Streptococcus mutans and closely related species. Two component systems (TCSs) composed of a signal sensing histidine kinase (HK) and a response regulator (RR) play key roles in pathogenicity, but have not been comparatively studied for these oral bacterial pathogens.Mutans streptococci are a group of gram-positive bacteria including the primary cariogenic dental pathogen S. sobrinus DSM20742, S. ratti DSM20564 and six S. mutans strains, were identified and compared to the TCSs of S. mutans UA159 and NN2025, two previously genome sequenced S. mutans strains. Ortholog analysis revealed 18 TCS clusters (HK-RR pairs), 2 orphan HKs and 2 orphan RRs, of which 8 TCS clusters were common to all 10 strains, 6 were absent in one or more strains, and the other 4 were exclusive to individual strains. Further classification of the predicted HKs and RRs revealed interesting aspects of their putative functions. While TCS complements were comparable within the six S. mutans strains, S. sobrinus DSM20742 lacked TCSs possibly involved in acid tolerance and fructan catabolism, and S. ratti DSM20564 possessed 3 unique TCSs but lacked the quorum-sensing related TCS (ComDE). Selected computational predictions were verified by PCR experiments.HKs and RRs of 8 newly sequenced mutans streptococci strains, including S. sobrinus and S. ratti in comparison to S. mutans, imply differences in their response mechanisms for survival in the dynamic oral environment. This genomic level study of TCSs should help in understanding the pathogenicity of these mutans streptococci strains.Differences in the TCS repertoires of mutans streptococci strains, especially those of  Streptococcus mutans is referred to as "mutans streptococci", and it includes in addition to S. mutans also S. ratti, and S. sobrinus among others. A common feature of the mutans streptococci is their ability to metabolize carbohydrates present in our daily diet to produce energy for their survival and to produce organic acids which erode and dematerialize the tooth enamel and dentin. These oral bacteria are able to grow in a community and colonize the oral environment by attaching to the tooth surfaces and forming biofilms. They can also tolerate and adapt to the harsh and rapidly changing physiological conditions of the oral cavity such as extreme acidity, fluctuation of nutrients, reactive oxygen species, and other environmental stresses [A group of oral streptococci which is closely related to the primary cariogenic dental pathogen stresses . They ocstresses . All togBacterial two-component signal transduction systems (TCS) play important roles for many bacteria by enabling them to detect and respond to diverse changes/stresses in the environment. The conspicuous absence of TCS proteins in mammalian genomes makes them interesting potential targets for the development of novel anti-bacterial drugs. A bacterial two-component system comprises in general a transmembrane sensor histidine kinase (HK) and a corresponding cytoplasmic response regulator (RR) encoded by genes located adjacently within the same operon, although stand-alone genes coding for HKs or RRs (without a corresponding cognate HK/RR in the same operon) have also been reported. In some cases, a HK and a RR are found to be merged in the same polypeptide, giving rise to a so called 'hybrid' HK protein. A HK protein is autophosphorylated at its conserved histidine (His) residue upon the recognition of a specific environmental stimulus. The phosphoryl group is then transferred to the aspartate (Asp) residue of the corresponding response regulator . While Hin silico studies have broadened our understanding of genomic repertoires essential for the growth and adjustment of the organisms to altering environmental challenges [S. mutans strain  has paved the way for researchers to carry out numerous molecular biological and functional genomic studies that help in understanding the robustness, genetic specificity and complexity of this bacterium as an oral pathogen [S. mutans UA159 [S. mutans isolates, namely S. mutans 5 DC8, KK21, KK23, AC4446, ATCC25175 and NCTC11060, as well as S. ratti DSM20564 and S. sobrinus DSM20742, we performed a systematic identification and classification of putative TCS proteins in the genomes of these eight mutans streptococci strains, using the well annotated genome of S. mutans UA159, as well as that of S. mutans NN2025, as references. Furthermore, we conducted a thorough comparative analysis of the identified TCS proteins among these strains which provides valuable insights into the conservation and divergence of TCS proteins in the mutans streptococci strains studied here. PCR experiments have been carried out to verify the presence of several predicted TCS genes of particular interest.With the advance of large scale sequencing technologies and bioinformatics tools, it has become possible to computationally predict the putative functions of genes/proteins from the whole genome of an organism. The prediction of TCS proteins using a whole genome-based computational approach has been carried out for different organisms. Such allenges . The genns UA159  and manyns UA159 -10. Aftehttp://134.28.64.65/www/index.php/genome/). The "high-quality draft" genomes obtained according to the definition by Chain et al. [S. mutans NN2025 and UA159 as references. By combining the Hidden Markov Model (HMM) profiling results and the information on putative operon organization, repertoires of potential TCS proteins (HKs and RRs) in the eight newly sequenced mutans streptococci strains were obtained, as shown in Table S. mutans NN2025 and UA159. The total numbers of TCS proteins identified are comparable among the 10 mutans streptococci strains.We have sequenced the genomes of eight mutans streptococci strains Table  using thn et al.  have an S. mutans UA159 and NN2025 as two reference strains. HKs and RRs are classified based on homology box analysis and output domain architecture, respectively. HPK1, HPK7, HPK8, HPK10, HPK11 represent HK families as found in the classification by Grebe et al. [Results are presented in comparison to e et al. .S. mutans strains. But across the species the conservation was clearly lower. Furthermore, it is obvious that, in most cases, putative HK alleles/orthologs within one TCS cluster exhibited a higher degree of diversity than the corresponding putative RR alleles/orthologs across the species. This could be attributed to the high variability of sensing/input domains harbored by the individual HKs, as will be discussed later in the classification of HKs and RRs. A clear clustering of putative HK and RR alleles/orthologs can be visualized in the phylogenetic trees shown in Figure By analyzing the putative operon organizations of genes encoding the identified TCS proteins 98.5% of the total putative HKs and 92.2% of the total putative RR were found to constitute HK-RR pairs. Ortholog analysis of the paired or non-paired TCS proteins among the 10 mutans streptococci strains revealed a total of 18 different TCS clusters, 2 orphan HKs and 2 orphan RRs Table . The numHPK, histidine protein kinase; C, E and M stand for cytoplasmic, extracytoplasmic and membrane sensing, respectively; N denotes that no sensing mechanism could be predicted either due to the complete absence or a truncation of the predicted sensory domain; SMU represents the proteins corresponding to the ORFs of S. mutans UA159, GI of S. mutans NN2025; smc of S. mutans 5 DC8; smd of S. mutans KK21; sme of S. mutans KK23; smf of S. mutans AC4446; smg of S. mutans ATCC 25175; smh of S. mutans NCTC 11060; sra of S. ratti DSM 20564; sob of S. sobrinus DSM 20742. The italic numbers in parentheses stand for similarities in percentage of the TCS proteins in S. ratti DSM20564 and S. sobrinus DSM20742 compared to the corresponding orthologs in S. mutans UA159. All other abbreviations are referred to in Table HKs are classified by two different approaches based on the homology-box analysis (classification denoted by C1) and the topology feature analysis (classification denoted by C2), respectively; RRs are classified based on the structure of their output domains (Abbreviations: S. ratti DSM20564 lacked any homologs of the sensor histidine kinase and the cognate response regulator of TCS-13 (known as ComD and ComE for S. mutans UA159). On the other hand, TCS-16, TCS-17 and TCS-18 were found to be unique to S. ratti DSM20564. TCS-15, a putative ortholog of a quorum sensing-related TCS cluster well studied in the gram-positive bacterium Bacillus subtilis, was uniquely identified in S. mutans NCTC11060, a serotype f blood isolate.Eight