Since the end of 2009, H9N2 has emerged in Tunisia causing several epidemics in poultry industry resulting in major economic losses. To monitor variations of Influenza viruses during the outbreaks, Tunisian H9N2 virus isolates were identified and genetically characterized.The genomic RNA segments of Tunisian H9N2 strains were subjected to RT-PCR amplifications followed by sequencing analysis.333 PARSSR*GLF341 which indicates the low pathogenicity nature of the Tunisian H9N2 strains and the potential to acquire the basic amino acids required for the highly pathogenic strains. Their neuraminidase protein (NA) carried substitutions in the hemadsorption (HB) site, similar to those of other avian H9N2 viruses from Asia, Middle Eastern and human pandemic H2N2 and H3N2 that bind to α -2, 6 -linked receptors. Two avian virus-like aa at positions 661 (A) and 702 (K), similar to H5N1 strains, were identified in the polymerase (PB2) protein. Likewise, matrix (M) protein carried some substitutions which are linked with increasing replication in mammals. In addition, H9N2 strain recently circulating carried new polymorphism, "GSEV" PDZ ligand (PL) C-terminal motif in its non structural (NS) protein.Phylogenetic analysis demonstrated that A/Ck/TUN/12/10 and A/Migratory Bird/TUN/51/10 viruses represent multiple reassortant lineages, with genes coming from Middle East strains, and share the common ancestor Qa/HK/G1/97 isolate which has contributed internal genes of H5N1 virus circulating in Asia. Some of the internal genes seemed to have undergone broad reassortments with other influenza subtypes. Deduced amino acid sequences of the hemagglutinin (HA) gene showed the presence of additional glycosylation site and Leu at position 234 indicating to binding preference to α  sialic acid receptors, indicating their potential to directly infect humans. The Hemagglutinin cleavage site motif sequence is Two new aa substitutions (I) and (V), that haven't been previously reported, were identified in the polymerase and matrix proteins, respectively. Nucleoprotein and non-structural protein carried some substitutions similar to H5N1 strains.Considering these new mutations, the molecular basis of tropism, host responses and enhanced virulence will be defined and studied. Otherwise, Continuous monitoring of viral genetic changes throughout the year is warranted to monitor variations of Influenza viruses in the field. Orthomyxoviridae; containing negative sense single stranded RNA [Avian Influenza virus (AIV) is a member of the family nded RNA . Based onded RNA . AIV subnded RNA ,4.Staphylococcus aureus, Avibacterium paragallinarum, Escherichia coli, or immune suppression [By 1997, H9N2 viruses have been isolated in multiple avian species including chickens, ducks, turkeys, quail, geese and pigeons, throughout Asia, the Middle East, Europe and Africa and for the first time from humans in Hong Kong and China, in 1999 -7. They pression .Two distinct lineages of H9N2 influenza viruses have been defined, the North American lineage and the Eurasian lineages which consists of at least three sublineages represented by their prototype strains: A/quail/Hong Kong/G1/97 (G1-like), A/duck/HongKong/Y280/9 (Y280-like), A/Chicken/Beijing/1/94 (BJ94-like), and A/chicken/Korea/38349-P96323/96 (Korean-like) ,12. The In addition, some of these currently circulating H9N2 strains have acquired human-like receptor specificity   and therSince the end of 2009, H9N2 has emerged in Tunisia causing several epidemics in poultry industry resulting in major economic losses. It should be noted that Tunisia was reportedly free from Avian Influenza infection .In the present study, we report the isolation of two Tunisian H9N2 influenza viruses that were identified, and their genome sequences were analyzed. Phylogenetic analysis characterization was performed in comparison to H9N2 sequences available in the Genbank database.2  and 17, 5 μl of H2O RNase free . The PCR program was 94°C for 2 min, 35 cycles of 94°C for 45 s, 50°C for 45 s [°C for HA1, NA, PB2, and NS) [°C for 1 min followed by 72°C for 10 min. PCR products were purified with gel DNA purification kit .Fecal and tissue samples were collected from affected commercial poultry and from migratory bird during the initial outbreak. H9N2 avian influenza viruses were isolated and identified using classical laboratory methods . The isofor 45 s -18 (58°C and NS) ,20, 72°CABI Big Dye Terminator version 1.1 sequencing Kit run on 3730 XL DNA Analyzed  sequencer was used for sequencing PCR products.The Bioedit program 5.0.6 software and ClustalW alignement algorithm (version 1.83) were initially used to compare and align nucleotide sequences. Phylogenetic trees were constructed using MEGA5.01 program version 3.65 with neighbor-joining method. The robustness of the groupings in the neighbor-joining analysis was assessed with 1000 bootstrap resamplings. The Blast software and Bioedit programs were used to determine the sequence similarity of the Tunisian strainsNucleotide sequence accession numbers: the nucleotide sequences obtained in this study were deposited in GenBank data library under accession numbers: JF323006 to JF323016.Number of amino acid residues: amino acid residues were numbered according to the HA sequences of Qu/HK/G1/97 (H9) with GenBank accession number AF156378.To determine evolutionary relationships between Tunisian H9N2 isolates and those selected from the Genbank Table .Phylogenetic analyses based on the HA Figure  and NA  genes n. Tree anFour internal genes  of Tunisian H9N2 strains showed more than 96% nucleotide identity with those of the Middle Eastern strains isolated from 2001 and 2008 Table .The phylogenetic studies of the PB2 genes were analysed using the 540- base-long nucleotide sequence (nucleotides 1591 to 2130) that codes the region of PB2 protein located between positions 530 and 710. Tree analysis of the PB2 gene showed that Ck/TUN/12/10 and Migratory bird/TUN/51/10 isolates are related to the Middle Eastern isolates (more than 97% similarity) and fall into a single group within the G1-like lineage Figure .In the NP gene tree Figure , represeThe matrix genes Figure  (regionsThe NS gene (nucleotides 1591 to 2130) of Ck/TUN/12/10 showed evidence of reassortment with other viral subtypes. It was 95% and 92.9% similar to those of A/gs/11/99 (H6N1) and CK/HK/8761/01 (H5N1), respectively Figure .To analyze the receptor binding site, the connecting peptide and the Hemadsorption site of neuraminidase specificity of the Tunisian isolates, we have compared the HA amino acid sequences (deduced from the nucleotide sequences) with those of representative viruses from GenBank.The molecular determinants of pathogenecity and virulence of the HA protein are the HA1/HA2 connecting peptide sequence, specific amino acids (aa) residues at the receptor binding site (RBS), and the presence or absence of glycosylation sites around the receptor binding site.333 PARSSR* GLF341 which is similar to other Asian H9N2 viruses including those from Hong Kong, Japan, Iran, Israel, Pakistan, and Saudi Arabia, Dubai. Residues at positions 110, 161, 163, 191, 198, 234, 235 and 236 are major components of the receptor binding site of the HA molecule. Tunisian viruses showed conservation of residues 110, W161, T163, H191, A198 Pand 235 Iin the receptor binding pocket. The left (aa residues at position 234-239) and right (aa residues at position 148-152) edge of the binding pocket motif were: NGL/QIGR and GTSKS, respectively.HA cleavage site motif sequence of the Tunisian isolates was ..3. 2link to a ..6. 2link between the sialic acid residues and galactose, whereas A/Migratory Bird/TUN/51/10 having Glu Q at this position. But, both strains had Gly (G) at position 228 at the RBS.The A/CK/TUN/12/10 H9N2 strain carried the amino acid substitution (Q234L), a mutation that correlates with a shift in affinity of the HA from avian type sialic receptors to human type and from a preference for a Analysis of HA protein sequences showed that Tunisian H9N2 isolates have many potential glycosylation sites with the N-X-T/S motif (X can be any amino acid expect proline): 82-84 (NPS), 105-107 (NGT), 141-143 (NVT), 218-220 (NRT), 298-300 (NST), 305-307 (NIS)  were substituted by KLA respectively, also D and Y substitution were found at residues 401, 406 respectively. Otherwise, the framework site contains RThe current Tunisian strains displayed Glu (E) at position 627 of the PB2 protein; therefore, a change to Lys could serve as a marker of the virulence phenotype. In addition, two avian virus-like aa at positions 661 (A) and 702 (K), similar to Ck/HK/8733/01 H5N1 strain isolated in 2001, were found (Table The nucleoprotein (NP) of Tunisian H9N2 strains retained conserved G1-lineage defining residues: R422, K430, T442, D455 and D480. However, Ser to Phe substitution at residue 402, found exclusively in Tunisian strains, similar to H5N1 strain circulated in 1997. Other aa specific for the nucleoprotein (NP) gene associated with host range, were not studied , 28 , related to the ion channel domain and associated with host range. In addition, Ile, Val and Pro substitutions were present in Tunisian H9N2 viruses at residue 15 (V15I), 46 (L46V) and 54 (S54P) of M1 protein, respectively. Avian virus like aa such as: VBased on the analysis of the non structural protein gene (NS), our recent isolates did not show the five amino acid (80TIAS84) deletions, resulting in an NS1 protein of 230 amino acid in length and containing the "GSEV" PDZ ligand (PL) C-terminal motif, and did not harbor the mutation of Asp (D) to Glu (E) at position 92, required for high virulence. Besides, The RNA-binding domain exhibited amino acid residues P31, D34, R35, R38, K41, G45, R46 and T49. However, the effector domain carried the F103L, R184G and K217N substitutions. Moreover, E227G mutation in C-terminal domain of the NS1 protein, introducing an S70I mutation into nuclear export protein (NEP), was also observed  have been frequently reported in China, other Asian and North American countries, since the late 1990s. They have been grouped in different sublineages on the basis of antigenic and genetic properties. Although, H9N2 viruses do not satisfy the criteria for highly pathogenic avian influenza, they are unique among this category, infecting a wide variety of species, including chickens ,22, quaiSince late 2009, H9N2 influenza A viruses have caused many outbreaks in Tunisian flocks. A second wave of AI was reported during July-October 2010. Here, we have provided the first comprehensive genetic data for the H9N2 subtype viruses circulating in Tunisian poultry flocks. Strains were collected from two different governorates situated in the northeastern part of Tunisia. Our findings revealed that H9N2 virus infection is well established in some Tunisian endemic areas. Therefore, understanding the genetic and the biological characteristics of H9N2 virus isolated from different species and regions can provide a comprehensive insight into the biology of H9N2, the ecology of AI virus, and the ability of migratory birds to disseminate influenza viruses.The Blast analysis (NCBI) of the nucleotide sequences of HA and NA genes showed that A/Ck/TUN/12/10 and A/Migratory Bird/TUN/51/10 were the most closely related to the Middle Eastern isolates belonging to the G1- like lineage in the H9N2 