, although tumours can be identified as early as 3 months of age , when compared to 129S6/SvEv +/-Men1 mice; pancreatic tumours occurring significantly earlier in male 129S6/SvEv than in C57BL/6 +/-Men1 mice (P < 0.05); adrenal tumours occurring significantly earlier in female 129S6/SvEv +/-Men1 mice than in C57BL/6 +/-Men1 mice (P < 0.005) and testicular tumours occurring significantly earlier in male 129S6/SvEv +/-Men1 than in C57BL/6 +/-Men1 mice (P < 0.005)  . The occ+/-Men1 mice at necropsy, from the age of 13 months in the 129S6/SvEv strain and from 15 months of age in the C57BL/6 strain (n = 4) from C57BL/6 +/-Men1 and 129S6/SvEv +/-Men1 mice and expression of prolactin, growth hormone and chromogranin A   (+/-Men1 mice between the two strains (Pituitary tumours were observed in the 6 strain . Histologranin A . The ant 0.0001) . In addi 0.0001) , whereas strains .Figure +/-Men1mice  and 15 from C57BL/6  +/-Men1mice were analysed by histology and immunohistochemistry, which confirmed loss of menin expression, but presence of chromogranin A expression   of the total PNETs, whereas glucagon-expressing tumours of C57BL/6 +/-Men1 mice accounted for only 2% (1/43) of all PNETs (P < 0.0001).Pancreatic neuroendocrine tumours (PNETs) were the most frequent tumour type observed at necropsy and were detected in >85% of 129S6/SvEv and C57BL/6 1+/-mice . The occ1+/-mice . A totalpression . This anglucagon , and one vs 93%) . However+/-Men1 mice, respectively, but from 19 and 18 months of age in male and female C57BL/6 +/-Men1 mice, respectively, thereby indicating a significant earlier age for development of adrenal tumours in the 129S6/SvEv +/-Men1 mice than the C57BL/6 +/-Men1 mice  indicated these tumours to arise from the adrenal cortex and confirmed loss of menin expression ) and dystrophia myotonica protein kinase ) genes, which were present in 129S6SvEv +/-Men1 mice but not C57BL/6 +/-Men1 mice. However, assessment of evolutionary conservation across mouse and human genomes revealed that the location of the variant in Coq7 only occurs in one transcript that is not present in humans and that the C terminal of the encoded protein from the mouse Dmpk transcript (547–582) only shares 14% identity with the human protein. This lack of conservation indicates that these variants are unlikely to be genetic modifiers of the MEN1 gene. In addition to the nonsense variants, 285 missense variants, 217 5′ UTR variants, 123 splice region variants and 1287 3′ UTR variants were identified within 216 different genes. PANTHER analysis was used to identify the functional classification of these genes, and this indicated that variants were present in many genes within tumour-associated cellular pathways including: cell cycle regulation (cyclin E2 (Ccne2)); Ras signalling ); wnt signalling (wingless-type MMTV integration site family member 2B (Wnt2b)); interleukin signalling ) and apoptosis signalling (TNF receptor superfamily member 10a (Tnfrsf10a)) (Supplementary Table 3). These, together with Coq7 and Dmpk, were therefore selected for further study. The expression of these genes was evaluated by qRT-PCR in pituitary, pancreatic islets and gonadal tissues from +/+Men1 C57BL/6 and 129S6/SvEv mice. This did not reveal significant differences in the expression of any of these genes in the pancreatic islets  is consistent with a previous C57BL/6 mouse study (77.8%)   . Our res (77.8%) , 29. ThuTable 4) , 31.Men1 knockout mouse model produced on a mixed genetic background that included the C57BL/6 mouse strain have reported that mice developed both glucagonomas and insulinomas  may help to clarify this. Another possibility is that the genetic background and any modifier genes may instead influence the cell types giving rise to the PNETs, and this may explain the absence of significant differences in the number of pancreatic tumours that occur at any age in the two mouse strains  variant (c.326T>G) and tumour multiplicity in MEN1 patients has been reported (Cdkn1b (encoding p27kip1) in our WGS data, we did observe variants in the cell cycle regulator, Ccne2, that is regulated by p27kip1. Ccne2 encodes cyclin E2 which, when in complex with cyclin dependent kinase 2 (Cdk2), is inhibited by p27kip1, and it has been demonstrated that cyclin E-Cdk2 substrates may vary in different cell types  signalling pathway by menin  programme grants G9825289 and G1000462  and a Wellcome Trust Clinical Research Training Fellowship grant 087332/Z/08/Z to M J. R V T is a Wellcome Trust Investigator.