of the 18 TCS clusters  were common to all the strains. Six clusters  were observed to be absent in one or several strains. An interesting observation was that S. ratti DSM20564  and S. sobrinus DSM20742 , respectively. In contrast, an orphan RR  was found to be common to all the 10 strains. S. sobrinus DSM20742 harbored an additional unique orphan RR .Two orphan HKs were identified as uniquely present in No hybrid HKs could be detected in all the genomes of the 10 mutans streptococci strains compared in this study. Also, the more complex variant of the two-component system, known as the phosphor-relay system comprised of an autophosphorylatable hybrid HK, one or more histidine phosphotransferases (HPT) and a terminal RR was totally absent, because no HPT domains were found in these strains.HKs are multi-domain homodimeric proteins consisting of a more variable N-terminal for recognition of diverse signals from different cellular localizations  and a more conserved C-terminal containing the conserved histidine residue for signal transduction to its corresponding RR protein. The N-terminal regions of most HKs are characterized by the presence of one to several (up to 20) transmembrane helices , whereaFive HK families  were recognized among the 10 mutans streptococci strains. For each of the 10 strains the distributions of their HKs within these families are highlighted in Table S. ratti DSM20564  were grouped into the HPK1 family, accounting for more than 50% of the total HKs identified. HPK1 family has been known as the most common type among the histidine kinase families, characterized by highly conserved aspartate/glutamate, lysine/arginine and proline residues downstream of the conserved histidine residue in the H-boxes  fell within this family. This family was characterized by the presence of a tyrosine located two residues downstream of the conserved histidine residue, the absence of the H-box proline and the lack of the D and F-boxes. HK of TCS-13 (ComD) was absent in S. ratti DSM20564, which lacked any orthologs of both ComD and its cognate response regulator ComE.Two other HK families found were the HPK8 and the HPK10 families. The HPK8 family comprised only HKs of the TCS-10 cluster. Known as LytS in s Figure . The idecus spp. . Indeed,S. ratti DSM20564. A similar strategy as that used for the classification of HKs of the HPK8 family was performed by using BLASTp homologs to recognize conserved homology boxes. The H-box of this family was quite distinct from the H-boxes of other families in that the conserved histidine was flanked at its left by another histidine residue, in addition to the downstream arginine (R) and asparagine (N) residues characteristic of this family  could not be classified into any of the 11 HK families.The HK of TCS-14 and the unique orphan HK of the strain The homology boxes-based classification of HKs described above focused solely on the conserved sequence motifs in the C-terminal transmitter domains of histidine kinases. Since the biological function of TCS-mediated signal transduction manifests itself in signal input and output rather than in the communication between its two components, it is necessary to characterize the N-terminal sensory regions with respect to the signal sensing mechanism of a particular HK protein. To this end, the classification method proposed by Mascher et al.  was applExtracytoplasmic sensing HKs represent the largest group of membrane-bound sensor kinases. Among all the HKs identified, HKs of TCS-2 to TCS-6 as well as TCS-16 and TCS-17 were recognized as extracytoplasmic sensing HKs by displaying at the N-terminal region an extracytoplasmic putative signal perception domain composed of 110-140 amino acids, which were flanked by (at least) two transmembrane helices (TMs). The cytoplasmic part of the HK proteins harboring the transmitter domain comprised either a HisKA-HATPase_c domain  or a HAMP-HisKA-HATPase_c domain (HKs of TCS-6 and TCS-17). HisKA and HATPase_c are domains essential for the functionality of a histidine kinase. HAMP functions as a linker to bridge the transmembrane helix and the transmitter domain [S. mutans UA159, is typified by the presence of only one membrane-anchoring transmembrane helix and a very short extracytoplasmic linker containing 30 amino acids in the N-terminal region. In addition, both HKs of TCS-1 and TCS-18 possess a PAS domain. PAS domains are known to play important roles as sensory modules for sensing oxygen tension, cellular redox state, or light intensity [Cytoplasmic-sensing HKs include either membrane-anchored or soluble proteins with their sensing domains located inside the cytoplasm . Only HKntensity , competentensity , virulenntensity  and biofntensity  in many ntensity . It has ntensity , as alsontensity . On the ntensity . A largentensity .S. sobrinus DSM20742 the unique orphan HK2 (sob|13200014) was also assigned to this group. Orphan HK2 shared a sequence similarity of about 37% with the HK (sob|8500005) of TCS-13, which is the predicted ortholog of ComD in S. sobrinus DSM20742.The highly diverse group of HKs with sensing mechanisms linked to transmembrane regions (membrane-sensing HKs) is marked by the presence of 2 to 20 transmembrane helices implicated in signal perception. Such HKs lack a recognizable extracellular input domain. Signal sensing is either membrane associated or occurs directly within the membrane interface . AlthougFor the highly diverse membrane-sensing HK family, six subfamilies have been additionally defined based on features such as the number of TM helices, the type and sequential arrangement of sensory domains, the length of the inter-helix linker regions and the presence or absence of extracytoplasmic linker regions . By applA RR protein, most commonly a transcription factor , comprisThe majority of the putative RRs identified in this study were classified into the following 4 families: NarL, LytTR, AmiR and OmpR, with RRs of the OmpR family constituting the largest group. For each of the 10 compared strains the distributions of their RRs within these families are highlighted in Table S. ratti DSM20564, contained a RNA-binding output domain and was found to be an AmiR-like response regulator. The AmiR family is a relatively newly defined response regulator family possessing RNA-binding activity. In contrast to the commonly known phosphorylation mechanism in DNA-binding RRs, AmiR is normally controlled by ligand-regulated activation, in keeping with the substitution of the conserved aspartate phosphoryl acceptor residue by serine, as well as the replacements of additional amino acid residues required for phosphoryl transfer [While most RRs contain a DNA binding domain, there were two exceptions: the putative RR of TCS-14 lacked any currently known output domains and therefore could not be assigned to any of the known RR families. The RR (sra|1400053) of TCS-18, a unique TCS cluster of transfer . HoweverRRs of the OmpR family constituted the largest group Table . ProteinStreptococcus mutans strains [vicK gene coding for the HK of TCS-1 could be obtained, the cognate response regulator VicR has been found to be essential for the survival of S. mutans UA159 [vicR knockout mutants could be obtained for Streptococcus pneumoniae, but knockout of the corresponding histidine kinase gene vicK is possible, because vicR can be phosphorylated in vivo by vanS, a gene coding for a TCS histidine kinase from Enterococcus faecium, which helps to suppress some effects of a vicK kinase disruption in S. pneumoniae [Enterococcus faecium belongs to HPK1 family like VicK. Thus, we speculate that such cross-phosphorylation of VicR by a non-cognate HK most likely also holds true in S. mutans UA159, and probably also for other mutans streptococci strains. Clearly, the systematic classification of HKs and RRs carried out in this study reveals potential cross-talking candidates that will be helpful in the future studies of two-component systems.The homology box-based classification of HK proteins has been shown to be able to reveal the co-occurrence relationships between the various kinase families and regulator families . In othe strains . Such a ns UA159 . It has eumoniae . AccordiS. mutans strain UA159. In case no knowledge is available about the functions of some TCSs, information about putative orthologs of the corresponding TCS genes/proteins in organisms other than S. mutans UA159 are provided  and has been found to play a vital role in sucrose-dependent adherence and cariogenesis [Proteins of the TCS clusters 1, 2, 4, 6, 8, 10, 11, 12 and the orphan RR1 are common to all the 10 