subtype (more than 96.5% similarity).The results of the phylogenetic analyses were basically in agreement with the blast data, and confirmed that our isolates fall, together with the Middle Eastern strains, into a distinct cluster, related to the G1 lineage; a finding that may indicate a common origin.The internal PB2, NP, M, NS genes were also similar to those of other Middle Eastern strains, which can be traced back to the same G1- like lineage. In addition, NP and NS genes seemed to have undergone broad reassortments with H5N1, H7N3, H7N1, H6N1 influenza virus subtypes as described for previous isolated strains ,29,30. TBased on the deduced amino acid sequences, the HA1-HA2 connecting peptides of the Tunisian avian H9N2 isolates did not harbor multiple basic amino acids: PARSSR/GL as found for recently isolated H9 viruses in Middle East  and AsiaMoreover, A/Migratory bird/TUN/51/10 possessed a Glu (Q) at position 234 in the HA1 portion , whereas A/Ck/TUN/12/10 showed a leu (L), a receptor binding site residue, typical for human influenza virus displaying human virus-like cell tropism through an association with a preferential binding of sialic acid (SA) to galactose in α 2, 6 linkage . A leu  at position 627 of the PB2 protein. Likewise, a single aa substitution in PB2 protein (E627K) can dramatically alter the virulence and enhance viral replication in mice  and otheThe main function of the NP is encapsidation of the viral genome to form a ribonucleoprotein particle for transcription and packaging; it interacts with other viral PB1, PB2, M1 and cellular proteins  for viral transcription and nuclear transportation controls . The NP It has been reported that aa residues at positions 15, 115, 121, 137, 240 in virus matrix protein are linked with increased replication in mammals or increased pathogenecity in small animal models ,50; an IA Val substitution at position 46 was also detected for the first time in our strains; however, all other H9N2 and other IA subtypes have leu at this position  of the dsRNA-binding domain (RBD (residues 1-73) of NS1 . It is dAn Asp (N) was found at residue 217 only in Ck/TUN/12/2010 strain that differs from the other H9N2 strains which have K at this position, but similar to those of H5N1 strain CK/HK/8761/01 Table . HoweverAlso, Ck/TUN/12/2010 didn't exhibit a five amino (80TIAS84) deletion observed in 2001 in poultry in Hong Kong and has, since, became the most common sequence found in the HP viruses. Long et al, demonstrated that viruses containing NS1 with 5 amino acid deletion (80TIAS84) residues showed increased virulence in both mouse and poultry  but the Fortunately, previous study, performed on large scale sequence analysis of viruses isolated from different birds and mammalian species, have identified that the C-terminal domain of NS1 functions as a species-specific virulence domain: the vast majority of avian influenza viruses have an NS1 protein with a PDZ ligand (PL) C-terminal ESEV domain, while typical human viruses have a conserved RSKV domain. NS1 proteins with C-terminal ESEV, KSEV, and EPEV domains were shown to bind to PDZ domains containing cellular proteins ,64. SoubIn the present study, we characterized two Tunisian H9N2 strains isolated from disease outbreaks in commercial chicken and migratory bird from different geographic areas. Our findings indicate that CK/TUN/12/10 isolate has evolved and reassorted with other influenza viruses and has acquired an affinity for the human receptor binding profile. Otherwise, the biological significance of new detected substitutions founded in some internal genes and the PDZ ligand polymorphism of the NS protein should be tested.Actually and face to the high mortality, we are preparing for an active program to develop and evaluate vaccines against H9N2 to control the further spread of the disease in the industrial poultry production sectors. Interestingly, continuous monitoring of viral genetic changes throughout the years is warranted to monitor variations of Influenza viruses in the field.aa: amino acid; AIV: Avian Influenza virus; HA: hemagglutinin; HB: heamadsorption; HPAIV: highly pathogenic type; LPAI: Low pathogenic type; M: matrix; NA: neuraminidase; NP: nucleoprotein; NS: non structural; PB2: polymerase; PL: PDZ ligand; RBS: receptor binding siteThe authors declare that they have no competing interests.WT is a PhD, who carried out the H9N2 avian influenza virus genes detection by RT-PCR and was involved in the sequencing assays, phylogenetic and amino acid analysis, coordinated the work described, and drafted of the manuscript. JN and IL were involved in its design and coordination. JLG involved in drafting the manuscript or revising it critically for important intellectual content. AG conceptualized the study, supervised all facets of the research, and involved in design of the trials, assisting in the writing process. All authors read and approved the final manuscript.


TRMT10A . TRMT10A mRNA and protein were absent in lymphoblasts from the affected siblings. TRMT10A is ubiquitously expressed but enriched in brain and pancreatic islets, consistent with the tissues affected in this syndrome. In situ hybridization studies showed that TRMT10A is expressed in human embryonic and fetal brain. TRMT10A is the mammalian ortholog of S. cerevisiae TRM10, previously shown to catalyze the methylation of guanine 9 (m1G9) in several tRNAs. Consistent with this putative function, in silico topology prediction indicated that TRMT10A has predominant nuclear localization, which we experimentally confirmed by immunofluorescence and confocal microscopy. TRMT10A localizes to the nucleolus of β- and non-β-cells, where tRNA modifications occur. TRMT10A silencing induces rat and human β-cell apoptosis. Taken together, we propose that TRMT10A deficiency negatively affects β-cell mass and the pool of neurons in the developing brain. This is the first study describing the impact of TRMT10A deficiency in mammals, highlighting a role in the pathogenesis of microcephaly and early onset diabetes. In light of the recent report that the type 2 diabetes candidate gene CDKAL1 is a tRNA methylthiotransferase, the findings in this family suggest broader relevance of tRNA methyltransferases in the pathogenesis of type 2 diabetes.We describe a new syndrome of young onset diabetes, short stature and microcephaly with intellectual disability in a large consanguineous family with three affected children. Linkage analysis and whole exome sequencing were used to identify the causal nonsense mutation, which changed an arginine codon into a stop at position 127 of the tRNA methyltransferase homolog gene  CDKAL1, which has recently been shown to be a tRNA modifying enzyme (methylthiotransferase). Genetic variants of different severity can generate a spectrum of monogenic and polygenic forms of diabetes. Here we describe a new syndrome of young onset diabetes, short stature and microcephaly  with intellectual disability in a large consanguineous family. By linkage analysis and whole exome sequencing we identified a nonsense mutation in TRMT10A, a gene that has hitherto not been studied in mammals. The yeast homolog TRM10 has been shown to be a tRNA modifying enzyme with methyltransferase activity. We demonstrate that TRMT10A mRNA and protein are absent in cells from the affected siblings. TRMT10A localizes to the nucleolus, where tRNA modifications occur. TRMT10A silencing induces cell death in insulin-producing pancreatic β-cells, suggesting that TRMT10A deficiency may reduce β-cell mass and the pool of neurons in the brain. This is the first study describing the impact of TRMT10A deficiency in man. Our findings may have broader relevance for the understanding of the pathogenesis of type 2 diabetes and microcephaly.The inherited predisposition to type 2 diabetes is attributed to common variants in over 60 loci. Among these risk variants is  CDK5 regulatory associated protein 1-like 1 (CDKAL1). Polymorphisms in this gene have been associated with T2D across ethnic populations CDKAL1 encodes a transfer RNA (tRNA) methylthiotransferase that catalyzes the methylthiolation of tRNALys(UUU) Type 2 diabetes (T2D) is a heterogeneous polygenic disease with dramatically increasing worldwide incidence as a consequence of the obesity epidemic tRNAs undergo modifications of their bases or sugar moieties that are crucial for proper cellular function. Mammalian cells have an average of 13–14 modifications per tRNA TRMT10A  in a new syndrome of young onset diabetes and microcephaly. The TRMT10A yeast ortholog YOL093w codes for the protein TRM10 that has tRNA methyltransferase activity. TRM10 specifically methylates tRNA-Arg, -Asn, -Gln, -Thr, -Trp, -Met and -Lys at position 9 (m1G9), using S-adenosylmethionine (SAM) as methyl donor 1G9 methyltransferase in yeast, but its knockout did not alter cell survival or growth 7G46 and m22G26, respectively TRM10 with TRM8, TRM82 or TRM1 induced growth arrest in S. cerevisiae exposed to high temperature, suggesting enhanced tRNA instability Here we identified a nonsense mutation in TRMT10A mutation. We followed this up with studies of TRMT10A expression in tissues and subcellular localization, and interrogated the functional consequences of TRMT10A deficiency.Here we describe the affected siblings and the identification of the 2; plasma glucose was 176 mg/dl and HbA1c 11.3%. Other features were a short neck, wide nose, low hairline, buffalo hump, retraction of the right 5th toe, scoliosis, and joint laxity. She also had osteoporosis, with dual-energy X-ray absorptiometry T-scores of -2.7 and -3.5 at the lumbar spine and femoral neck, respectively. A skeletal survey revealed no epiphyseal dysplasia or other bone abnormality , microcephaly  and mental retardation (IQ 52). His head circumference at birth was reportedly normal (36 cm). He was diagnosed with diabetes at 14 years of age, with a plasma glucose of 251 mg/dl and HbA1c 11.1%. His BMI was 20.6 kg/m2. None of the patients had ketoacidosis and all three were treated with insulin at diagnosis. They were negative for anti-insulin, anti-GAD65, anti-IA2 and islet cell autoantibodies and had a HLA genotype that did not confer risk for type 1 diabetes. Endogenous insulin secretion persisted, shown by C-peptide measurements for up to 20 years of follow-up. The insulin requirements were moderate with an average insulin dose of 0.4–1.2 U/kg/day; glycemic control ranged from good to insufficient (HbA1c 6.5–8.5%). After 18 years of diabetes, the proband's ophthalmologic examination revealed bilateral diabetic retinopathy and cortical cataract. The parents and non-affected siblings had normal size  and head circumference . The parents developed diabetes at age 58 years  and were treated with metformin and a sulphonylurea. One grandfather and two aunts had adult onset diabetes  homozygous genomic region that was common to the three affected siblings. It was located on chromosome 4q22-23 and spanned 12.4 Mb between heterozygous SNPs rs4128340 and rs10516462. In this segment, we genotyped 15 microsatellite markers, which confirmed homozygosity and biparental inheritance of a haplotype shared by both parents . The mulWe initially sequenced the following genes located in the 3.1 Mb segment and considered as candidates: H2AFZ , LAMTOR3 , DDIT4L (DNA-damage-inducible transcript 4-like), RAP1GDS1  