A line of evidence has shown that ATP is released from many different types of cells including neurons, endothelial cells, and muscle cells. In this review, we described the distribution of P2 receptor subtypes in the cardiac cells and their physiological and pathological roles in the heart. So far, the effects of external application of ATP or its analogues, and those of UTP on cardiac contractility and rhythm have been reported. In addition, specific genetic alterations and pharmacological agonists and antagonists have been adopted to discover specific roles of P2 receptor subtypes including P2X4-, P2X7-, P2Y2- and P2Y6-receptors in cardiac cells under physiological and pathological conditions. Accumulated data suggest that P2X4 receptors may play a beneficial role in cardiac muscle function, and that P2Y2- and P2Y6-receptors can induce cardiac fibrosis. Recent evidence further demonstrates P2Y1 receptor and P2X4 receptor as important mechanical signaling molecules to alter membrane potential and Ca ATP is now widely accepted as a key extracellular chemical messenger released from many cell types including neuronal cells, endothelial cells, muscle cells, and it significantly regulates different cell functions via P2 purinergic receptors . Extrace2+ increase that are activated by ATP [− channel [Early studies have demonstrated ATP exocytosis using new bioluminescence methods with cell surface attached firefly luciferase ,5, or atd by ATP ,11,12,13d by ATP ,11,14 and by ATP , cystic d by ATP ,19,20,21 channel , and exo channel ,24. The  channel ,26. A li channel . In the  channel . In addi channel ,30,31, c channel ,33,34,35 channel ,37,38, s channel ,39,40, p channel ,42,43, a2+ waves [2+ waves depend on P2 receptor subtypes , mainly2+ waves . It has e below; ,11). Atre below; .+, K+, and Ca2+ across the cell membrane -β followed by inositol 1,4,5-trisphosphate (IP3) generation from phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ mobilization from intracellular stores [i proteins that inhibit adenylate cyclase followed by a reduction of cAMP level in the cytosol [The P2Y receptors are G-protein coupled receptors, which, in turn, activates intracellular second messenger systems to modulate the physiological function of the cells. In addition to ATP and ADP, P2Y receptors bind to the pyrimidines UTP and UDP. A group of P2Y receptor subtypes  are metabotropic receptors mainly coupled with Gr stores ,66. In pr stores . Remaini cytosol ,67.Studies have demonstrated several subtypes of P2X receptors in the heart ,68,69,7050 to P2X3-, P2X5-, and P2X6-receptors. However, they have higher EC50 compared with P2X1- and P2X3-receptors. P2X4 receptor has 1 magnitude lower EC50 for ATP compared with P2X7 receptor. Unique property of P2X4 receptor is its resistance to suramin and PPADS, the well-known P2 receptor antagonists [The P2X4 receptor is structurally similar to others in the P2X receptor family and binds to ATP with similar ECagonists ,54. In aagonists . This suagonists . This P2Abundant P2Y receptor subtypes expressed in cardiac tissues include P2Y receptor-1, -2, and -6 . P2Y1 reIt has been found that there are differences in the expression profiles of P2Y receptor subtypes within the heart and among the species. It has been reported by Musa et al. (2009)  that theIn the heart, extracellular ATP exerts both negative and positive inotropic effects (For review see ). Extrac2+ current and Ca2+ transient [There are some controversies among the previous reports on the role of P2X receptor on ATP-mediated negative or positive inotropy among the species and heart regions . In rat ransient . The ATPransient ,150. In ransient . In miceransient . In addiransient . Consistransient .