mutans streptococci strains compared here, indicating probably the functional importance of these TCS clusters for the adaptation and survival of these mutans streptococci  and a four-gene cluster levDEFG, which encode a fructose/mannose sugar:phosphotransferase system located immediately downstream of levQRST [S. sobrinus was also found to lack the levQ, levT and levDEFG genes. Taking together, these findings indicate dramatic differences in the regulation of fructan catabolism and the acid tolerance response of S. sobrinus DSM20742 in comparison to the S. mutans strains.The TCS-3 (CovSR) cluster was predicted to be absent in s UA 159 , and hass UA 159 . TCS-9  [S. mutans KK23 and S. mutans NN2025 carried two and three genes, respectively, which encode putative bacteriocin precursor peptides sharing more than 60% similarity with SAFF22. In addition, a putative bacteriocin biosynthesis protein coding gene was also found downstream of the scnKR operon in the two S. mutans strains. We therefore infer that TCS-5 (ScnKR) might be involved in the regulation of mutacin production at least in S. mutans KK23 and S. mutans NN2025.TCS-5 (ScnKR) could be neither found in the two (SAFF22) . By a clS. mutans strains. In S. mutans UA159, an insertion mutant of the gene encoding PhoR displayed no significant difference to the wild type with respect to growth under various stress conditions [TCS-7 (PhoR/YcbL) was only shared by the eight nditions . The cleS. ratti DSM20564. This finding was also supported by the PCR experiment , it is involved in multiple stress responses and has been implicated in competence development, bacteriocin production, virulence, biofilm formation, and cariogenicity [comC gene, whose product is the precursor of the signal peptide CSP sensed by ComDE in S. mutans, was absent in S. ratti. Putative orthologs of comD and comE were found in S. sobrinus DSM20742. However, with a similarity of merely 37% and 43%, respectively, to the comD and comE of S. mutans UA159  was absent in genicity ,45,64-66S. mutans strains, namely 5 DC8, KK23, KK21, UA159 and NN2025  residue.Putative alleles of the HK and RR proteins of TCS-14 are present in five of the S. mutans NCTC11060  of TCS-15 is also the only histidine kinase found in this study that contains a PDZ domain  of a TCS from Lactococcus lactis subsp. lactis KF147. Furthermore, we found that the cognate response regulator of TCS-15 showed a sequence identity of around 78% with the corresponding response regulator (YP_003353660.1) from the same L. lactis subsp. lactis strain. In our study, the RR (smh|00177) of TCS-15 was termed as CmpR  at its N-terminal side and 9 TMs at its C-terminal side, which is characteristic of a ComP-like HK. ComP-like HKs are a group of sensors of another peptide-dependent quorum sensing system related to cell density-responsive regulation other than ComDE in gram-positive bacteria. In molecule . The mosase YP_00353659.1 S. ratti DSM20564  and the RR protein (sra|3500014) are from Listeria species. Since the HK of TCS-18 possesses a PAS domain that is commonly involved in sensing intracellular signals such as redox potential, similar to the sensing mechanism described for the HK (VicK) of TCS-1, the function of TCS-18 might also be related to the sensing and response to signal(s) originated in the cytoplasm. The unique presence of TCS-18 in S. ratti DSM20564 was also confirmed by the PCR experiment  in the only serotype f blood isolate, S. mutans NCTC11060. S. sobrinus DSM20742 shows differences to the S. mutans strains in the signal perceptions possibly related to fructan catabolism and acid tolerance response. With its 3 unique TCS clusters, S. ratti DSM20564 seems even more distinct to the S. mutans strains. One of the unique TCS clusters is homologous to the DltSR TCS in Streptococcus agalactiae strains that is probably involved in the regulation of the biosynthesis of D-alanyl-lipoteichoic acid, an important cell wall component. Another one has an AmiR-like RR protein, which is the only RR found in this study to contain a RNA-binding output domain instead of a DNA-binding output domain.In the present study we conducted a genome-wide identification, classification, and ortholog analysis of the TCS proteins in eight newly sequenced mutans streptococci strains in comparison with two previously sequenced In addition to highlighting putative functions of the individual histidine kinases and response regulators, our detailed classification of the TCS proteins also confirms that specific HK families are preferentially associated with specific RR families. Such associations might play significant roles in the phenomenon of cross-talking between unpaired HKs and RRs.S. mutans, S. sobrinus and S. ratti might employ different sensing and response mechanisms for their survival in the fast changing oral environment, mostly in a symbiotic lifestyle. We believe that the results from this genomic level study will be certainly helpful for the design of physiological studies and mutation analysis towards the verification of some hypotheses proposed in this study, which in turn will lead to a better understanding of signal transductions involved in the pathogenicities of mutans streptococci.The asymmetric distribution of TCS among the mutans streptococci strains compared in this study implies that http://134.28.64.65/www/index.php/genome.We have recently sequenced eight mutans streptococci strains as given in Table S. mutans strains, the S. mutans UA159 and S. mutans NN2025, were used as reference strains for comparison. To this end, the same identification procedure was carried out on the genomes of S. mutans NN2025 and UA159 to ensure that the same search criteria were applied for all the strains included in this study so that a reasonable comparison can be achieved. The genome sequences of the two reference strains were obtained from the genome database at the National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov/sites/genome. Approaches for identifying HKs and RRs were similar to those described previously [http://pfam.sanger.ac.uk/ as templates [http://hmmer.org/[The identification of histidine kinases (HKs) and response regulators (RRs) of putative two-component systems (TCSs) of the eight mutans streptococci strains was carried out based on computational domain analysis of the predicted protein sequences. Two previously sequenced eviously  with slimmer.org/ was usedmmer.org/.TCS proteins that are common or unique among the mutans streptococci strains were identified through ortholog analysis. The ortholog groups were constructed by using the OrthoMCL program . OrthoMCFunctional domains of the TCS proteins, which were needed for the classification of both HKs and RRs were obtained by subjecting the predicted proteome of each strain to a search against the protein sequence analysis and classification database InterPro using the protein signature recognition software InterProScan . In addihttp://smart.embl-heidelberg.de provided a user-friendly web interface and excellent illustration features, the visualization of HK transmembrane helices, conserved domains and their organization was realized by using the SMART web interface with small manual modifications for the pictorial depictions.The HK protein classifications were carried out by two different approaches: a) homology box based analysis through the recognition and alignment of characteristic sequence motifs (homology boxes) of different HK families, as described previously by Grebe et al. ; b) domaAll the putative HKs and RRs identified in the mutans streptococci strains compared in this study were used for establishing the phylogenetic relationships between the TCS proteins. Using the ClustalX program multiple alignments were performed with the following parameter settings: residue-specific penalties: on, hydrophilic penalties: on, gap separation distance: 4 and the phylogenetic relationships with a bootstrap value set at 1000 were constructed . The resS. mutans NCTC11060 and to exclude the possibility of contamination with e. g. human DNA during the process of genome sequencing, PCR amplification with original DNA from this strain (and from strain S. mutans UA159 as negative control) using two different forward primers was performed. The primers used were: 5'-TTGCTTGCTGTTGTTGTG-3' (forward primer), 5'- GGCTACCATTTAGTAGAAAAGAGG -3' , 5'-TGTTACCATCTTCGGAAGG-3' (reverse primer), which were designed by using Primer Premier 6 http://www.premierbiosoft.com/primerdesign/index.html and Vector NTI 9.0 (InforMax) respectively. Conditions for this conventional PCR were: 94°C, 2 