and METAP1 (methionyl aminopeptidase 1), but no mutation was identified.TRMT10A (tRNA methyltransferase 10 homolog A (S. cerevisiae) at position 379 of the coding DNA sequence, predicted to replace an Arginine residue with a premature termination codon at position 127 of the polypeptide . Sanger sequencing confirmed the mutation (Exonic sequences-enriched DNA (whole exome) sequencing was performed in one proband and results were analyzed for variants that were not found in: dbSNP135 database, the Thousand Genomes database, the Exome Variant Server, or in-house exome sequencing on 51 individuals. There was only a single candidate mutation in the 3.1 Mb critical linkage segment, a homozygous G to A transition in exon 4 of gene mutation , which wmutation . OutsideTRMT10A in 26 patients with non-autoimmune diabetes with onset before 25 years and a positive family history of diabetes, in whom no mutation was identified in known MODY-associated genes, but did not identify any mutation in TRMT10A.We sequenced the 8 exons and flanking intronic sequences in 20 patients with a similar phenotype of young onset diabetes associated to intellectual disability, microcephaly, epilepsy, developmental delay and/or short stature, five of whom were born to consanguineous parents, but failed to identify another patient with biallelic disease-causing mutations. We furthermore sequenced To examine the outcome of the TRMT10A nonsense mutation on TRMT10A protein and mRNA expression, we performed Western blot and real-time PCR on lymphoblasts from two patients, a heterozygous carrier of the mutation, and three healthy controls. TRMT10A protein was absent in lymphoblasts from patients homozygous for the Arg127Stop mutation . TRMT10AWe next evaluated TRMT10A transcript and protein expression in rat tissues. TRMT10A was ubiquitously expressed with similar mRNA abundance in liver, kidney, spleen, lung, fat, and brain. Heart and muscle showed lesser TRMT10A mRNA expression, while pancreatic islets were enriched in TRMT10A transcripts . TRMT10AIn situ hybridization studies were performed in human embryonic brain samples at 8, 11, 17 and 19 gestational weeks (GW). TRMT10A was expressed throughout the whole thickness of the dorsal telencephalon  at 8 and 11 GW, with higher expression in the ventricular zone and marginal zone . In the rest of the patients' exome, we found no potentially damaging, rare biallelic variants shared by the three patients that might have qualified for a causal mutation.In a large consanguineous family of Moroccan origin, we identified a new syndrome of severe insulinopenic young onset diabetes and microcephaly with intellectual disability. We used linkage analysis and whole exome sequencing to identify the causal mutation. We found only one region of homozygosity by descent shared by the three affected patients, and only one potentially damaging rare genetic variant in this region, located in the Among the family members, four were heterozygous carriers of a mutant allele. Of these, the parents developed diabetes in their fifties, one sister had gestational diabetes, and one brother had normal plasma glucose levels at the age of 23 . Other fTRMT10A contains 8 exons, the first exon being non-protein coding. The mutated codon 127 is in exon 4. The protein environment of Arg127 is extremely conserved across species. Little is known about the role of TRMT10A in mammals. A single study suggested altered TRMT10A mRNA expression in colorectal cancer S. cerevisiae TRM10, previously shown to be involved in guanine 9 tRNA methylation m1G9The Arg127Stop mutation is predicted to block the expression of the five coding human TRMT10A isoforms. The nonsense mutation abolished TRMT10A protein expression, and also significantly reduced its mRNA expression , probablAlterations in tRNA modification are expected to affect protein translation. We showed that TRMT10A knockdown in rat β-cells enhances total protein biosynthesis . TRMT10APrimary microcephaly refers to a congenitally small but otherwise normally structured brain, with a head circumference later in life that remains 3 SD below the mean for age and gender. Primary microcephaly is a very rare disorder affecting approximately 1/100,000 live births, mainly inherited as an autosomal recessive trait, and is associated with a high rate of parental consanguinity Microcephaly in our patients was associated with intellectual disability and no other neurological feature, except for a history of petit mal seizures in the proband. This clinical presentation fits with the phenotype of primary microcephaly The expression pattern of TRMT10A in the ventricular zone of the developing cortex is consistent with its influence on neural progenitor properties, including control of survival that is known to affect brain size. In addition it may act in subsets of differentiated neurons, as suggested by its expression in cortical marginal zone and cerebellum.IER3IP1 gene encoding the immediate and early response 3 interacting protein 1 result in infantile diabetes and congenital microcephaly with simplified gyration, hypotonia, intractable seizures, and early death EIF2AK3 encoding translation initiation factor 2-α kinase-3 Early onset diabetes has been associated with microcephaly in other genetic disorders. Homozygous mutations in the TSEN) or mitochondrial arginyl-tRNA synthetase (RARS2) NSUN2, encoding a methyltransferase that catalyzes the intron-dependent formation of 5-methylcytosine at C34 of tRNA-leu(CAA) NSUN2 mutations resulted in nonsense-mediated mRNA decay Other human diseases are caused by mutations in genes encoding tRNAs and tRNA modifying enzymes. Pontocerebellar hypoplasia, characterized by hypoplasia and atrophy of ventral pons, cerebellum and the cerebral cortex, is caused by mutations in genes encoding tRNA splicing endonuclease subunits  In addition to causing microcephaly and short stature, the TRMT10A gene in a new syndrome of young onset diabetes and microcephaly. Based on its cellular localization and by homology with its yeast counterpart, we propose that TRMT10A has methyltransferase activity. We show that TRMT10A is expressed in human fetal brain; TRMT10A silencing does not impair β-cell function but induces apoptosis, suggesting that TRMT10A deficiency may negatively affect β-cell mass and the pool of neurons in the developing brain. Our findings may have broader relevance for the understanding of the pathogenesis of T2D and microcephaly.In conclusion, we describe a nonsense mutation in the The ethics committee of the Erasmus Hospital, Université Libre de Bruxelles approved of the study. The three patients, their parents, and two unaffected siblings gave informed consent. Human fetal brain was collected and used according to the guidelines of the local ethics committees on research involving human subjects . Adult male Wistar rats were housed and used following the rules of the Belgian Regulations for Animal Care, with approval of the ethics committee of the Université Libre de Bruxelles.http://genome.ucsc.edu/cgi-bin/hgGateway). A multipoint LOD score was computed using the MAPMAKER/HOMOZ software A peripheral blood sample was obtained for genetic analysis from the three patients, their parents, and two unaffected siblings. Leukocyte DNA was extracted using proteinase K digestion followed by phenol-chloroform extraction and ethanol precipitation www.1000genomes.org) database, nor in the Exome Variant Server (http://evs.gs.washington.edu/EVS/), and that were not found in our other in-house exome sequencing results.Genomic DNA from the proband , arrow whttp://ihg.helmholtz-muenchen.de/ihg/ExonPrimer.html). All exons and flanking intronic regions of the candidate genes were sequenced by the Sanger method using the Big Dye Terminator cycle sequencing kit v2 , and analyzed on a 3130 Genetic Analyser sequencing machine (Applied Biosystems). Sequences were analyzed in silico for mutations using the SeqScape software V.2.0. (Applied Biosystems).PCR primers for all exons and flanking intronic sequences were designed using the Exonprimer software  as previously described CCAAGCTAATACGACTCACTATAGGGAGATGTGAACCAATATCTAAACGACAAA – R: GGATCCATTAACCCTCACTAAAGGGAGAGATTTTCCTTATCCTGCTTTTCTTC.2) were isolated by collagenase digestion and density gradient purification Clonal rat INS-1E cells  were cultured in RPMI medium as previously described TRMT10A open reading frame (ORF), using primers F GAATTCATGTCATCTGAAATGTTGCCCG and R CTCGAGGTGTGGCAGAGAGTTCACTGCG. The restriction sites EcoRI and XhoI (underlined) were added to facilitate the directional cloning into the expression vector pGEX-6P-1 . This vector allows the expression of recombinant proteins fused to glutathione-s-transferase (GST) at its N-terminus. E. coli BL21 cells were transformed with the pGEX-6P-1-TRMT10A plasmid by electroporation. Positive clones were selected by colony PCR and sequenced. For recombinant protein expression, a single colony was grown overnight at 37°C in LB medium containing 100 µg/ml ampicillin. Cells were then diluted 1∶50 in the same medium and grown at 37°C until an optical density of 0.6 at 600 nm was reached. Isopropyl-β-D-thiogalactoside (0.25 mM) was then added and cells were grown at 28°C for 3 h to induce recombinant protein expression. Cells were harvested by centrifugation at 3000×g for 10 min, lysed by sonication in 20 mM Tris buffer pH 8 containing 0.5% Triton ×100, 10 mM dithiothreitol, 0.1 mM PMSF and protease inhibitor cocktail (Roche), and centrifuged for 15 min at 15,000×g at 4°C. The supernatant was applied to 1 ml glutathione spin columns (Pierce) and washed with ice-cold lysis buffer. The recombinant hTRMT10A was separated from the GST moiety by in column site-specific proteolysis using PreScission protease  following the manufacturer's instructions. The purified recombinant hTRMT10A was used for rabbit polyclonal antibody production (Eurogentec).hTRMT10A was amplified by PCR from lymphoblast cDNA using oligonucleotides spanning the start and stop codons of the CCCGGGAATGTCATCTGAAATGTTG-3′5′-AAAAAA (start codon is indicated in bold), and R GGATCCTGAGTGTGGCAGAGAGTT-3′5′-AAAAAA in which the restriction sites SmaI and BamHI (underlined) were added to facilitate the directional cloning into the mammalian expression vector Vitality phrGFP-1 (Stratagene). The stop codon of the TRMT10A ORF was removed to allow the production of the recombinant TRMT10A fused to the N-terminus of hrGFP. The PCR product was purified using the Wizard SV Gel and PCR clean-up system (Promega), sequentially digested with SmaI and BamHI (New England Biolabs), and cloned into the Vitality phrGFP-1 vector digested with the same restriction enzymes. The plasmid was introduced into electrocompetent One Shot E. coli (Invitrogen) by electroporation, and positive clones were identified by colony PCR. A single colony containing hrGFP (empty vector) or TRMT10A-hrGFP plasmid was grown overnight at 37°C in LB medium with 100 µg/ml ampicillin. Plasmids were purified with PureYield Midiprep (Promega) and quantified by NanoDrop (Thermo Scientific). Expression of recombinant TRMT10A-hrGFP in rat β-cells was confirmed by Western blot.hTRMT10A was amplified by PCR from HeLa cDNA using the oligonucleotides F Cells were transfected overnight with 30 nM of a control siRNA (Qiagen), or two single siRNAs targeting rat or human TRMT10A using