+ channels (KACh) [2+ concentrations. In these cells, Ju et al. (2003) [2+ transient amplitudes, which is followed by decrease in the rate of beating [2+ increase by 2-MeS-ADP in toad SAN cells seems to be similar to caffeine-induced Ca2+ release. The secondary negative chronotropy by ATP in these cells has been suggested to be associated with partial sarcoplasmic reticulum (SR) Ca2+ depletion [In clinic, ATP is used to treat supraventricular arrhythmias mainly in children, because adenosine degraded from ATP in serum activates P1 receptors . In frogs (KACh) ,153. How. (2003)  have sho beating . They al beating  or by thepletion .Xenopus oocytes have provided evidence that activation of P2X4 receptors leads to permeation of various cations (mainly Na+) through the cell membrane [+-Ca2+ exchanger (NCX) by activation of P2X4 receptor similar to the action of digitalis via Na+-K+ pump inhibition has been suggested to suppress SAN beating rate [In SAN-containing beating atrial strip from rat, ATP has decreased heart rate and contractility . The negmembrane ,158. Theing rate . Further2+ channel [+ through P2X4 receptors can increase intracellular Ca2+ concentration via affecting the NCX [2+ transient and SR Ca2+ loading, of which effects were larger in P2X4 receptor transgenic myocytes, providing a mechanism for P2X4 receptor-mediated increase in contractility in this mice ventricle [Extracellular ATP has been thought to have beneficial effects on the heart via its metabolic product adenosine ,159,160. channel . It has  the NCX . In factentricle .It has been shown that the P2X4 receptor knock-out mice develop worse heart failure phenotype after coronary artery ligation or pressure overload by transverse aortic constriction, such that it depresses contractile function faster and more significantly in pressure overload or MI-induced heart failure in mice . The carAnother pathway of beneficial effect exerted by ATP itself is P2X7 receptor. It has been reported that activation of P2X7 receptors by ATP can also protect cardiac muscle under ischemia-reperfusion injury in the heart. The cardiac ischemia-reperfusion injury is prevented with appropriate treatments initiated either before (preconditioning) or immediately after (postconditioning) the index ischemia. Ischemia preconditioning or postconditioning has been shown to induce release of endogenous cardioprotectants from cardiomyocytes via the opening of a channel formed by the interaction of a P2X7 receptor with a pannexin 1 hemichannel ,162. It In mouse atrial myocytes, it has been shown that caveolin 1 and 3 are co-localized with PX7 receptors . The absANP, MLC-2) expression in the norepinephrine- or phenylephrine-treated cells [3Rs localized in the perinuclear region [3R1 is not expressed in ventricular myocytes. These previous findings suggest distinct ATP signaling and/or receptor context and distinct role of ATP in hypertrophic growth of atrial and ventricular myocytes.Prolonged exposure of cardiac muscle to neurohumoral factors, such as norepinephrine and endothelin-1, induces muscle hypertrophy ,168,169.ed cells . The UTPed cells . Interes12,13 signaling has been shown to mediate pressure-overload induced cardiac fibrosis [12 family G protein (Gα12/13) in cardiomyocytes suppressed pressure overload-induced fibrosis without affecting hypertrophy. The mRNA for P2Y6 receptors increases in pressure-overloaded mice having decreased ejection fraction and Gα12,13 signaling [13 and stimulated by upstream ATP and UTP releases through pannexin-1 in this pressure-overloaded mice ventricles [It has been suggested that inhibition of P2Y2 receptors may diminish fibrotic remodeling and turnover of extracellular matrix in the heart, because the nucleotide, UTP, induces a profibrotic response via P2Y2 receptor in cardiac fibroblast . P2Y6 refibrosis . Transgeignaling . This sintricles . Regardintricles .2+ and subsequent production of the potent vasodilator NO. Blood vessel dilation induced by acute increases in blood flow is markedly suppressed in P2X4 receptor knock-out mice.Pressure overload and volume overload in the heart are associated with cardiac myocyte remodeling and dysfunction, leading to arrhythmogeneis and failure. Such mechanical forces are clinically related to hypertension, heart failure, and valvular heart diseases and include stretch, shear stress, and afterload increase. Enlarged cardiac chamber has been observed under heart failure and stretch signaling has been thought to play an important role in the pathogenesis of such congestive heart failure and subsequent arrhythmias ,173. Str2+ waves. In fact, the same shear force elicits action potential (AP)-involved transverse Ca2+ wave in most of RA myocytes, but it induces slow longitudinal Ca2+ wave in a majority of LA myocytes [2+ waves in atrial myocytes have been discovered to be dependent on distinctly distributed P2 receptor subtypes between LA myocytes and RA myocytes [2+ wave in LA myocytes under shear stress