min; followed by 32 cycles of 94°C for 30 s; annealing temperature 49°C for 30 s; and 72°C for 90 s; final extension at 72°C for 5 min; length of amplicons: 1624 bp and 504 bp, respectively.To verify the unique presence of TCS-15 in S. ratti, the unique absence of TCS-13 in S. ratti, and the unique absence of TCS-9 as well as TCS-3 in S. sobrinus, PCR amplification using original DNA from strains S. mutans UA159, S. ratti DSM20564, and S. sobrinus DSM20742 was performed. The primers used, the annealing temperatures and the lengths of amplicons were as follows : TCS-18 F 5' CACTGTTCCTCCTGTATCC 3', TCS-18R 5' ATGCTGGCTATGATGTTGT 3' ; TCS-13 F 5' RAKTTYATGCCYCTMACYTTYCAG 3', TCS-13R 5' GATTCRWWRGCMGCCTC 3' ; TCS-9 HK-F 5' ATACAGTCAATATGCYAAGC 3', TCS-9HK-R 5' GRATAACACGGAAAA 3' ; TCS-9 RR-F 5' TGCTGARGACCAAGA 3', TCS-9RR-R 5' TTAGCTGCAATTTCTT 3' ; TCS-3 HK-F 5' CAYGAYYTIMGIAAYCC 3', TCS-3 HK-R 5' GTDATIACIGTICCC 3' .To verify the unique presence of TCS-18 in TCS: Two component system; HK: Sensing histidine kinase; RR: Response regulator; HTH: Helix-turn-helix; HMM: Hidden Markov Model; HPT: Histidine phosphotransferases; TM: Transmembrane helix; HGT: Horizontal gene transfer; PCR: Polymerase Chain Reaction.The authors declare that they have no competing interests.LS carried out the major bioinformatics analysis and participated in drafting the manuscript. PS was involved in the bioinformatic analysis and contributed significantly to drafting the manuscript. WW participated in the conception and coordination of the study and contributed significantly to drafting the manuscript. GC and AB performed the PCR verification experiments. JS was involved in genome sequencing and annotation. GC and IWD contributed to the microbial aspects and valuable discussions. AZE conceived of and supervised the study and revised the manuscript. All authors read and approved the final manuscript.S. mutans KK21; sme|00049 from S. mutans KK23; smc|00046 from S. mutans 5DC8; SMU.44 and SMU.45 from S. mutans UA159; GI|290579565 from S. mutans NN2025. The splitting of the alleles into two separate proteins in the strains S. mutans KK21 and S. mutans UA159 can be observed.Multiple sequence alignment of the allelic HK proteins belonging to the TCS-14 cluster. smd|00044 and smd|00045 from Click here for file


Aim. To compare targeted neonatal hearing screening (TNHS) and universal neonatal hearing screening (UNHS) since many developing countries, including Egypt, implement selective screening for high-risk neonates. Methods. 150 neonates were assessed; 50 full terms consecutively admitted to the well-baby nursery and 100 neonates consecutively admitted to neonatal intensive care unit (NICU), Ain Shams University. Patients were further subdivided into high-risk group which included 50 neonates with multiple risk factors for hearing loss and low risk group which included 50 neonates with only one risk factor. Transient evoked otoacoustic emissions (TEOAEs) were used for hearing screening. Auditory brain response (ABR) was performed 3 months later for failed TEOAEs. Results. The most frequent risk factor was consanguinity (46%). In the well-baby population, 16% failed TEOAEs. In the NICU, 30% of the low risk and 38% of the high risk groups failed TEOAEs. Regarding ABR, failed results were 12%, 10%, and 8% in the high-risk, low-risk, and healthy groups, respectively. Conclusion. The use of TNHS would have missed 8% of neonates from the well-baby group who actually had PCHL . The use of UNHS would identify all cases with PCHL, allowing for early intervention and follow-up. Significant hearing loss is one of the most frequent congenital diseases present at birth occurring in about 1 to 3 of every 1,000 healthy neonates and 2 to 4 of every 100 neonates in the neonatal intensive care unit (NICU) population [Many etiological factors may result in severe hearing loss in neonates. Genetic causes account for at least 50 to 60 percent of childhood hearing loss. Environmental causes of hearing loss includes, for example, rubella embryopathy, prematurity, bacterial meningitis, and head trauma .The initial signs of hearing loss are very subtle, and systematic neonatal hearing screening is the most effective means of early detection. The early identification of congenital hearing loss is necessary to minimize the consequences of hearing impairment on the future communication skills of a baby , 5.The Joint Committee on Infant Hearing (JCIH) has set 3 goals: screening should be completed by 1 month of age, diagnosis should be made by 3 months, and intervention and treatment should commence by 6 months . This waTransient Evoked Otoacoustic Emissions (TEOAEs) are the most adequate hearing screening tests because they are accurate, economic, and of simple execution. In recent years, automated auditory brain stem response (AABR) instruments have been developed for screening neonates in hospital setting in order to identify patients with auditory neuropathy, and it is also important to reduce the number of false-positives . In Egypt, there is a relatively high prevalence of consanguineous marriage together with poor antenatal care and lack of public awareness about the importance of hearing screening. Therefore, despite the presence of reliable screening methods  and effective methods of management, UNHS programs are still not available. On the other hand, targeted neonatal hearing screening (TNHS) or risk-based hearing screening of neonates with risk factors for hearing loss is considered a compromise between no screening at all and UNHS.Accordingly, this study was conducted to compare the targeted, risk-based hearing screening and screening of the well-baby populations. A secondary aim was to investigate the frequency and the relative importance of each risk factor for hearing loss among the at-risk neonates.This prospective cohort study was conducted at the NICU and well-baby nursery populations, Ain Shams University Hospital, between March 2009 and March 2010 on 150 neonates. One hundred consecutively admitted neonates with high-risk registry for hearing loss as listed by JCIH [All neonates were subjected to the following: detailed medical, perinatal, and family history and thorough clinical examination with an emphasis on estimation of gestational age and assessment of Apgar score at 1 and 5 minutes. Otological examination included external examination of auricles and otoscopic examination for patency and structure of external auditory canal, tympanic membrane structure, and consistency. Laboratory investigations included complete blood count (CBC) with differential leucocytic count using Coulter Counter GEN-S . Diagnosis of hyperbilirubinemia was done by measuring total serum bilirubin level using Hitachi 911 Analyzer  and interpretation of the results according to the age-specific nomogram  which re Neonates were classified into 3 groups; group A (high-risk group) included 50 neonates with multiple (3-4) risk factors for hearing loss; group B (low-risk group) included 50 neonates with only one risk factor, and group C (control group) included 50 healthy neonates.  It was done by the same observer  using a two-staged TEOAEs screening tests, where initial TEOAEs were done immediately after birth followed by a retest prior to hospital discharge, and then failures of this second stage were referred for the diagnostic ABR assessment after 3 months. Referral was done through a follow-up card given to the parents. The study protocol was approved by the local Ethical Committee of Ain Shams University. The TEOAEs were elicited using click stimuli with intensity ranging between 79 and 90 dB SPL with a mean of 83 ± 3 dB SLP using a portable TEOAEs screener, vivosonic integrity 500. Better stimulus levels were associated with better probe fit and quiet state of the infant. The software determined the amplitude of the TEOAEs in five frequency bands: 1, 1.5, 3, 4, and 5 KHz. Testing was done while infants were naturally sleeping or were alert and calm (no sedatives were used). If during the test infants were active or crying, the test could not be completed, and retesting was performed on another day. The results of TEOAEs were interpreted according to the Rhode Island criteria ; pass: r Recordings were done in the Audiology Unit, Ain Shams University Hospitals, using the evoked response audiometry Ampliad, model MK 12. Infants were naturally sleeping. Three surface electrodes were placed so that the active electrode was applied to the forehead and the reference and ground electrodes to the ipsilateral and contralateral mastoids, respectively. Acoustic rarefaction unfiltered clicks of 0.1 msec. duration were presented at a rate of 21 p/sec. at 90 dB nHL and down to threshold. At least two recordings were obtained for each intensity. A total of 1024 sweeps were differentially amplified through a band pass filter of 300–3000 Hz with 10 msec. analysis time.  