Lipofectamine RNAiMAX (Invitrogen). siRNA-lipid complexes were formed in Opti-mem (Invitrogen) for 20 min as previously described hrGFP and TRMT10A-hrGFP plasmids were introduced by lipofection in INS-1E cells or dispersed rat and human islet cells using Lipofectamine 2000 (Invitrogen). siRNA sequences, plasmid and Lipofectamine concentrations are described in TRMT10A subcellular localization was examined by expressing recombinant hTRMT10A fused to GFP, or by immunolabeling endogenous TRMT10A in INS-1E cells and dispersed rat and human islet cells. Cells plated on poly-lysine coated cover slips were transfected or not with hrGFP and TRMT10A-hrGFP plasmids, fixed with 4% formaldehyde + mRNA was isolated from INS-1E cells, dispersed human islets, or primary rat β-cells using the Dynabeads mRNA DIRECT kit (Invitrogen). For total RNA purification, rat tissues and pancreatic islets were resuspended in RNeasy Minikit lysis buffer (Qiagen), homogenized using a T10 basic ULTRA-TURRAX disperser (IKA) or lysed by sonication in a Bioruptor NGS (Diagenode), respectively. Total RNA was purified with the RNeasy Minikit and quantified by NanoDrop. mRNA and total RNA were reverse transcribed as previously described Poly(A)Rat tissues and pancreatic islets were resuspended in ice-cold PBS containing protease inhibitor cocktail and homogenized as described above. Total protein was measured in the lysates using the Protein Assay Dye Reagent (BIO-RAD). INS-1E cells and human lymphoblasts were lysed with Laemmli buffer Insulin secretion was performed as previously described FFA treatment was performed in RPMI 1640 containing 0.75% FFA-free BSA (Roche). Oleate and palmitate  were dissolved in 90% ethanol and diluted 1∶100 to a final concentration of 0.5 mM Apoptotic cell death was detected and counted by fluorescence microscopy after Hoechst 33342  and propidium iodide (5 µg/ml) staining as described 3H)-leucine (Perkin Elmer). Cells were then washed with Krebs-Ringer solution containing 10 mM non-radioactive leucine. Cells were collected in ice-cold water and lysed by sonication. Total protein was precipitated with 10% trichloro-acetic acid. The content of 3H-labeled proteins was determined in a liquid scintillation analyzer (Packard) 72 h after transfection INS-1E cells were cultured for 2 h in Krebs-Ringer buffer containing 11 mM glucose, 1% BSA and 10 µCi/ml L- is observed in the three affected siblings. A critical recombination event (boxed) is observed distal to D4S1628 in an unaffected brother, who is otherwise homozygous for all microsatellite markers. The TRMT10A gene (arrowhead) is at chr4:100,467,864–100,485,189.(TIF)Click here for additional data file.Figure S2Species comparison shows very high conservation of TRMT10A Arginine 127 and surrounding amino acids.(TIF)Click here for additional data file.Figure S3TRMT10A expression profile in fetal brain at 19 GW. (A) Cresyl Violet. (B) TRMT10A antisense (AS) probe showing expression in the presumptive cerebellar cortex. (C) TRMT10A sense (S) probe as a negative control.(TIF)Click here for additional data file.Figure S4TRMT10A has a nucleolar localization in islet cells. Dispersed rat (A) and human islet cells (B–C) were transfected with a vector encoding hrGFP alone (hrGFP) or fused to TRMT10A (TRMT10A-hrGFP). 48 h after transfection TRMT10A subcellular localization was examined by fluorescence microscopy. Nuclei were stained with Hoechst 33342. Nucleolus was stained with anti-fibrillarin antibody. Pictures were taken at 40× magnification and are representative of two independent experiments.(TIF)Click here for additional data file.Figure S5TRMT10A knockdown in INS-1E cells. INS-1E cells were transfected with control siRNA (siCT) or two siRNAs targeting rat TRMT10A (siTRMT10A #1 and #2). 72 h after transfection TRMT10A mRNA and protein expression was examined by real-time PCR and Western blot. (A) TRMT10A mRNA expression corrected for the reference gene GAPDH. (B) Representative Western blot and (C) densitometry of TRMT10A protein expression corrected by α-tubulin or β-actin. Data are means ± SE (n = 4). § p<0.05 by paired t test.(TIF)Click here for additional data file.Figure S6t test.TRMT10A knockdown does not modify insulin secretion or content in β-cells. INS-1E cells were transfected with control siRNA (siCT) or two siRNAs targeting rat TRMT10A (siTRMT10A #1 and #2). (A) 72 h after transfection insulin secretion was induced by 1.67 or 16.7 mM glucose or 16.7 mM glucose +10 µM forskolin (FK). (B) Insulin content corrected by total protein. (TIF)Click here for additional data file.Figure S7t test.Endoplasmic reticulum stress enhances TRMT10A expression in INS-1E cells and human islets. TRMT10A mRNA expression was examined by real-time PCR in INS-1E cells and human islets exposed or not (CT) to oleate (OL), palmitate (PAL), 28 mM glucose (G28), CPA, thapsigargin (THAP), tunicamycin (TU) or brefeldin (BR). (A) Time course of TRMT10A mRNA expression in FFA-treated INS-1E cells (n = 3–4). Data is expressed as fold of untreated control. (B–C) INS-1E cells exposed for 24 h to FFAs, high glucose or synthetic ER stressors. Data was normalized to the geometric mean of GAPDH, tubulin and OAZ1 mRNA expression (n = 4–11). (D) Human islets exposed for 24 h to synthetic ER stressors. Expression data was normalized to β-actin (n = 5). * Treated vs CT. One symbol p<0.05, two p<0.01, three p<0.001, by paired (TIF)Click here for additional data file.Figure S8t test.Expression of ER stress markers after exposure to synthetic ER stressors. XBP-1s, CHOP, BiP and ATF-3 mRNA expression was examined in INS-1E cells and human islets exposed or not (CT) to CPA, thapsigargin (THAP), tunicamycin (TU) or brefeldin (BR). Expression was normalized to GAPDH or β-actin (n = 4–5). * Treated vs CT. One symbol p<0.05, two p<0.01, three p<0.001, by ratio (TIF)Click here for additional data file.Figure S9t test.Endoplasmic reticulum stress modulates TRMT10A expression via PERK but not IRE1 activation. (A–C) INS-1E cells were transfected with control siRNA (siCT), or siRNA targeting rat PERK. 48 h after transfection cells were exposed or not to palmitate (PAL) or CPA for 16 h. (D–F) INS-1E cells were exposed for 16 h to palmitate (PAL) or CPA alone or combined with the IRE1 inhibitor 4μ8C. TRMT10A, ATF3 and XBP-1s mRNA expression was examined by real-time PCR and normalized to GAPDH expression (n = 4–6). * Treated vs CT, § siPERK vs siCT, ## IRE1 inhibitor vs CT. One symbol p<0.05, two p<0.01, three p<0.001, by ratio (TIF)Click here for additional data file.Table S1Sequence of rat and human siRNAs.(DOCX)Click here for additional data file.Table S2Transfection reagents used in INS-1E cells, primary rat β-cells and human islets.(DOCX)Click here for additional data file.Table S35′-3′ primer sequence used for standard and real-time PCR.(DOCX)Click here for additional data file.


The expressions of STAT3, BcL-2, cyclin D1 and C-myc in these cells were detected by Western blot and Northern blot. The cell cycle and the growth were determined by flow cytometry (FCM) and MTT assay, respectively. Cell apoptosis was determined by TUNEL staining.Three pairs of DNA template (stat3-3 markedly suppressed the protein expression of stat3 in SKOV3 cells; MTT assay and FCM showed that transfection of stat3-3 siRNA could significantly suppress the growth of SKOV3 cells and arrest the cell cycle in vitro. TUNEL staining also showed massive apoptosis in SKOV3 cells transfected with stat3-3 siRNA.Of the three siRNAs, only siRNA targeting stat3-3 can significantly inhibit the STAT3 expression in human ovarian cancer cells resulting in the inhibition of the cancer growth and the increase of apoptosis of cancer cells.pSilencer2.1-U6-siRNA- Recent studies demonstrate the blockade of STAT3 expression in human cancer cells that suppresses the in vitro proliferation of cancer cells and the in vivo tumorigenicity. Attempts to suppress STAT3 expression have been made using tyrosine kinase inhibitorsIn vitro studies have shown that the inhibition of STAT3 activity in human cancer cells can induce the apoptosis and/or the growth arrest. In human head and neck squamous carcinoma cells, prostate cancer cells, and laryngeal cancer cells, blocking of stat3 by decoy oligonucleotides or antisense oligonucleotides or siRNA abrogates the production of transforming growth factor (TGF) and suppresses the oncogenic growth of these cells.Ovarian cancer, such as serous carcinoma, mucinous carcinoma, endometrioid carcinoma, metastatic carcinoma etc., is one of the common gynecological malignant tumors with high mortality. It is highly malignant and its incidence is increasing.RNAi is triggered by the presence of double-stranded RNA (dsRNA) in cells and results in rapid degradation of targeted mRNA with homology to double strand RNA leading to potent and selective silencing of genes. RNAi provides a novel approach to inhibit gene expression, and to date, RNAi with siRNA has been applied as a functional genomic tool.20stat3 gene was synthesized and siRNA-stat3 expression vectors were constructed with pSilencer 2.1-U6, which was then used to transfect human ovarian cancer cells (SKOV3 cells) aiming to inhibit the STAT3 expression and induce apoptosis of cancer cells. SKOV3 cells transfected with siRNA-stat3 were subcutaneously injected into nude mice and the growth and the apoptosis of ovarian cancer cells were observed.In the present study, siRNA targeting Twenty-five ovarian cancer samples and twenty fresh normal ovary tissues were collected for the determination of STAT3 expression. These tissues were embedded in paraffin and cut into 5-μm sections. After deparaffinization, the endogenous peroxidase was inactivated by 3% hydrogen peroxide in methanol for 10 min. The sections were treated with rabbit anti-human STAT3 polyclonal antibody  and then with goat anti-rabbit IgG conjugated with horseradish peroxidase. The development was done at room temperature, using an avidin-biotin-peroxidase complex method . The criteria for grading of STAT3 expression were as follows: negative (−): ≤5% positive cells; low (+): 5∼25% positive cells; moderate (++): 25∼50% positive cells; strong (+++): 50∼100% positive cells).stat3  were designed according to the sequence of human stat3 gene (Genebank: NM003150). These oligonucleotides contain a sense strand with 19 nucleotides followed by a short spacer (TTCAAGAGA). The reverse complement of the sense strand has five thymines as a stop signal of RNA polymerase III transcription. The sequences of oligonucleotides were as follows:Three pairs of double stranded siRNA oligonucleotide against stat3-1:5′ - GATTGACCTAGAGAC-CCACTTCAAGAGAGTGGGTCTCTAGGT-CAATCTTTTT-3′ (forward) and 5′-AATTAAAAAG ATTGACCTAGAGACCCACTCTCTTGAAGTG GTCTCTAGGTCAATCGGCC-3′ (reverse); stat3-2:5′-GAGTCGAATGTTCTCTATCTTCAAGAGAGATAGAGAACATTCGACTCTTTTT-3′ (forward); and 5′-AATTAAAAAGAG TCGAATGTTCTCTATCTCTCTTGAAGATAGA GAACATTCGACTCTGGCC-3′ (reverse); stat3-3:5′-GCAGCAGCTGAACAACATGCATGTTCAAGAGACATGTTGTTCAGCTGCTGCTTTTT-3′ (forward), and 5′-AATTAAAAAGCAGCAGCTGAACAACATGTCTCTTGAACATGTTGTTCAG CTGCTGCTGCGGCC-3′ (reverse).Hind III-BamH I sites of pSliencer 2.1-U6 vectorsHind III-BamH I restriction enzymes. The products with the double-stranded structure were used to form a recombinant plasmid with T4 DNA ligase followed by annealing. A negative control scrambled siRNA , which