has been known to be due to activation of P2Y1 receptor-PLC-IP3R type 2 signaling with subsequent Ca2+-induced Ca2+ release via ryanodine receptor type 2 (RyR2)  (. The P2Yrection (; Figure rection (,176, sucirection ,178,179.2+ signaling with shear stress. UTP signaling appears to be involved in cardiac remodeling and fibrosis via P2Y6 receptor, and also modulate atrial contraction although there are still contradictory findings with regard to P2Y6 receptor. Accumulated findings strongly suggest that ATP release and subsequent activation of P2 receptors are major signaling pathway activated by mechanical stimulus in the ventricular and atrial muscles, and that different responses between differnt sides of cardiac chamber can be achived by adopting distict P2X- or P2Y-receptor subtypes. In the pathologic hearts a signaling between ATP release pathway and purinergic receptor activation may occur in a compartmentalized microdomains in cardiac myocytes more significantly because of larger mechanical stresses and they could play a role in myocytes remodeling, functional alterations, and fibrosis. Role of P2 receptor subtypes in different cardiac pathogenesis with distict environmental changes needs to be further discovered considering cardiac regions.Cardiac myocytes express several types of P2X- and P2Y-receptor subtypes and themselves release ATP under various stimuli. So far, the effects of ATP or UTP on cardiac contraction and rhythm have been studied. There is some consensus on the role of P2X4 receptors in positive inotropy in ventricular tissue and negative chronotropy in beating atrial tissue based on a line of evidence with pharmacological and genetic interventions. A role of P2Y1 receptor subtype, one abundant P2Y receptor in cardiac myocytes, has been found in the positive regualtion of rhythm in SAN preparation and in pathologic alteration in LA cell Ca


Breast cancer (BC) is a heterogeneous disease consisting of various subtypes, with different prognostic and therapeutic outcomes. The amino acid transporter, SLC7A8, is overexpressed in oestrogen receptor-positive BC. However, the consequence of this overexpression, in terms of disease prognosis, is still obscure. This study aimed to evaluate the biological and prognostic value of SLC7A8 in BC with emphasis on the intrinsic molecular subtypes.n = 1980), and proteomic, using immunohistochemistry and TMA (n = 1562), levels in well-characterised primary BC cohorts. SLC7A8 expression was examined with clinicopathological parameters, molecular subtypes, and patient outcome.SLC7A8 was assessed at the genomic, using METABRIC data  . Expression of SLC7A8 mRNA was higher in luminal tumours compared to other subtypes (P < 0.001). High expression of SLC7A8 mRNA and SLC7A8 protein was associated with good patient outcome (P ≤ 0.001) but only in the low proliferative ER+/luminal A tumours (P = 0.01). In multivariate analysis, SLC7A8 mRNA and SLC7A8 protein were independent factors for longer breast cancer specific survival (P = 0.01 and P = 0.03), respectively.SLC7A8 appears to play a role in BC and is a marker for favourable prognosis in the most predominant, ER+ low proliferative/luminal A, BC subtype. Functional assessment is necessary to reveal the specific role played by SLC7A8 in ER+ BC.The online version of this article (10.1007/s10549-020-05586-6) contains supplementary material, which is available to authorised users. Many cancer cells alter their metabolism to provide energy and cellular building blocks required for their rapid proliferation. Amino acids, particularly glutamine and leucine, are essential for cancer cell growth, as they are critical for controlling protein translation and driving cell cycle progression through regulation of the mammalian target of rapamycin complex1 (mTORC1) pathway . This piSolute Carrier Family 7 Member 8 (SLC7A8) and Member 5 (SLC7A5) are sodium-independent amino acid exchangers (antiports), which transport small and large neutral amino acids, such as alanine, serine, threonine, cysteine, phenylalanine, tyrosine, leucine, and glutamine . Both soAlthough the function of SLC7A8 and SLC7A5 are similar, the former displays relatively lower affinity for its substrates, glutamine, and serine. SLC7A5 has been extensively studied in a variety of cancers and it is regulated by the oncogene c-MYC –13. We hSLC7A8 gene