Assessment of an ABR pass was based on the detection of “wave V” at its expected latency (5.4 milliseconds) according to our norms and down to threshold (30 dB nHL). Patients with elevated thresholds underwent low probe tone (256 Hz) tympanometry. Neonates with type (B) tympanometry received medical management until the resolution of middle ear effusion and ABR was retested when immittancemetry revealed type (A) or (C) tympanogram. Abnormal results of ABR were interpreted as Permanent congenital hearing loss (PCHL) with failed test of a ≥40 dB nHL in the presence of normal middle ear function. A recall of the diagnosed neonates for reevaluation was done using air and bone conduction ABR. For bone conduction, ABR bone vibrator was placed on the mastoid with effective masking applied to the other ear while testing.χ2) test or Fischer's exact test when frequencies were below five. Significant risk factors of hearing loss were entered into a multivariate analysis to determine the magnitude of the association between these factors and hearing loss and identify its odds ratio. A P value < 0.05 was considered significant in all analyses.The data were recorded, coded, entered, and processed on computer using SPSS computer software (version 15). Comparison between categorical variables was performed using chi-square (P > 0.05). Multivariate analysis of the most important risk factors for hearing loss revealed that all factors were non significantly associated with impaired ABR results (P > 0.05).Hearing loss is twenty times more prevalent in neonates than other disorders that are routinely screened for, including hypothyroidism, phenylketonuria, and sickle cell anemia. Until just a decade ago, late diagnosis was the norm, with the average age at identification of congenital hearing loss reported as 2.5 to 3 years or even later . A valua In the current analysis, TEOAEs test revealed failed response in 42/150 (28%) infants bilaterally. Failed results were 38%, 30%, and 16% in the high-risk, low-risk, and healthy groups, respectively. The results of TEOAEs screening varied widely in the literature according to the characteristics of the screened population; In Egypt, El-Gamal et al.  examinedIt was assumed that failure in the first hearing screening using TEOAEs in this study might be secondary to external ear obstruction and/or middle ear effusion. That is why our hearing screening protocol associated TEOAEs with otoscopic examination and external ear cleaning or suction when necessary. Additionally, two-staged TEOAEs test was performed in order to reduce the false positives. This protocol was in agreement with other studies , 11. On It is very important to know the exact numbers of neonates who failed screening and actually had hearing loss confirmed in subsequent assessments. This is a weakness of most similar studies in the literature. However, in the current study, diagnostic ABR confirmed that bilateral PCHL in 15 neonates out of the 42 (36%) neonates failed TEOAEs screening. The reason for such high PCHL rate in our study could be attributed to the characteristics of the studied group of neonates admitted to the NICU at a University Hospital that is a regional excellence center and therefore provides care to highly complex cases with an expected higher prevalence of hearing loss in the population treated at this facility. Attempts to screen school children for hearing loss in Egypt, mostly secondary to middle ear conditions, documented a higher prevalence of hearing loss than that in the developed countries –15.Consanguineous marriage is common in some developing communities including Egypt. It could be considered as a risk factor for hearing loss because of unrecognized higher prevalence of genes responsible for heridofamilial hearing loss . In thisNevertheless, there is a consensus that the levels of PCHL are greater in underdeveloped countries, with Davidson et al.  estimatiAnother finding worth discussing in the current analysis is that if hearing screening was limited to the at-risk neonates, 4/50 (8%) neonates from the well-baby group who had actually failed the ABR test would have been missed. This finding suggests that UNHS is far preferable because many hearing impaired infants do not have any risk factors. Thus, UNHS is strongly needed putting in mind not the cost of the screening but the cost it would be to rehabilitate a deaf-mute child.Traditionally, hearing screening has been targeted to high-risk populations which represent less than 10% of births and can potentially identify between 50% and 59% of those with congenital hearing loss. In the literature, Wroblewska-Seniuk et al.  reportedAlthough risk factors for hearing loss have been used for screening neonates in many countries worldwide, there is no consensus with regard to the importance of each of these factors and the level of relative risk. In the present study, we tried to define prospectively the risk demarcators that could predict hearing impairment in at-risk neonates. However, no significant interaction was found between any of the studied risk factors and the impaired ABR results. Risk factors for hearing loss are constantly refined by the JCIH ; howeverFinally, the potential limitations inherent in this study include lack of a standard method to track children who failed screening tests and missed diagnostic ABR. However, we believe that our findings provide new insights into neonatal hearing screening in a developing country like Egypt.In conclusion, universal neonatal screening for congenital hearing loss would detect more cases than targeted, risk-based screening, allowing for early intervention. Designing hearing screening policy, refining risk factors, and implementing follow-up and treatment protocols are of paramount importance in Egypt.


This period coincides with photoreceptor terminal differentiation in a normal retina. We have used the rd1 retina as a model to investigate early molecular defects in developing rod photoreceptors prior to the onset of degeneration.The rd1 and wild type (wt) retinas at four time points starting at P2, prior to any obvious biochemical or morphological differences, and concluding at P8, prior to the initiation of cell death. Of the 143 identified differentially expressed genes, we focused on Rab acceptor 1 (Rabac1), which codes for the protein Prenylated rab acceptor 1 (PRA1) and plays an important role in vesicular trafficking. Quantitative RT-PCR analysis confirmed reduced expression of PRA1 in rd1 retina at all time points examined. Immunohistochemical observation showed that PRA1-like immunoreactivity (LIR) co-localized with the cis-Golgi marker GM-130 in the photoreceptor as the Golgi translocated from the perikarya to the inner segment during photoreceptor differentiation in wt retinas. Diffuse PRA1-LIR, distinct from the Golgi marker, was seen in the distal inner segment of wt photoreceptors starting at P8. Both plexiform layers contained PRA1 positive punctae independent of GM-130 staining during postnatal development. In the inner retina, PRA1-LIR also colocalized with the Golgi marker in the perinuclear region of most cells. A similar pattern was seen in the rd1 mouse inner retina. However, punctate and significantly reduced PRA1-LIR was present throughout the developing rd1 inner segment, consistent with delayed photoreceptor development and abnormalities in Golgi sorting and vesicular trafficking.Using a microarray approach, we performed gene profiling comparing rd1 retina at early time points, which may give insights into developmental defects that precede photoreceptor cell death. This is the first report of PRA1 expression in the retina. Our data support the hypothesis that PRA1 plays an important role in vesicular trafficking between the Golgi and cilia in differentiating and mature rod photoreceptors.We have identified genes that are differentially regulated in the  In recent years, progress has been made in the identification of genetic defects and molecular mechanisms that underlie RP ,2. The rls of RP ,4. It isls of RP . Cone cels of RP ,6. The r6b) gene ,8. The de by P13 .rd1 retina, photoreceptors degenerate just as the outer segment begins to form. Although the significance of cGMP in phototransduction is well established, little is known about the role of cGMP in developing photoreceptors