has no evident homology to mouse or human stat3 sequences, was also designed aiming to exclude non-specificity.These Oligos were annealed in the annealing buffer  and cloned into the stat3 siRNA group. LipofectAMINE 2000  was used in the cell transfection, and enhanced green fluorescent protein vector  was cotransfected with either pSilencer 2.1-U6-stat3 siRNAs or pSi-lencer 2.1-U6-scrambled siRNA at a volume ratio of 1:20 to label the positive transfected cells. The transfection was performed for 5∼20 h and then the medium was refreshed with medium containing 10% FBS followed by lysis for 24∼72 h after the transfection.Human ovarian cancer cells (SKOV3 cells) were grown in Iscove’s Modified Dulbecco’s Medium (IMDM)  containing 10% fetal bovine serum (FBS). When the cell confluence reached 80∼90%, SKOV3 cells were washed three times with serum-free medium and divided into three groups: mock group, pSi-scramble siRNA group and pSi-N membranes . Hybridization was performed using the express Hyb buffer  with 32P-labeled cDNA of Survivin and actin as probes. Blots were exposed to Kodak MS film and then quantitated using a Molecular Dynamics Phosphorlmager.Total RNA was extracted from tissues with Trizol  following the manufacturer’s instructions. For Northern blot analysis, 20 μg of total RNA were separated by 1.2% agarose-formaldehyde gel, and blotted onto Hybridization-570) was determined with a microplate reader. The growth inhibition rate was calculated according to the following formula:SKOV3 cells were incubated in 96-well plates. The cell proliferation was determined by 3--2,5-diphenyltetrazolium bromide  assay and the number of viable cells were counted with a hemocytometer at 72 h after the transfection. The absorbance at 570 nm  containing 4 mmol/L ethylenediaminetetraacetic acid (EDTA), and fixed in cold 70% ethanol followed by centrifugation. The supernatant was removed and cells were washed once with PBS containing 4 mmol/L EDTA. Cells were then re-suspended in PBS containing 4 mmol/L EDTA, 20 ml/L of propidium iodide , 0.2% Triton X-100, and 40 mg/L RNase A followed by incubation for at least 30 min at 4°C. The cell cycle was detected with a flow cytometer  followed by the analysis with Cell Quest software. For the determination of apoptotic cells, 95 μl of floating cells were mixed with 0.1% AO/EB  followed by the observation under a microscope.6) were subcutaneously inoculated into the back of 18 nude mice. The tumor volume  was measured every 2∼3 d until the tumors reached about 50.56±36.45 mm3 (by day 12) in volume. Then, these nude mice were randomized into three groups (n=6): (1) mock transfection group ; (2) scrambled vector group (20 μg/mouse); (3) pSilencer2.1-U6-stat3-siRNA group (20 μg/mouse). The plasmids were diluted in 50 μl of PBS and injected percutaneously into the tumors by using a syringe with a 27-gauge needle. Immediately after the injection, tumors were pulsed with an electroporation generator . Pulses were delivered at a frequency of 1/sec and 150 v/cm for 50 ms. This process was repeated once a week, mice were sacrificed on day 33. The tumor volume was determined and then tumors were collected for H&E staining and terminal deoxynucleotidy1 transferase-mediated nick end labeling (TUNEL) assay.SKOV3 cells . Paraffin-embedded tissues were cut into 3-μm sections, deparaffinized and hydrated according to the standard protocol. After the incubation with proteinase K (200 μg/ml) for 30 min at 21°C, the TUNEL reaction mixture containing bromodeoxyuridine triphosphate, terminal deoxynucleotidy1 transferase, and reaction buffer was added onto the sections which were incubated in a humidified chamber for 60 s at 37°C, followed by washing and incubation with a FITC-labeled anti-bromodeoxyuridine monoclonal antibody for 30 min at room temperature. The reaction was visualized under a fluorescence microscope. TUNEL-positive cells present green fluorescence. The apoptotic index was calculated as follows: apoptotic index =  ×100%.P<0.05 was considered statistically significant.The statistical analysis was done with SPSS 13.0 statistic software package. The significance of the differences between various samples was determined using the student’s two-tailed t test. The comparisons between medians were performed with the two-tailed Mann-Whitney test. A value of stat3 expressions in 85% of ovarian cancers were classified as ++ to +++, which were significantly different (P<0.001) from that in normal ovarian tissues (+).The STAT3 expression in normal ovarian tissue (n=20), ovarian cancer tissues (n=25) and ovarian cancer cells (SKOV3 cells) was determined by using immunohistochemistry. Normal ovarian tissues showed low to moderate expression of Stat3. In contrast, primary ovarian cancer tissues and ovarian cancer cells had moderate to high expression of STAT3 . Immunohstat3 gene can significantly suppress STAT3 protein expression.stat3 gene were designed previously which inhibited STAT3 expression significantly in prostate cancer cells.stat3-3 could decrease the STAT3 expression significantly at both mRNA   . To detec tissue  and sevec tissue , which wstat3 plays an important role in maintaining physiological functions of cells. STAT3 is a critical element in the epidermal growth factor receptor (EGFR), interleukin-6 (IL-6)/Janus kinase (JAK) and other carcinogenic tyrosine kinase signaling pathways. Evidence reveals STAT3 is expressed in a variety of human malignancies, including leukemia, multiple myeloma, multiple melanoma, squamous cell carcinoma of the head and neck (SCCHN), breast cancer, prostate cancer, ovarian cancer and lung cancer. The activated stat3 plays crucial roles in the occurrence, growth, apoptosis inhibition of cancer cells.et al.stat3 can be used as a novel target for the anti-cancer therapy and may become a novel cancer marker.Under physiological conditions, STAT3 activation is a rapid process and activated STAT3 has short half life. The study has demonstrated stat3, only siRNA targeting stat3-3 could markedly inhibit the STAT3 expression in SKOV3 cells demonstrated by Western Blot and Northern Blot. Then, siRNA-stat3-3 was used to transfect SKOV3 cells for further experiments. Results revealed transfection with siRNA-stat3 significantly decreased the expressions of BcL-2, cyclin D1 and c-Myc in SKOV3 cells. In addition, stat3 siRNA inhibits the in vitro growth and survival, induces apoptosis of SKOV3 cells and arrests SKOV3 cells in G1 phase. Furthermore, the anti-tumor effects of stat3 siRNA were also confirmed in ovarian cancer bearing nude mice. Taken together, these findings demonstrated the in vitro and in vivo anti-tumor effects of stat3 siRNA.Our results showed the STAT3 expression was significantly increased in human ovarian cancer when compared with that in normal ovary tissues. Among the three pairs of double stranded siRNA oligonucleotide against et al.stat3 siRNA in vitro and in vivo. However, in the present study, we designed three pairs of double stranded siRNA oligonucleotide against stat3, and the most effective siRNA in the inhibition of STAT3 expression was selected. In addition, the suppressed expression of stat3 was confirmed by Western Blot and Northern Blot at protein and mRNA levels, respectively. In the in vivo experiment, we detected not only changes of the tumor size but also the apoptosis of cancer cells in the tumors. In 2008, Huang et al. applied shRNA to knockdown stat3 expression in CAOV3 ovarian cancer cell line demonstrating similar results.stat3 expression in the treatment of ovarian cancer.Our results were similar to that of Cai stat3 may be related to the cell cycle arrest and promotion of the apoptosis of cancer cells.Bcl-2 is the founding member of the Bcl-2 family of apoptosis regulator proteins. The Bcl-2 gene has been implicated in a number of cancers. It is also thought to be involved in resistance to the conventional cancer treatment.stat3 siRNA treatment can significantly inhibit the growth of ovarian cancer cells and promote their apoptosis. Thus, we postulate that STAT3 can be used as a therapeutic target for ovarian cancer patients and RNA interference with siRNA targeting stat3 may become an effective strategy for the treatment of ovarian cancer.In summary, our results indicate


S. cerevisiae to Drosophila and human, and is required for the initiation of DNA replication. Knockdown of Drosophila Mcm10 (dMcm10) by RNA interference in eye imaginal discs induces abnormal eye morphology (rough eye phenotype), and the number of ommatidia is decreased in adult eyes. We also observed a delay in the S phase and M phase in eye discs of dMcm10 knockdown fly lines. These results show important roles for dMcm10 in the progression of S and M phases. Furthermore, genome damage and apoptosis were induced by dMcm10 knockdown in eye imaginal discs. Surprisingly, when we used deadpan-lacZ and klingon-lacZ enhancer trap lines to monitor the photoreceptor cells in eye discs, knockdown of dMcm10 by the GMR-GAL4 driver reduced the signals of R7 photoreceptor cells. These data suggest an involvement of dMcm10 in R7 cell differentiation. This involvement appears to be independent of the apoptosis induced by dMcm10 knockdown. Together, these results suggest that dMcm10 knockdown has an effect on DNA replication and R7 cell differentiation.Mini chromosome maintenance 10 (Mcm10) is an essential protein, which is conserved from  Xenopus laevis suggested that Mcm10 is recruited to chromatin after origin licensing but before the initiation step of DNA replication in vitro studies in yeast have shown that the recruitment of Mcm10 to the origin occurs after the assembly of the Cdc45-Mcm2-7-GINS complex (CMG) in vivo studies demonstrated that Cdc45 loading onto chromatin was independent of Mcm10 Minichromosome maintenance protein 10 (Mcm10) is needed for DNA replication in eukaryotes A direct or indirect role of Mcm10 in remodeling the CMG helicase to promote DNA unwinding has been debated for years. Some groups have suggested that Mcm10 works as an active helicase activator Xenopus showed that the NTD has a role in homodimerization Mcm10 has three functional domains: an N-terminal domain (NTD), the internal domain (ID) and a metazoan-specific C-terminal domain (CTD). The N-terminal domain is not highly conserved across species. This domain was reported to be responsible for self-association of Mcm10 protein S. cerevisiae implicate Mcm10 in transcriptional repression of the mating type loci, and link DNA replication proteins to heterochromatin formation S. cerevisiae showed that mutations of the sin3 and sds3 genes, encoding components of the Rpd3 complex, improved the viability of Mcm10 mutants Drosophila cultured cells leads to under-condensed metaphase chromosomes Drosophila Mcm10 (dMcm10) demonstrates that the protein has a role in heterochromatic silencing and chromosome condensation, while those with a C-terminal truncation allele of dMcm10 indicate that the CTD of dMcm10 is important for DNA replication in vivo studies with Drosophila have been performed on the salivary glands and wing discs Although extensive studies have been carried out on the role of Mcm10 in initiation of DNA replication, few studies have been reported that address the involvement of Mcm10 in the regulation of chromatin structure. Several studies in Drosophila eye imaginal disc has