copy number (CN) and mRNA expression, alongside SLC7A8 protein expression in large and well-characterised cohorts of BC to determine its clinicopathological and prognostic value with emphasis on the different molecular classes.In this study, we aimed to assess SLC7A8 gene copy number and gene expression were evaluated using the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort of invasive BC (n = 1980) [SLC7A8 mRNA was achieved using X-tile , based on prediction of Breast Cancer Specific Survival (BCSS). SLC7A8 mRNA expression was associated with clinicopathological parameters, molecular subtypes and patient outcome. = 1980) . In this = 1980) . Patienthttps://bcgenex.centregauducheau.fr), was used for external validation of SLC7A8 mRNA expression.The online dataset, Breast Cancer Gene-Expression Miner v4.0  and BCSS.Supplementary Table 1 summarises the clinicopathological parameters for the Nottingham and METABRIC series.The specificity of anti-SLC7A8 primary antibody  was validated using Western blotting in BC lysates  as previously described . A singlTumour samples, 0.6 mm cores, were arrayed as previously described , 19. ImmStained TMA sections were scanned using high resolution digital images , at × 20 magnification. Modified histochemical score (H-score) was applied to evaluate SLC7A8 immunostaining. This includes a semi-quantitative assessment of both the percentage and the intensity of stained cells [Immunhistochemical staining and dichotomisation of the other biomarkers included in this study were as per previous publications , 18–22. P values were adjusted using Bonferroni correction for multiple testing, whenever applicable. A P value < 0.05 was considered significant. The study endpoints were 10-year BCSS or distant metastasis free survival (DMFS). This study complied with reporting recommendations for tumour marker prognostic studies (REMARK) criteria [SPSS 24.0 statistical software  was applied for statistical analysis. The Chi-square test was carried out for inter-relationships between categorical variables. One-way ANOVA with post hoc Tukey multiple comparison test and Pearson’s correlation coefficient was performed to analyse the association between continuous variables. Survival curves were examined by Kaplan–Meier with Log Rank test. Cox’s proportional hazard method was performed for multivariate analysis to identify the independent prognostic/predictive factors. criteria .This study was approved by the Nottingham Research Ethics Committee 2 under the title ‘Development of a molecular genetic classification of breast cancer’ and the North West—Greater Manchester Central Research Ethics Committee under the title ‘Nottingham Health Science Biobank (NHSB)’ reference number 15/NW/0685.SLC7A8 mRNA expression was observed in almost two-third (67%) of the METABRIC BC cases. A total of 90/1,980 (4.5%) of cases showed SLC7A8 copy number (CN) gain, whereas 45/1,980 (2.3%) cases showed a CN loss. A significant association was observed between SLC7A8 copy number variation (CNV) and SLC7A8 mRNA expression .High SLC7A8 protein expression was observed, predominantly in the cytoplasm of invasive BC cells, with expression levels varying from absent to high Fig. a and b. SLC7A8 mRNA expression was significantly associated with good prognostic parameters, including smaller tumour size , lower tumour grade , and good Nottingham Prognostic Index (NPI) . These associations were confirmed using the Breast Cancer Gene-Expression Miner .High P = 0.03), low tumour grade (P < 0.001), and good NPI (P < 0.001).Similar associations were observed with SLC7A8 protein expression. Table SLC7A8 mRNA was significantly associated with hormone receptor positive (ER+ and PgR+) and HER2− BC  compared with TN tumours  and although it was expressed primarily in hormone receptor positive tumours this did not reach significance (Table High expression of SLC7A8 CN and mRNA with the intrinsic (PAM50) subtypes, SLC7A8 CN gain was mainly observed in luminal B tumours , whereas high mRNA expression was observed primarily in luminal A and B tumours and to lesser extent in HER2+BC .Regarding the association of Although the result of the association of SLC7A8 protein in the defined IHC subtypes were not nominally significant, it also showed higher SLC7A8 expression in the ER+ low proliferation tumours compared with the other