or how it leads to degeneration in the rd1 retina.cGMP is an important second messenger involved in regulation of many functions including phototransduction as well as neuronal differentiation, smooth muscle contractility, and olfactory stimulation . In the rd1 and wt mouse retinas during the period preceding cell death from P2, prior to any identified morphological or biochemical differences, through P8, when early degenerative changes are present but prior to onset of cell death. During this period, 143 differentially expressed genes were identified. We confirmed two genes to be differentially expressed at all 4 time points: the mutant gene, PDE6b, and Rab acceptor 1 (prenylated) (Rabac1). Rabac1 codes for an integral membrane protein, PRA1, that interacts with numerous small prenylated GTPases in the Rab family [We have used microarray analysis to investigate differences in gene expression between the b family -14, consrd1 mouse retinas. We demonstrate that its expression in photoreceptors is significantly decreased and mislocalized in rd1 retina compared to wt, prior to rod photoreceptor degeneration and consistent with a role of PRA1 in rod differentiation.Here we present the first description of PRA1 in the retina, establishing the localization of PRA1 protein in developing wt and rd1 mouse retina were compared to those of wt retina at four time points: P2, P4, P6, and P8. This time span was selected such that the earliest time point precedes any reported morphological or biochemical changes in rd1 retina compared to wt and the latest time point precedes onset of cell death. At least four samples were examined for microarray analysis at each time point. Genes that showed 1.5 fold or greater change, with False Discovery Rate Confidence Interval (FDR-CI) p-values less than 1 were included, resulting in the identification of 143 differentially expressed genes in rd1 compared to wt retina between P2 and P8  terms Figure . Analysird1 retina [Functional analysis shows differences in genes identified at P2 compared to P8 . PRA1 has not previously been investigated in the retina and was selected for further studies.Quantitative real-time PCR (qPCR) was used to validate microarray results for 18 genes at one or more time points  was observed when the primary antibody was omitted  and more diffusely in the inner plexiform layer (IPL).At P8, in both wt and e Figure E, F. PRAe Figure E. No comn Figure F. As obsn Figure E while ie Figure F. The inrd1 age-matched tissue. Intense PRA1-LIR could be observed in the proximal half of the IS layer with more diffuse label extending throughout the distal portion of the IS layer  of rd1 retina at P21 appeared to have less intense PRA1-LIR in the perinuclear region compared to wt  at each developmental time point. In this analysis we found that the average intensity of PRA1-LIR was very similar in wt and rd1 retinas with some difference seen only at P8 . A virtrd1 whole retina during this time window have shown that the cytoplasmic cGMP level is twice the wt level by P6 with an increase of nearly 10-fold by P13 [rd1 retina, including retardation of IS growth as early as P4 [rd1 retina [Previous studies in d by P13 . Electrod by P13 ,23,24 haly as P4 , Golgi dly as P4 . The mitly as P4 ,23,24. Ard1 retina at later time points, focusing on initiation of cell death, have been previously reported [Our microarray analysis of differentially expressed genes during this early time frame identified only 2 of 143 genes associated with apoptosis, consistent with our effort to target the earliest molecular changes that may provide the initial trigger for degeneration. Several studies using microarray analysis of reported -27. Usinreported ,26. Hack2+ and Na+ entry in response to light. Efforts to measure Ca2+ in the degenerating rd1 retina have provided support for the hypothesis that the loss of function of Pde6β induces apoptosis as a result of high levels of Ca2+ influx through the CNG ion channel [Pde6b mutants [cGMP regulates the cGMP-gated (CNG) ion channel in the mature retina, which regulates Ca channel ,28,29. T mutants ,31.Pde6b is expressed at embryonic day 12 (E12) in the mouse retina, much earlier than other genes involved in phototransduction, such as rhodopsin expression that becomes apparent at P5. Pde6b is even expressed prior to Nrl, a transcription factor that activates expression of rod-specific genes and is expressed at P1 [Pde6a and Pde6g are first expressed more than a week after Pde6b at P1 [Several observations raise the question as to whether Pde6β may play an unexplored role in photoreceptor development, independent of its role in phototransduction in the mature retina, and whether such a role could contribute to the initiation of cell death. First, ed at P1 . Interes6b at P1 , suggestrd1 retina by P6 [rd1 retina compared to the wt retina at P11, consistent with multiple signaling roles for cGMP in the retina [Secondly, the doubling of cGMP levels in whole na by P6 ,9 indicana by P6 . In addina by P6 -37. cGMPe retina . Althouge retina , signaliRabac1, which was of particular interest as the only gene in the dataset other than the mutant Pde6b gene, identified as downregulated at all four time points examined.Together, these observations support the hypothesis that Pde6β plays a role in regulation of cGMP signaling during pre- and/or early postnatal photoreceptor differentiation that is independent of its role in phototransduction in the mature outer segment. Our analysis of functional groups of the differentially regulated genes supports this hypothesis. First, only 7 of the differentially expressed genes, excluding the mutant gene, were associated with visual transduction, and most of these were only expressed at the later time points. Secondly, the differentially expressed genes are predominantly in functional groups that are consistent with a role in development and differentiation, including development, transport, cell cycle, signal transduction, transcription, and metabolism. Transport is particularly important during photoreceptor differentiation as the cell must transport membranes and proteins in order to elongate both its axon and inner segment, followed by assembly of the outer segment. Within this category, we identified Rabac1, is comprised of 185 amino acids and an estimated molecular mass of 20.6 kDa [PRA1, the protein product of 20.6 kDa ,12,40. S20.6 kDa , althoug20.6 kDa ,14,41,4220.6 kDa -14 and hMartincic et al.  hypothesrd1 mouse retinas. In the developing wt retina, colocalization with GM-130-LIR indicates that PRA1 is localized to the photoreceptor Golgi apparatus as it translocates from the perikarya to the proximal IS during photoreceptor differentiation. PRA1 is also inferred to be in proximity with Rab6 during photoreceptor differentiation. Diffuse PRA1-LIR that does not colocalize with the Golgi marker is seen in the distal IS of wt photoreceptors starting at P8. In the mature retina, PRA1 positive punctae extend up to, but not overlapping with, the proximal end of the OS axoneme as labeled by RP1-LIR. Only very sparse punctae are seen in the OS. Both plexiform layers also contain PRA1 positive punctae independent of GM-130 staining during postnatal retinal development. In the wt OPL, PRA1-LIR appears less intense at P21, suggesting that it may play a role in neurite outgrowth and/or synapse formation that is not required for maintenance in the adult. This observation is of particular interest in light of the failure of the rd1 rod photoreceptors to form a triad synapse with bipolar dendrites [We present here the first report describing the expression and localization of PRA1 protein in the developing and mature wt and endrites . Togetherd1 compared to age-matched, wt retinas. Mislocalization of PRA1-LIR in the IS layer is seen in the rd1 retina at all ages examined. Compared to wt, rd1 retinas display a less intense, diffuse pattern of staining in the IS with some large punctae distributed throughout. Colocalization of PRA1- and GM-130-LIR is seen in some, but not all, of these IS punctae. This observation is consistent with EM pathology in the rd1 retina showing defects characteristic of vesicular trafficking [By both Western blot at P2-P4 and immunohistochemistry at P6-P21, PRA1 expression appears less intense in fficking ,23,24.rd1 IS layer compared to wt is apparent at all ages examined, and is statistically different by P8. Staining in the residual rd1 outer retina at P21 suggests that PRA1-LIR is also present in cone photoreceptors. The PRA1-LIR intensity in the perinuclear region of the rd1 GCL is also reduced at