been widely used for the study of DNA replication due to the highly synchronized mitotic waves which pass across the disc. In third instar larvae, the morphogenetic furrow (MF) appears at the posterior end of the eye imaginal discs and slowly moves in the anterior direction. Cells in front of the MF proliferate asynchronously, while those on the MF are arrested synchronously in the G1 phase. Cells behind the MF either leave the cell cycle and differentiate into the photoreceptors of the adult ommatidium, or undergo one more cell division. This cell cycle is a final synchronous round and produces an S-phase band (the second mitotic wave-SMW), after which these cells form a reservoir of cells for subsequent differentiation events. Mitotic cells are not normally present in the SMW The Drosophila eye discs are also useful for development studies as defects in particular cell lineages can be easily observed. The Drosophila compound eye consists of nearly 800 ommatidia. Each ommatidial unit is surrounded by a hexagonal lattice of 12 interommatidial cells which are comprised of bristles, and secondary and tertiary pigment cells. It contains eight photoreceptor cells, four cone cells, and two primary pigment cells. The photoreceptors (R cells) are divided into three different types depending on their genetic and morphological function. The outer (R1–R6) lies in a ring surrounding two central receptors; R7, the distal, or outer, central cells; and R8, the proximal, or inner, central cells. R cells have been found to be generated sequentially: R8 is generated first, with movement posterior from the MF, then cells are added pair wise , and R7 is the last photoreceptor to be added to the precluster. Identification of each photoreceptor cell can be determined using enhancer trap lines expressing a nuclear-localized form of E. coli β-galactosidase controlled by the specific enhancer-promoter located nearby the P-element.Drosophila Mcm10 during eye development. A small and rough eye phenotype was observed in the dMcm10 knockdown flies. We showed that the knockdown of dMcm10 caused a delay in the S phase and subsequently lead to a delay in M phase. Furthermore, we found that knockdown of dMcm10 also resulted in DNA damage and then induced apoptosis in cells. Surprisingly, a significant reduction of R7 photoreceptor cells in the eye imaginal discs was found in dMcm10 knockdown flies crossed with enhancer trap lines. These defects in R7 cell differentiation appeared to be independent of apoptosis induction. Thus, dMcm10 knockdown has an effect on both DNA replication and R7 cell differentiation.Here we analyze the role of 3-117dMcm10IR were obtained from VDRC stock center carrying dsRNA that targets the region of dMCM10 from aa3 to aa117 klingon), P82 (inserted into deadpan) were obtained from Dr. Y. Hiromi. These lines express the β-galactosidase marker in photoreceptor cells (R) of R7 or R3/R4/R7. All other stocks used in this study were obtained from Bloomington Drosophila stock center or Kyoto Drosophila Genetic Resource Center.Fly stocks were reared at 25°C on standard food. Either yellow white flies or Canton S flies were used for the wild-type strain. Transgenic flies carrying UAS-Drosophila whole bodies of adult flies. The fragment was cloned into the pWIZ vector 633-700dMcm10IR. The full length dMcm10 cDNA fragment was amplified with the primers 5′-EcoRI-HA-dMcm10 (5′-CGAATTCATGGCTTACCCATACGATGTTCCAGATTACGCTATGGGTCCTGCTGAGAAATC) and 3′-XhoI-dMcm10-2 (5′-CAACTCGAGTCACTCTTGATCGGGTACCA) and inserted into EcoRI and XhoI sites of pUAST HA-dMcm10. P element-mediated germ line transformation was carried out as described earlier dMcm10IR. These lines in which the transgene was inserted in the second and third chromosome, respectively showed essentially same phenotype. In the knockdown studies we mainly used the line carrying the UAS-633-700dMcm10IR gene on the third chromosome. Only one line was established for the over-expression line carrying UAS-HA-dMcm10 gene on the X chromosome.The dMcm10 cDNA fragment encoding amino acids (aa) 633-700 was obtained by RT-PCR with total RNA isolated from E. coli BL21. Lysates of cells were prepared by sonication in PBS containing 1 mM PMSF, and 1 μM each of pepstatin and leupeptin. The recombinant protein was purified with a glutathione-Sepharose column . Then, the purified Mcm10 protein was used to elicit polyclonal antibody production in rabbit. Polyclonal antibodies reacting with dMcm10 were affinity-purified from anti-serum using the NHS-activated Sepharose HP  coupled with GST-dMcm10 fusion protein after passing through the GST-conjugated Sepharose HP.To generate pGEX-dMcm10, a cDNA fragment was amplified by RT-PCR and the PCR product was inserted into pGEX-6T-1 . The recombinant GST-dMcm10 protein was expressed in yw, +, Act5C-GAL4/UAS-HA-dMcm10; and yw, +, Act5C-GAL4/UAS-633-700dMcm10IR were washed in PBS and homogenized in an extraction buffer containing 50 mM Tris-HCl (pH7.5), 5 mM MgCl2, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 0.1% NP-40, 10 μg/ml each of aprotinin, leupeptin, pepstatin A and 1 g/ml each of antipain, chymostatin and phosphoramidon. Homogenates were centrifuged, and 20 μg of protein from each of these extracts was electrophoretically separated on 10% polyacrylamide gels containing 10% SDS and transferred to Polyvinilidenedifluoride (PVDF) membrane (BIO-RAD) in a solution containing 25 mM Tris-HCl, 192 mM glycine and 20% methanol at 25°C for 2 h. The blotted membranes were blocked with a blocking buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl and 5% skimmed milk at 25°C for 1 h and then incubated with a rabbit anti-dMcm10 antibody at 1∶5000 dilution or mouse anti-α-tubulin monoclonal antibody (Sigma-Aldrich) at 1∶8000 dilution at 4°C for 16 h. After washing with TBS containing 0.05% Tween20, the blots were incubated with horseradish peroxidase-labeled rabbit anti-IgG  at 1∶20,000 dilution or mouse anti-IgG  at 1∶10,000 dilution at 25°C for 1 h. Detection was performed with ECL Western blotting analysis system  with LumivisionPro HS II image analyzer (Aisin seiki).Third instar larvae of flies with the following genotypes: Canton S; Adult flies were anesthetized, mounted on stages and observed with a VE-7800 (Keyence Inc.) scanning electron microscope or JSM-6510A (JEOL) analytical scanning electron microscope. In every experiment, at least five adult flies of each line were chosen for scanning electron microscopy observation to assess the eye phenotype, and these experiments were repeated 3 times. In the experiments, no significant variation in eye phenotype among the five individuals was observed.Drosophila polymerase α (dpolα) , samples were blocked with PBS containing 0.15% Triton X-100 and 10% normal goat serum for 30 min at 25°C and incubated with diluted primary antibodies in PBS or in PBS containing 0.15% Triton X-100 and 10% normal goat serum for 16 h at 4°C. The following antibodies were used as primary antibodies: rabbit anti-dMcm10 (diluted at 1∶100), mouse anti-) 1∶200, , rabbit Detection of cells in S phase was performed using an EdU-labeling kit from Invitrogen (Click-iT EdU Alexa Fluor 594 Imaging Kit). Third instar larvae were dissected in PBS and the imaginal discs were suspended in Grace's insect medium in the presence of 10 μM EdU for 60 min at 25°C. The samples then were fixed with 3.7% Formaldehyde in PBS for 15 min at 25°C. After fixing, samples were washed with 3% BSA in PBS and were permeabilized in 0.5% Triton X-100 in PBS for 20 min at 25°C. Then, samples were washed with 3% BSA in PBS and incubated with Click-iT reaction cocktails for 30 min at 25°C (following the manufacturers' instructions). After further washing with 3% BSA in PBS and PBS, samples were stained with Hoechst 33342 (Invitrogen) for labeling DNA, and finally samples were mounted and observed as described in immunostaining section.hs-flp; Act5C>FRT y FRT>GAL4, UAS-GFP were crossed with UAS-dMcm10IR and clones were identified by the presence of green fluorescent protein (GFP) expressed under control of the Act5C promoter. Flip-out was induced by heat shock (60 min at 37°C) 24–48 h after the eggs were laid.RNAi clones in eye discs were generated with a flip-out system The phospho-H2AvD, phospho-Histone H3 positive cells and EdU signals in the region posterior to the MF were counted and measured from six eye imaginal discs by using Meta Morph software. The experiments were repeated three times. Then, statistical analysis was conducted, as indicated in the figure legends, using GraphPad Prism 6. Every single set of data was calculated by using Welch's t-test. Significance is as described in the figure legends with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.Act5C-GAL4>UAS-HA-dMcm10 flies . Many dMcm10 signals appear as dots in the posterior region of the eye discs in 4 driver . Howeverwn flies . FurtherdMcm10 knockdown on the cell cycle progression in eye imaginal discs was examined. An EdU incorporation assay was used to monitor S phase, and immunostaining with the anti-phospho-histone H3 (PH3) antibody to monitor M phase cells. In third instar larvae, the morphogenetic furrow moves from posterior to anterior in eye discs. Cells in the region anterior to the morphogenetic furrow proliferate at random. All cells in the morphogenetic furrow are arrested in G1 phase and then they undergo one round of cell cycle consisting of G1, S, G2, M phases in a highly synchronized fashion before entering G0. The GMR-GAL4 driver expresses GAL4 in cells from morphogenetic furrow to the posterior end in eye discs. The EdU incorporation assay detected the synchronized S phase cells behind the morphogenetic furrow (MF) in the eye discs  to visualize DNA damage sites in the chromatin. The immunostaining data showed that more phospho-H2AvD positive cells were detected in the posterior region of dMcm10 knockdown eye discs  and B38 (inserted in klingon). P82 and B38 specifically express β-galactosidase in the photoreceptor cells of R3/R4/R7 and R7, respectively. The eye imaginal discs of third instar larvae were immunostained with anti-β-galactosidase antibody. R3/R4/R7 signals were detected in the eye discs of P82 control flies carrying GMR-GAL4>UAS-GFPIR  were used to rule out the possibility of off-target effect of the RNAi. These also showed similar reduction of the R7 photoreceptor cells in the eye discs ; Canton S (lane 2); yw, +, Act5C-GAL4/UAS-dMcm10IR (lane 3). The blots were probed with anti-dMcm10 antibodies . Anti-α-tubulin was used as a loading control. White arrowhead shows the position of dMcm10. M indicates marker.(TIF)Click here for additional data file.Figure S2Two different dMcm10 knockdown lines induced a similar delay in S phase in eye imaginal discs. The eye imaginal discs were labeled with EdU (Red). (A) GMR-GAL4/+; +; +; (B) w; +; UAS-633-700Mcm10IR; (C) w; UAS-3-117Mcm10IR; +; (D) GMR-GAL4/+; +; UAS-633-700Mcm10IR/UAS-633-700Mcm10IR; (E) GMR-GAL4/+; UAS-3-117Mcm10IR/UAS-3-117Mcm10IR; +. The arrows show the positions measured. The bars indicate 10 μm. The flies were reared at 28°C. White arrowheads indicate morphogenetic furrow (MF). (a) indicates anterior, (p) indicates posterior.(TIF)Click here for additional data file.Table S1Summary of effects of expression of dMcm10 dsRNA with several GAL4 driver lines.(DOCX)Click here for additional data file.