subtypes Table .SLC7A8 mRNA with other relevant genes were investigated using the METABRIC dataset , which mediate the conversion of glutamine to glutamate. While the correlation with GLS was negative, it was positive with GLS2. There was also a positive association with the enzymes which mediate conversion of glutamine to proline, namely, ALDH4A1 and PRODH (P < 0.001). In contrast, some glutamine transporters were negatively correlated with SLC7A8 expression (P ≤ 0.009), including SLC1A5, SLC7A5, SLC7A6, SLC7A7, and SLC38A3, while others showed a positive association, including SLC7A9, SLC38A1, SLC38A2, and SLC38A7 (P ≤ 0.003). The associations between SLC7A8 and glutamine metabolic enzymes and transporters were primarily observed within luminal A tumours and to lesser extent in luminal B, HER2+ and TN subtypes. High SLC7A8 mRNA expression was associated with tumours which showed wild-type TP53 expression  within the ER+ low proliferation class  but not with other subtypes . The relationship between high SLC7A8 mRNA expression and good patient outcome was verified using Breast Cancer Gene-Expression Miner .Likewise, high SLC7A8 protein expression was associated with longer distant metastases-free survival (DMFS)  which are associated with tumours of good prognosis and favourable patient outcome , 41. SomIt is noteworthy that different transcriptional pathways such as c-MYC oncogenic transcription, hormone receptors and nutrient starvation responses regulate the expression of amino acid transporters in human cancers , 35. ThiThis study has revealed that the solute carrier SLC7A8 is an independent good prognostic marker in BC. Overexpression of SLC7A8 appears to have tumour suppressive characteristics especially in the low proliferative ER+ subtype, thus it could act as a potential prognostic factor. Functional assessment is necessary to reveal the specific role played by this amino acid transporter in the low proliferative ER+ tumours.Supplementary file1 (DOCX 1064 kb)Below is the link to the electronic supplementary material.


The parity of the buffaloes ranged from 4 to 8 with an average 5.8±0.2. Subclinical endometritis was diagnosed by the percentage of polymorphonuclear cells (PMNs) in uterine cytology obtained from endometrial cytobrush at W5 and W7. The cut-off point of PMNs% in buffaloes for SCE was ≥ 6% at W5 or ≥ 4% at W7. According to PMNs%, buffaloes were divided into SCE group (n=27) and non-SCE group (n=12). Ovarian cyclicity was monitored by rectal palpation, ultrasonography and progesterone assay at W3, W5 and W7. Serum and endometrial TNF-α, IL-8 and serum CRP were estimated at W5 and W7. Buffaloes with SCE (55.6%) showed delayed ovarian activity as compared to non-SCE (16.7%) animals (P=0.036). Significant increase in serum cytokines and CRP levels were detected at W5 (P ˂0.05) and W7 (P <0.01) in SCE buffaloes as compared to non-SCE. Endometrial levels of cytokines were significantly (P ˂0.05) elevated in SCE buffaloes. Serum and endometrial cytokines showed significant positive correlation. Furthermore, levels of TNF-α, IL-8 and CRP exhibited significant positive correlation with PMNs%. In conclusion, SCE delayed postpartum ovarian cyclicity in buffaloes. Moreover, TNF-α, IL-8 and CRP assessments could be efficient tools in prediction of SCE in buffaloes.The study was carried out to find the relation between subclinical endometritis (SCE) and postpartum (pp.) ovarian resumption as well as to evaluate serum and endometrial TNF-α, IL-8 and serum CRP in buffaloes with and without SCE. Thirty-nine pluriparous buffaloes at the 3 Identification and recognition of biomarkers help in early diagnosis and assessment of the animal’s pathophysiological condition of some diseases . Among tUterine infection altered endometrial and postpartum (pp.) ovarian functions  that subImpairment of peri-partum immune function is a causal factor for the progress of pp. uterine infections in bovines . Innate Pro-inflammatory cytokines from monocytes and macrophages are effective stimulators for the production of acute phase proteins (APPs) such as C-reactive protein (CRP), haptoglobin and serum amyloid A . Acute pDiagnosis of SCE depends mainly on histological examination and counting of PMNs in endometrial cytology . HoweverPluriparous postpartum buffaloes in Educational Farm of Suez Canal