P21, consistent with a possible role of PRA1 in neurite remodeling and sprouting. Virtually all of the proteins altered in PRA1-depleted nasopharyngeal carcinoma cells that are linked to changes in cell migration [Rabac1 mRNA measured by qPCR in whole retina is largely due to loss of expression in photoreceptors, with less overall change in protein expression in the inner retina.In developing mouse retina, the decrease in PRA1-LIR in the igration  are alsoXenopus as a model system, four members of the Rab GTPase family, Rab3, Rab6, Rab8, and Rab11, have been identified in vesicular trafficking of rhodopsin from the Golgi to the connecting cilium at the base of the outer segments [Rod photoreceptors are highly specialized cells that exhibit unambiguous cellular polarity. Polarity of these cells is established during cell differentiation and maintained by the sorting of lipid membranes and proteins to their appropriate targets through a process of vesicular trafficking. Using segments . Two of segments . Rab prosegments  and of tsegments . These srd1 mouse. PRA1 has been proposed to regulate the recruitment of Rab effector proteins as well as proteins involved in proper tethering and fusion of vesicles to their target membrane, such as VAMP2 [rd1 retinas [Defects in vesicular trafficking have been implicated in retinal degenerative diseases. Mutation in Rab escort protein 1, a protein responsible for modifying small GTPases, has been linked to choroideremia, a disease characterized by degeneration of the choroid followed by photoreceptor degeneration . Defectsas VAMP2 ,55. A de retinas ,56.rd1 mouse retina.Alternatively, Figueroa and colleagues  have proRabac1 gene expression and in PRA1 protein expression by P2 in the rd1 retina. These data support the hypothesis that PRA1 plays an important role in organization of the Golgi and vesicular trafficking during the early stages of rod photoreceptor cell differentiation and suggest that the decrease in PRA1 expression in the rd1 retina may serve as a link coupling the genetic mutation in Pde6b to the very early defects in membrane trafficking and the delay in rod photoreceptor cell differentiation that are subsequently followed by cell death. The interaction of Pde6β and cGMP signaling pathways with PRA1 is an important area for future investigation.In summary, our results are consistent with the hypothesis that Pde6β may have a function during photoreceptor development distinct from its phototransduction role in mature OS. Our data demonstrate a significant downregulation in rd1/le) linked mutations on a C57BL/6 background were used for these experiments. Runts or noticeably underdeveloped pups were excluded. Animal colonies were housed in 12/12 light/dark conditions and were handled by the same staff throughout the period of microarray sample collection. Animals were handled in accordance with the National Institutes of Health Guidelines on Laboratory Animal Welfare using procedures that were approved by the Saint Louis University Institutional Animal Care and Use Committee.Mice homozygous for either the wt allele or the retinal degeneration 1/light ear  were used for first-strand and second-strand cDNA synthesis. Double-stranded cDNA was cleaned with Phase Lock Gels-Phenol/Chloroform extraction and ethanol precipitation. Biotin-labeled antisense cRNA was produced by an in vitro transcription reaction . Fifteen micrograms of cRNA was incubated with fragmentation buffer . Target hybridization to MOE430A Gene Chips was performed using the standard Affymetrix Eukaryotic Target Hybridization protocol [Total RNA was harvested from flash-frozen retina tissue using TRIzol  extraction followed by cleanup with the RNeasy kit . Ten micrograms of total RNA was used for cDNA synthesis. A T7-(dT)protocol ,59. Washprotocol ,59 and sAll microarray data is MIAME compliant. Raw data have been deposited in the NCBI GEO database (accession #GSE41821). Expression scores were obtained using Robust Multichip Average to do background correction, quantile normalization and summarization . NormaliTotal RNA was extracted using TRIzol (Invitrogen) followed by further purification using RNeasy (Qiagen). Reverse transcription was performed using 2.5 μg of total RNA and Superscript II reverse transcriptase (Invitrogen). qPCR was performed in triplicate with the iCycler IQ system  using Sybr Green I (Invitrogen). qPCR normalization was performed using the housekeeping gene hypoxanthine guanine phosphoribosyl transferase (HPRT).Retinal tissue was rapidly dissected and 2–8 retinas were pooled in RIPA buffer with protease inhibitors , 1% phosphatase inhibitor cocktail (Sigma P2850), and 1mM PMSF), flash frozen on dry ice, followed by sonication. Samples were spun at 13,000 rpm at 4°C for 10 min. Protein content of the supernatant was determined using the BCA Protein Assay . Samples of either 6.5 or 15.8 μg/lane were electrophoresed on 4-20% Precise Protein gels  according to manufacturer's recommendations. Proteins were transferred to nitrocellulose membranes and blocked for 1 hour at room temperature in 5% nonfat dry milk in TBST. Membranes were then probed with a primary antibody to PRA1  overnight at 4°C. Protein was visualized using HRP-conjugated anti-rabbit IgG secondary antibody  and enhanced chemiluminesence reagents (Sigma). Blots were stripped in 0.2M NaOH and probed with a primary antibody to β-actin  and an HRP-conjugated anti-mouse IgG secondary antibody  to control for protein loading. Densitometry was performed using Image J software and represents the average of two gels.rd1 reverse 5'-GTAAACAGCAAGAGGCTTTATTGGGAAC-3' and wt reverse 5'-TACCCACCCTTCCTAATTTTTCTCACGC-3' (Sigma). PCR products were separated on 1.5% agarose gels resulting in bands of approximately 400bp and 550bp for the wt and rd1 alleles, respectively.Genotyping was performed using the RedExtract Kit (Sigma). The following primers were used: Forward 5'-TGACAATTACTCCTTTTCCCTCAGTCTG-3', rd1/le and wt littermates from heterozygous crosses at P6, 8, 10, and 21 and handled in the same fashion for all subsequent steps. Pups between P2-8 were anesthetized on ice and decapitated; older pups were injected intraperitoneally with an overdose of pentobarbital. Tissue was maintained on ice while the eyes were enucleated and the anterior segment was removed. Eyecups were fixed in 4% paraformaldehyde in 0.1M phosphate buffer for one hour, on ice. Following fixation, eyecups were washed 3 times for 15 minutes in 0.1M phosphate buffer and cryoprotected in 30% sucrose overnight, at 4°C. The following day, eyes were embedded in OCT  and frozen in 2-methylbutane on dry ice. At least three samples were analyzed at each time point.Eyecups were harvested from age-matched Cryostat sections were cut at a thickness of 12 μm. Each section was post-fixed with 4% paraformaldehyde in 0.1M phosphate buffer for one minute. Slides were then washed 3 times for 15 minutes in phosphate buffered saline . All successive washes were performed in the same manner. All blocking and antibody incubations were performed in a moist chamber with each tissue section receiving 40 μl of the indicated solution. Slides were covered with parafilm during incubation. All sections were incubated in blocking solution comprised of 2% normal donkey serum  and 0.3% TritonX (Sigma) in PBS, for 20 minutes. Sections were incubated in polyclonal rabbit anti-PRA1 primary antibody (Proteintech Group), diluted 1:50 in blocking solution. For double label experiments, slides were concurrently incubated with monoclonal mouse anti-GM-130 , diluted 1:1000 in blocking solution; Rab6 , diluted 1:500; or RP1 , diluteSlides were washed and each section was then incubated in blocking solution for 20 minutes. Sections were incubated in donkey anti-rabbit Alexa 488 secondary antibody  diluted 1:400 in blocking solution in the dark for 1 hour. For double label experiments with GM-130, slides were concurrently incubated in donkey anti-mouse Alexa 555 secondary antibody (Molecular Probes) diluted 1:400 in blocking solution. For double label experiments with RP1, slides were concurrently incubated in goat anti-chicken Alexa 555 secondary antibody (Molecular Probes) diluted 1:50 in a blocking solution with 2% NDS. Slides were washed and coverslipped with Vectashield  and stored at -20°C.Labeled sections were imaged using a Zeiss LSM 510 Meta confocal microscope. Each image consisted of a stack of slices having a thickness of 0.44 μm per slice. All images were adjusted for brightness and contrast using Image J software. All photomicrographs from a single experiment were adjusted with precisely the same