Bombyx mori) and evaluated the immunogenicity of the expression products. Four of five cattle vaccinated with these proteins developed high titers of FMDV-specific antibody and were completely protected against virulent homologous virus challenge with 10,000 50% bovine infectious doses (BID50). Furthermore, the 50% bovine protective dose (PD50) test was performed to assess the bovine potency of the empty capsid subunit vaccine and was shown to achieve 4.33 PD50 per dose. These data provide evidence that silkworm pupae can be used to express immunogenic FMDV proteins. This strategy might be used to develop a new generation of empty capsid subunit vaccines against a variety of diseases.Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that inflicts severe economic losses in the livestock industry. In 2009, FMDV serotype A caused outbreaks of FMD in cattle in China. Although an inactivated virus vaccine has proven effective to control FMD, its use may lead to new disease outbreaks due to a possible incomplete inactivation of the virus during the manufacturing process. Here, we expressed the P1-2A and the 3C coding regions of a serotype A FMDV field isolate in silkworm pupae ( Aphthovirus genus, family PicornaviridaeFoot-and-mouth disease (FMD) is a highly contagious disease of livestock that causes severe economic loss in susceptible cloven-hoofed animals, such as cattle, swine, sheep, and goats. FMD virus (FMDV) is a positive-sense single-stranded RNA virus belonging to the in vitro culture using cells is commonly used but insect larvae and pupae may also be used for protein production To overcome these issues, a number of different types of vaccines have been developed including synthesized peptide vaccines Bombyx mori) and assembled into empty capsids. Subsequent studies evaluated the ability of this serotype A FMDV empty capsid subunit vaccine to induce specific antibodies and protect cattle against homologous challenge after a single vaccination.FMDV consists of seven serotypes, named O, A, C, Asia I, SATI, SAT II, and SAT III, where viral infection or vaccination with one serotype does not confer protection against the other serotypes Bm-N cells was analyzed by immunofluorescence assay (IFA). Fluorescence microscopy revealed that Bm-N cells infected with rBmNPV (P1-2A3C) produced specific fluorescence, while only very weak background fluorescence appeared in the uninfected control cells (Bm-N cell.Recombinant baculovirus rBmNPV (P1-2A3C) was generated by transfection of Bm-N cells with a recombinant bacmid that contained consecutive P1-2A and 3C coding regions of serotype A FMDV. The expression of the P1-2A3C polyprotein in ol cells . These dSimilar to the FMDV antigen used as positive control, antigen-capture sandwich-ELISA indicated that the signal detected in the pupae infected with rBmNPV (P1-2A3C) decreased with dilution . FurtherThe expressed empty capsid-like particles were further purified by sucrose gradient centrifugation and collected fractions were analyzed by antigen-capture sandwich ELISA. The results revealed that the maximum amount of empty capsid- like particles was found in fractions 4–9 . From thCattle were vaccinated with extracts prepared from silkworm pupae infected with rBmNPV(P12A3C). As directed by the method recommended by the OIE/FAO World Reference Laboratory (WRL) for FMDV , we evaluated the specific anti-FMDV antibody response by the liquid-phase blocking enzyme-linked immunosorbent assay (LPB-ELISA) 50 FMDV A/WH/CHA/09. Body temperature, mouth, and feet were observed consecutively for 10 days to evaluate the incidence of disease  developed a detectable level anti-FMDV antibody response at 14 dpv, and some reached a titer of 256. By 21 dpv, the antibody level was maintained at the same level or higher. In contrast, the antibody level in control cattle was not significant .50 FMDV A/WH/CHA/09. Body temperature was recorded, and mouth and feet were examined for 10 days to determine the incidence of disease. Four of the five cattle were considered protected in the 1 dose immunized group, three of five cattle were protected in the 1/3rd dose group, and two of five cattle were protected in the 1/9th dose group. The vaccine potency of the batch immunized with the expressed P1-2A3C antigens reached 4.33 PD50 per dose . Production of FMDV empty capsid requires the proteolytic processing of P1 by protease 3C to generate the structural proteins VP0, VP1, and VP3, which self-assemble to form the viral capsid In March 2009, an FMDV serotype A outbreak in Wuhan, China resulted in the slaughter of approximately 9000 infected and susceptible animals. Prior to this outbreak there had been no previous outbreaks of serotype A FMDV in China and little research on a serotype A FMDV vaccine had been undertaken. Although vaccination with inactivated viruses has been shown to be efficient, it has been associated with several problems related to its safety and the discrimination between vaccinated and naturally infected animals in vitro.Veterinary products obtained using gene expression techniques are typically constrained by the final price of the antigen due to production costs. Thus, the production of a safe, low-cost source of antigens is required for feasible vaccine design. The silkworm expression system is an innovative and simple approach to achieve high protein expression levels Extracts of silkworm pupae infected with rBmNPV(P1-2A3C) were used to vaccinate five cattle and two controls. The anti-FMDV antibody response developed in cattle was evaluated using the LPB-ELISA at 14dpv, 21dpv and 28 dpv. The LPB-ELISA has previously been proved to correlate well with the standard virus neutralization test (VNT) for measuring the serum antibody titers of FMD vaccinated animals 50 per dose for cattle. Thus, when employed for routine prophylactic use, the vaccine should contain at least 3 PD50 per dose for cattle as recommended by the OIE. In conclusion, the use of silkworm pupae (Bombyx mori) as bioreactors is feasible for the expression of immunogenic proteins to develop a next generation FMDV empty capsid subunit vaccine.Using the data obtained from challenge experiments, the bovine potency test protocol described by the OIE was used to evaluate the efficacy of the empty capsid subunit vaccine. The empty capsid subunit vaccine potency was determined to be 4.33 PDAll the experiments involving animals were approved by the animal ethics committee of Lanzhou Veterinary Research Institute (Permit Number: 09–26) and followed the national guidelines for the use of animals in scientific research.Bm-N cell line, and silkworm variety JY1 were maintained at the Biotechnology Research Institute of the Chinese Academy of Agriculture Sciences. The BmBacPAK-6 and recombinant viruses were maintained in Bm-N cells at 27°C in TC-100 insect medium  supplemented with 10% heat-inactivated fetal bovine serum .FMDV A/WH/CHA/09 strain was propagated in the BHK-21 cell line which is extensively used for the diagnosis and identification of FMDV 18 primer  at 42°C for 1 h following the recommended protocol. PCR was used to amplify the intact P1-2A and 3C protease coding regions from the cDNA using two pairs of specific primers: forward primer for P1-2A, AGATCTACCATGGGGGCAGGGCAATCTAGCC-3′5′-ATA (BglII restriction site underlined); reverse primer for P1-2A, TCTAGATGACATGTCCTCTTGCATCTGGTTA-3′5′-GCG (XbaI restriction site underlined); forward primer for 3C, TCTAGAAAGAAACCTGTCGCTTTGAAAGT-3′5′-GCG (XbaI restriction site underlined); reverse primer for 3C, AGATCTCTACTCGTGGTGTGGTTCGGGAT-3′5′-ATA (BglII restriction site underlined). The target fragments of P1-2A and 3C were sequentially cloned into BamHI/XbaI and XbaI/BglII sites, respectively, of the baculovirus transfer vector, pVL1393, and named pVL-P12A3C. The pVL-P12A3C baculovirus transfer plasmid was co-transfected with linearized Bm-BacPAK6 DNA into Bm-N cells via a liposome-mediated method Viral RNA was extracted from cell culture supernatants using RNeasy  and used immediately for cDNA synthesis. The cDNA was synthesized using AMV reverse transcriptase and Oligo(dT)Bm-N cells infected with rBmNPV(P1-2A3C) was analyzed by IFA. The rBmNPV (P1-2A3C) was multiplied in Bm-N cells. Bm-N cells (2.0×105) were cultured on cover slips and inoculated with Bm-P12A3C at a multiplicity of infection (MOI) of 10. At 48 h post-infection (hpi), IFA was conducted to analyze the expression of FMDV proteins. Cells were then rinsed twice phosphate-buffered saline (PBS) and fixed in 100% cold acetone (−20°C for 30 min). Samples were incubated with rabbit anti-FMDV serum (37°C for 30 min) in a humid box, washed five times with PBS, and then stained with fluorescein-conjugated goat anti-rabbit serum at 37°C for 30 min. Cover slips were coated with glycerin and observed on a fluorescence microscope. Bm-N cells infected with BmBacPAK-6 were used as negative controls for these analyses.The expression of FMDV P1-2A3C polyprotein in 5 plaque-forming units (pfu) per pupae. Approximately 50 g of silkworm pupae infected with rBmNPV(P1-2A3C) were homogenized in 50 mL of homogenization buffer  using a polytron PT2100 homogenizer  at 20,000 rpm. The homogenate was clarified by centrifugation at 10,000×g for 15 min at 4°C. After removal of the lipid layer with a spatula, the supernatant was collected on ice and stored at −20°C. An antigen-capture sandwich enzyme-linked immunosorbent assay (ELISA) and western immunoblot were used for detection of P1-2A3C polyprotein.The silkworm pupae were infected with the recombinant virus at approximately 102O2 plus orthophenylene diamine) was added, allowed to react for 15 min and stopped by the addition of 1M H2SO4. The absorbance at 492 nm was determined for each well.The antigen-capture sandwich ELISA was carried out as described by the World Organization for Animal Health, Office International desEpizooties (OIE) standard for FMDV serotyping. In brief, microtiter plates were coated with anti-FMDV type A rabbit sera at a dilution of 1∶1000 in carbonate coating buffer overnight at 4°C. All subsequent incubations were performed at 37°C for 1 h, using 50 µL of reagent added to each well. The extracts of silkworm pupae infected with rBmNPV(P1-2A3C) were serially diluted two-fold and added to the wells, and inactivated FMDV A/WH/CHA/09 virions and mock-infected antigen were added as positive and negative controls, respectively. Plates were incubated with anti-FMDV serotype A guinea pig sera, followed by horseradish peroxidase-conjugate rabbit anti-guinea pig IgG  at 1∶10,000 dilution. Substrate  and passed through a 0.45 µm filter . The filtrate was then purified by gel filtration using a Sepharose 4 Fast Flow (FF) column  with elution buffer . The first elution peak containing the empty capsid-like particles was collected and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were electro-transferred onto a nitrocellulose membrane, blocked, and then incubated with rabbit FMDV antiserum. Rabbit primary antibodies were detected using IRDye 700DX-conjugated anti-rabbit antibody , and the membrane was scanned using the LiCor Odyssey fluorescent scanning system .g for 2.5 h at 4°C. The resulting gradients were collected in 20 500 µL-fractions and analyzed by antigen-capture sandwich ELISA. Fractions containing empty