University, Ismailia, Egypt were used. Buffaloes were examined regularly by farm veterinarians. All the used buffaloes were apparently healthy. They were free from mastitis, general health diseases and reproductive disorders. Before beginning of the study, buffaloes with clinical endometritis (n=7) were excluded after vaginoscopic examination and these animals were not used in the experiment.Days in milk (DIM) of the previous lactation period was calculated for the studied animals. Buffaloes calved in winter and spring seasons. The mean parity of these buffaloes was 5.8 ± 0.2 (ranged from 4 to 8). Animals without clinical endometritis (n=39) were used throughout the study. Buffaloes were followed idealistic management practices and settled freely in an open yard. The study followed research ethical committee of the faculty guidelines (protocol # 2018056).rd (W3), 5th (W5) and 7th (W7) pp. weeks. They were collected from jugular vein in plain tubes and transported on ice to the laboratory. Sera were obtained by cooled centrifugation of blood samples at 3000 rpm for 15 minutes. The harvested sera were stored at -20 °C until analysis.Blood samples (n=117) were collected at the 3At W5 and W7 pp., duplicate of aseptic endometrial cytology were collected from each buffalo using autoclaved cytobrush  according to Fixed endometrial cytological slides were transferred to the laboratory within an hour to be stained by Diff-Quik stain  according to the manufacturer’s instructions. Assessment of uterine cytology was performed in order to determine PMNs% via counting 200 cells using light microscope (X400). Based on the percentage of PMNs , buffaloes (n=39) were categorized into SCE and non-SCE groups .The cytobrush was carefully weighed before collection of endometrial sample and after scratching of endometrium. The difference between the two weights was considered the actual weight of the endometrial scrape which was diluted by 10% (w/v) cold PBS (pH= 7.4) centrifuged at 3000 rpm for 20 minutes at 4 °C . The sup4) were performed at W3, W5 and W7 for monitoring of ovarian resumption. Serum P4 was assessed by bovine progesterone ELISA kit . Buffaloes with P4 levels ≥1.0 ng/mL were considered to have luteal activity  as well as assessment of serum progesterone (Pactivity . While t.0 ng/mL . The ani.0 ng/mL .Bovine enzyme-linked immunosorbent assay (ELISA) kits were used for assessment of TNF-α and IL-8  at W5 and W7 in sera and endometrial samples. Briefly, pre-coated microplate with specific bovine antibody conjugates for either TNF-α or IL-8 were employed for competitive inhibition enzyme immunoassay technique. Both of standards and samples were added to the microtiter plate wells. A competitive inhibition reaction was launched with Horseradish Peroxidase (HRP) labeled target antigen and unlabeled antigen with the antibody. Substrate color development was inversely proportional to the amount of cytokines in the sample. The reaction was stopped and the color intensity was measured by ELISA reader . Serum CRP was evaluated by using bovine CRP ELISA kit  at W5 and W7 according to the manufacturer’s guidelines.4 levels in W3, W5 and W7 as well as DIM on PMNs% (as indicator for SCE).The obtained data are presented as means with SEM. Significance was designated at probability value of P < 0.05.Differences in serum and endometrial levels of TNF-α, IL-8 and serum CRP between SCE and non-SCE groups at W5 and W7 were assessed by Mann-Whitney nonparametric analysis. The PMNs% and parity of SCE subgroups and non-SCE buffaloes were analyzed by nonparametric Kruskal-Wallis test. Further comparisons among different groups were done by Dunn’s test for multiple comparisons. The prevalence of normal and delayed ovarian cyclicity at W7 pp. in buffaloes with and without subclinical endometritis was analyzed by Fisher’s exact test. The correlation coefficient between serum and endometrial TNF-α, IL-8 and serum CRP and their correlation with PMNs% were analyzed by Spearman’s test . Logistic regression analysis  was applied to evaluate the effect of calving season, serum PAccording to PMNs %, buffaloes (n=39) were divided into SCE group (n=27) and non-SCE group (n=12). Twelve out of 39 buffaloes did not exhibit SCE neither at W5 nor W7, while 3 exhibited SCE at W5 not W7, six exhibited SCE at W7 not W5 and the remaining 18 exhibited SCE at both W5 and W7 .P > 0.05) were recorded in parity between the two groups of buffaloes. At W5, SCE W5 group as well as SCE W5 and W7 group showed significant (P< 0.001) elevation in PMNs% as compared to non-SCE and SCE W7 groups. While at W7, SCE W7 and both SCE W5 and W7 groups showed significant (P < 0.001) increase in PMNs% compared to that in those without SCE and SCE W5 buffaloes  in buffaloes; 10 (83.3%) out of 12 non-SCE buffaloes, 2 (66.7%) out of 3 SCE buffaloes at W5, 3 (50.0%) out of 6 SCE buffaloes at W7 and 7 (38.9%) out of 18 SCE buffaloes at W5 and W7 were assumed to resume the luteal activity (Based on serum Pactivity .P = 0.036) delay in resumption of pp. luteal activity compared with that in non-SCE buffaloes at W7  and IL-8 (P = 0.036) in SCE buffaloes when compared to that in non-SCE group at W5. The same trend was observed (P= 0.009) in all parameters at W7 (P < 0.001) and at W7 (P < 0.01) compared to non-SCE buffaloes  elevations in SCE group as compared to non-SCE group at W5 and W7, respectively  and at W7  as illustrated in Serum TNF-α and IL-8 were positively correlated to their respective endometrial levels at W5 (P > 0.05) with serum and endometrial TNF-α, IL-8 and serum CRP levels at W5 and W7. While, PMNs% of SCE group at W5 showed significant correlations with both serum and endometrial levels of TNF-α (P< 0.05), IL-8 (P < 0.01) and serum CRP (P < 0.05). Moreover, SCE buffaloes revealed significant correlations in cytological PMN% at W7 with serum and endometrium TNF-α  and IL-8  as well as serum CRP (P < 0.01)  .4 levels at W3, W5 and W7 as well as DIM on PMNs%  that observed in our study could be attributed to the high liability of buffaloes to uterine infections due to the poor hygienic conditions in the rearing areas. Moreover, buffaloes behave improper practices as vaginal stimulation for milk let-down as well as their wallowing habit .Some buffaloes were positive for SCE at W5 but not at W7. This might be explained by the capability of uterus to eliminate the infection. On the other side, the buffaloes that were negative to SCE at W5 but acquired it at W7. This could be attributed to the lowered immune response of endometrium postpartum that was well consolidated in cattle as recorded by 4 that adversely influence the resumption of ovarian cyclicity. Genital infections had deleterious effects on bovines' reproductive pattern through suppressing secretion of pituitary LH as well as outfaces postpartum ovulation (4 in SCE buffaloes. Endometrial cytokines which produced during uterine inflammation could obscure ovulation due to low concentrations of plasma estradiol and slow growth of the dominant follicle as well as reducing P4 secretion (4 and hence gonadotropin supported P4 production. This led to delayed resumption of ovarian activity in SCE buffaloes where uterine and serum TNF-α were elevated (4 production (The delay of postpartum resumption of ovarian cyclicity in SCE group was in accordance with vulation . Our stuecretion . Moreoveecretion  to enhanecretion  and endoecretion  thus inhelevated . Actualloduction . On the After parturition, the physiological barrier of the genital system becomes loosen and the pathogenic opportunistic microorganisms invade the uterus. The endometrial immunity responds to bacterial agents via production of pro-inflammatory cytokines  which coThe observed elevation in IL-8 levels augmented the elevation of TNF-α that induced its production and further uterine PMNs chemotaxis . SimilarThe congruent positive correlation between PMNs% and serum as well as endometrial levels of TNF-α and IL-8 in SCE buffaloes invigorated the permeation of neutrophils into uterus for withdrawal of the pathogenic micro-organisms . This coC-reactive protein is an essential element of innate system defense that regulates the inflammatory response and acts as a safeguard against infection . MoreoveIn conclusion, SCE was implicated in delay of postpartum ovarian resumption in Egyptian buffaloes. The elevated concentrations of endometrial TNF-α and IL-8 could be reflected on their serum levels that might have a diagnostic importance for SCE in Egyptian buffaloes. Hence, the complications met by field conditions to obtain endometrial sample for SCE diagnosis, the serum levels of TNF-α, IL-8 and CRP could introduce a logic alternate to diagnose SCE in buffaloes.