capsid-like particles were collected and stored at 4°C for subsequent analysis. For immunogold labeling of the empty capsid-like particles, 20 µL of each fraction was placed onto grids and incubated for 5 min. Grids were blocked with 3% bovine serum albumin in PBS for 30 min and incubated with anti-FMDV type A rabbit sera for 30 min at room temperature. Grids were then washed three times with PBS and incubated with gold-conjugated secondary antibodies (10-nm gold-conjugated anti-rabbit antibody) for 30 min at room temperature. After secondary-antibody incubation, grids were washed with PBS three times, followed by negative staining with 1% phosphotungstic acid. Transmission electron micrographs were captured.The elution peak containing empty capsid-like particles were layered onto a 15–45% (w/v) sucrose gradient in NET buffer  and centrifuged at 35,000×http://www.oie.int/eng/norms/MMANUAL/A_00024.htm). A detection kit was prepared by the National Foot and Mouth Disease Reference Laboratory (OIE Reference laboratory for Foot and Mouth Disease), Lanzhou Veterinary Research Institute. Cattle with an antibody titer <8 were housed in the animal biosafety level (ABSL)-3 laboratory at Lanzhou Veterinary Research Institute. Five cattle were immunized (2 mL per cattle) by intramuscular inoculation in the neck with extracts of silkworm pupae infected with rBmNPV(P1-2A3C). Two control cattle were vaccinated with the same dose of extracts from silkworm pupae infected with Bm-BacPAK6. Cattle sera were collected at 14, 21, and 28 dpv. Anti-FMDV antibody was detected using the LPB-ELISA, as previously described.Silkworm pupae extracts were prepared by homogenizing 50 g of silkworm pupae infected with rBmNPV(P1-2A3C) in 250 mL PBS mixed with Montanide ISA 206 oil adjuvant. In order to determine the antibody titer for screening of candidate cattle for vaccination, liquid-phase blocking (LPB)-ELISA was performed according to the standard OIE method (http://www.oie.int/eng/norms/MMANUAL/A_00024.htm), all animals were challenged at 28 dpv by intradermal inoculation at two sites in the tongue with 10,000 bovine infectious doses (BID50) of A/WH/CHA/09. The body temperature of the animals was monitored daily. Restrained animals were carefully examined in the mouth and feet every day for the first 10 dpc.According to the standard protocol described by the OIE , 1/3th dose (0.66 mL) or 1/9th dose (0.22 mL) of the empty capsid subunit vaccine. Cattle sera were collected at 14 and 21 dpv, and determined by LPB-ELISA. All animals were challenged 21 dpv with 10,000 BID50 of A/WH/CHA/09 administered by intradermal inoculation into two sites on the upper surface of the tongue. The cattle were observed daily for clinical signs of FMD for 10 dpc. Control cattle developed lesions on at least three feet. Unprotected animals showed lesions at sites other than the tongue. The PD50 of the vaccine was calculated based on the Karber method.The OIE PD


Worldwide emergence of variant viruses has prompted a change in the 2005–2006 H3N2 influenza A vaccine strain. In July 2004, an outbreak of influenza A (H3N2) was detected at 3 Bhutanese refugee camps in southeastern Nepal. Hemagglutination inhibition showed that ≈40% of the viruses from this outbreak were antigenically distinct from the A/Wyoming/3/03 vaccine strain. Four amino acid differences were observed in most of the 26 isolates compared with the A/Wyoming/3/2003 vaccine strain. All 4 substitutions are located within or adjacent to known antibody-binding sites. Several isolates showed a lysine-to-asparagine substitution at position 145 (K145N) in the hemagglutinin molecule, which may be noteworthy since position 145 is located within a glycosylation site and adjacent to an antibody-binding site. H3N2 viruses continue to drift from the vaccine strain and may remain as the dominant strains during the 2005–2006 influenza season. Thus, the 2005–2006 Northern Hemisphere vaccine strain was changed to A/California/7/2004, a virus with all 4 amino acid substitutions observed in these Nepalese isolates. The 2003–2004 influenza season was severe in terms of its impact on illness because of widespread circulation of antigenically distinct influenza A (H3N2) Fujian-like viruses. These viruses first appeared late during the 2002–2003 influenza season and continued to persist as the dominant circulating strain throughout the subsequent 2003–2004 influenza season, replacing the A/Panama/2007/99-like H3N2 viruses . Of the The HA1 segment of the influenza HA protein is the most rapidly evolving gene product  and playIn July 2004, an outbreak of influenza A (H3N2) was detected in patients at 3 Bhutanese refugee camps in southeastern Nepal. To elucidate the molecular mechanism underlying the emergence of this H3N2 outbreak, we conducted a molecular analysis of the HA1 region of the HA protein. In this report, we describe the epidemiologic and molecular aspects of isolates obtained from this off-season influenza A (H3N2) outbreak.Sixty-four patients in Nepal that met US Department of Defense enrollment criteria  for infl4, 1× enzyme mixture, and 400 nmol/L primers  by using the SuperScript III One-Step RT-PCR System . PCR thermocycling consisted of an RT step at 50°C for 30 min, hot start activation at 95°C for 3 min, followed by 40 amplification cycles of 95°C for 30 s, 52°C for 15 s, and 68°C for 1 min, with a final extension cycle at 68°C for 7 min. All PCR products were visualized after electrophoresis in 2% precast gels stained with ethidium bromide (Invitrogen) under UV illumination. PCR products were purified by using the PCR Purification Kit . The HA1 amplicon (1177 bp) was sequenced by using the H3-F7 and H3R-1184 PCR primers (described above) and 2 additional internal oligonucleotides, H3R-466 (5´-GGT-GCA-ACC-AAT-TCA-ATC-3´) and H3F-282 (5´-CAG-CAA-CTG-TTA-CCC-3´).RNA was extracted from 48-hour shell vial cultures  by usingUnincorporated fluorescent nucleotides were removed by using a Dye Ex 96-well plate kit (Qiagen) according to the manufacturer's recommendations. Nucleotide sequencing was performed by using the Big Dye Terminator v3.1 Kit and analyzed by using an ABI 3100 Genetic Analyzer  according to the manufacturer's specifications. Multiple sequence alignments, protein translation, and phylogenetic analysis were performed with the DNAStar  software package. Three-dimensional HA protein structures were generated by using MOLMOL  and the Clinical evaluations and throat specimens were obtained from 64 patients from 3 refugee camps in southeastern Nepal . Of the HI was performed by using postinfection ferret antisera with reference antigens that included the 2004–2005 H3N2 vaccine seed strain (A/Wyoming/03/2003) and the 2005–2006 Southern Hemisphere H3N2 vaccine strain (A/Wellington/1/2004). When compared with A/Wyoming/03/2003, 4 of 9 Nepal isolates showed 4-fold lower titers  than that allowed for homologous titer of the reference antisera. This indicated that these 4 isolates were antigenically distinct. Six of 9 Nepal isolates were antigenically distinct when compared with the A/Wellington/1/2004 strain and showed a 4-fold (1:160 versus 1:640) reduction in titer to ferret antisera .RT-PCR-based molecular subtyping showed that all 42 specimens were the H3N2 influenza subtype. Twenty-six of the 42 influenza A–positive samples were randomly selected for molecular characterization using direct nucleotide sequencing of the HA gene. The 26 Nepal isolates exhibited 99.8% nucleotide sequence identity and contained the Fujian-like amino acid substitutions at positions 155 (H155T) and 156 (Q156H) in the HA protein . AlignmeOf the 26 Nepal strains examined, 24 exhibited a novel lysine-to-asparagine substitution at position 145 in the HA protein (K145N). This substitution is noteworthy because most strains characterized in 2003–2004, including the Fujian/411/2002 vaccine strain, contained a lysine (K) at this position. Prior to this outbreak, the US Department of Defense had only observed K145N substitutions in 6 strains obtained from Ramstein, Germany, (data not shown) in June 2004. Additionally, all 26 Nepal sequences exhibited a serine-to-asparagine substitution at position 189 (S189N) that had also been observed in the 6 isolates from Germany, as well as in a few isolates from Asia characterized at the end of the 2003–2004 influenza season.Two other substitution mutations in the HA1 hemagglutinin, i.e., valine to isoleucine at position 226 (V226I) and serine to proline at position 227 (S227P), were also observed in 24 (92%) and 26 of 26 of the Nepal isolates, respectively. Both substitutions differ from most influenza A H3N2 field isolates collected in 2003–2004, including the Fujian and Wyoming vaccine strain for 2004–2005 .The phylogeny of H3N2 HA proteins indicates a drifting of the Nepal isolates from the A/Fujian/411/03 and A/Wyoming/03/03 vaccine strains and shows that these outbreak isolates have a higher genetic homology to A/Wellington/1/04, a prototype strain selected as the 2005–2006 Southern Hemisphere H3 vaccine strain . The A/WThree-dimensional views of influenza HA proteins highlighting amino acid changes in a representative Nepal isolate and the A/Wyoming/3/03 vaccine strains are shown in The 4 substitutions described represent a growing lineage of influenza A (H3N2) viruses characterized since July 2004. Three amino acid changes are confined within known antibody-binding sites, i.e., the S189N change within antibody-binding site B  and the The K145N mutation represents a change from a charged to uncharged amino acid R group. This change may affect protein-protein interactions since it is immediately adjacent to antibody-binding site A, where neutralizing antibodies have been shown to bind . AdditioCollectively, the clinical isolates obtained from this outbreak in Nepal cannot be considered antigenically distinct from the A/Wyoming/3/03 vaccine strain because only 4 of 9 isolates evaluated exhibited 4-fold lower titers by HI . FurtherWith the exception of A/Nepal/1670/2004 and A/Nepal/1672/2004, all isolates evaluated by HI  exhibiteIn this report, we describe the genetic analysis of the HA proteins from viruses obtained from an early season outbreak and compare them to current vaccine strains. Three amino acids changes  were noted in known ,5 antiboThe K145N substitution change has become a marker for an increasingly large subset of the Fujian-like viruses. CDC and the US Department of Defense have recently characterized viruses with the K145N change in Singapore, Taiwan, China, Australia, Canada, and the United States. In February 2005, WHO reported the emergence of a new influenza H3N2 strain in the United States. The A/California/7/2004 strain, which was first identified in the United States in September 2004, contains all 4 changes observed in isolates from this Nepalese outbreak. The A/California/7/2004 strain differs by only 1 amino acid in HA1 (which is of no immunologic importance) from most isolates from the outbreak in Nepal. All viruses characterized  subsequent to the preparation of this report (March 2005) by the US Department of Defense are genetically similar in amino acid sequence to these Nepalese strains . Most of the isolates (80%) analyzed by CDC since October 2004 are antigenically related to A/California (