settings.rd1 age-matched littermates. Average intensity was obtained for each of the three regions of the IS layer and these values were normalized as follows: the P6 wt average intensities were arbitrarily set to one, within each pair the rd1 intensity was normalized to the wt value, and across time points the average wt values were normalized against the P6 wt value. A paired t-test was used to compare the average of rd1 IS intensity to that of wt IS. The same procedure was used to analyze intensity of the inner plexiform layer (IPL).Image J software was used to process and analyze images. Analysis of pixel density for regions of interest (ROI) was obtained at each developmental time point examined. First, small stacks of six slices were generated. Next, a ROI from the central and each peripheral regions of the IS layer was obtained for n = 3 eyecups of wt and Rabac1: Rab acceptor 1; rd1: Retinal degeneration 1; ROI: Regions of interest; RP: Retinitis pigmentosa; wt: Wild type.CNG: cGMP-gated; E: Embryonic day; FDR-CI: False Discovery Rate Confidence Interval; GCL: Ganglion cell layer; GDI: Guanine nucleotide dissociation inhibitor; GDF: GDI displacement factor; HPRT: Hypoxanthine guanine phosphoribosyl transferase; INL: Inner nuclear layer; IPL: Inner plexiform layer; IS: Inner segment; LIR: Like immunoreactivity; PDE6b: cGMP phosphodiesterase 6 β-subunit; ONL: Outer nuclear layer; OPL: Outer plexiform layer; NDS: Normal donkey serum; P: Postnatal day; PBS: Phosphate buffered saline; PKG: Phosphokinase G; PRA1: Prenylated rab acceptor 1; qPCR: Quantitative real-time PCR; The authors declare that they have no competing interests.Conceived and designed the experiments: VMD, AMR, AJM, AS, JMO. Performed the experiments: VMD, AMR, JGM, SCEH, AAI, AYC, MJB, MIO, JMO. Analyzed the data: VMD, AMR, RK, AJM, AS, JMO. Wrote the paper: VMD, AMR, JMO. All authors read and approved the final manuscript.rd1 compared to wt retina for one or more time points. Table S2. includes the eighteen genes that were analyzed by qPCR. Microarray analysis of fold-change is indicated in the final column for comparison.includes the 143 genes that showed significant differential expression by microarray analysis in Click here for file


The 5 G/5 G genotype of PAI-1 polymorphism is linked to decreased plasminogen activator inhibitor-1 (PAI-1) levels and it has been suggested that lower PAI-1 levels may provide protective effects on inflammation, local microcirculatory disturbance, and fibrotic changes, which are likely associated with development of sudden sensorineural hearing loss (SSNHL).via a case control study. 103 patients with SSNHL and 113 age and sex-matched controls were enrolled at University of Ferrara, Italy and hearing loss outcome was measured at least 3 months after the onset of hearing loss. DNA was isolated from peripheral blood using the QIAamp kit and the 4 G/5 G polymorphism in the −675 promoter region was genotyped with an allele-specific PCR. Genotype distribution was tested in patients and compared to controls by chi-square and odd-ratio analysis. The codominant and recessive models were used for the multiple logistic regression analyses of the PAI-1 gene allele.The association of the 4 G/5 G PAI-1 polymorphism with the development and clinical outcome of SSNHL is evaluated p = 0.005). In addition, the patients with 5 G/5 G genotype showed a trend of more than 2 times higher ratio of hearing recovery (> 20 dB) after systemic corticosteroid treatment compared to 4 G/5 G genotype , suggesting a better clinical outcome.In this population, 5 G/5 G genotype had a two-time lower frequency in SSNHL patients compared to healthy controls (15.5% vs 30.1%) and was associated with decreased odds compared to 4 G/5 G genotype (OR 0.37, 95% CI 0.19-0.75, The 5 G/5 G genotype of PAI-1 may be associated with a reduced risk of SSNHL in the Italian population. Sudden sensorineural hearing loss (SSNHL) is defined as a rapid onset sensorineural hearing loss occurring over a 72-hour period with a decrease in hearing of > 30 decibels (dB) affecting at least 3 consecutive frequencies . The majPlasminogen activator inhibitor-1 (PAI-1) is the principal inhibitor of tissue plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), which actively facilitate plasminogen, and hence fibrinolysis. PAI-1 is a key molecule for thrombus formation and inflammation . ElevatiThe PAI-1 gene has variation in the promoter region on the basis of a single guanosine insertion-deletion (5 G or 4 G) . PreviouIn this study we investigated the 4 G/5 G polymorphism of PAI-1 gene in the Italian patients with SSNHL to see if this polymorphism is a risk factor in developing SSNHL and can be used as a prognostic indicator for clinical outcome in patients with this disease. We demonstrated a significant contribution of 5 G/5 G polymorphism to lowering the risk of developing SSNHL and a trend of improved hearing recovery in follow up evaluation after treatment of SSNHL.One hundred and three patients, 54 females and 49 males , were evaluated at the Department of Audiology at the University of Ferrara and Modena with a diagnosis of SSNHL and gave their consent to participate in the study. They were referred to the department from January 2005 to December 2009. All the patients underwent audiological examinations including pure tone audiometry, speech recognition threshold, immittance measurements such as tympanogram and acoustic reflex; other audiometric tests for the differential diagnosis of cochlear vs retrocochlear pathologies were performed in selected cases. Auditory brainstem response (ABR) was always performed except when hearing loss exceeded 80 dB in the acute frequencies. Audiometric inclusion criterion was a decrease in hearing of at least 30 dB in 3 contiguous frequencies. The following scale of hearing loss degree was used: mild, ≥ 30 to < 40 dB hearing loss; moderate, ≥ 40 to < 70 dB hearing loss; severe, ≥ 70 to < 90 dB hearing loss; and profound, ≥ 90 dB hearing loss. Patients did not receive vestibular evaluation because exclusion criteria were the presence of vertigo or suspected Meniere’s disease. Acoustic neuroma was excluded by performing MRI or by serial audiometry in selected cases. Any participant with a history of head trauma was also excluded from the study.All the subjects provided their informed consent for the study and blood was taken as usual for routine laboratory exams, in the morning before starting the pharmacological therapy. Medical treatment consisted of betamethasone 4 mg i.m. for 3 days and 1.5 mg i.m. for 3–4 days, and oral prednisone was administered for at least 7 days afterwards, depending on clinical evolution. Pure tone audiometry was repeated daily according the history of the patient. We examined follow-up audiometry at least 6 months after the initial episode of hearing loss and considered a hearing improvement more than 20 dB as a significant recovery. The practice guidelines suggest <10 dB changes as no recovery [Blood was taken from each patient or control for genomic DNA extraction, which was performed using the QIAamp kit  as directed to obtain 3 to 12 μg of DNA from 200 μl whole blood.TM Thermal Cycler . The reaction mixture contained 10 mmol/L Tris–HCl (pH 8.0), 2.5 mmol/L MgCl2, 200 μmol/L deoxyribonucleoside triphosphates, and 25 pmol of each primer. For each PCR, 2.5 U of Taq polymerase  was used. Electrophoresis was performed in 2% agarose with 1 x TAE buffer. The gels were photographed after ethidium bromide staining. As a control of this PCR technique, PCR analysis was performed on DNA samples of known genotypes.The PAI-1 4 G/5 G genotype was analyzed with an allele-specific PCR modified from that of Falk et al. , using a2) and odd-ratio analysis using GraphPad Prism for Windows version 4.03 . We also used chi-square test (χ2) and odd-ratio analysis for the association between genotype distribution and hearing outcome. A p value of less than 0.05 was considered to be statistically significant. All odds ratios (OR) are given with their 95% confidence interval (CI). In the multiple logistic regression analyses for each PAI-1 gene allele, we used multiple inheritance models, including codominant model  and recessive model . 5 G was considered the minor allele in our case.Genotype distribution and allele frequencies were tested in patients and compared to controls by chi-square test (χp < 0.005), which is the most prevalent genotype. When we compared the 5 G/5 G genotype with combined non-5 G/5 G genotypes (4 G/5 G + 4 G/4 G), the 5 G/5 G genotype had two times lower risk of developing SSNHL than non-5 G/5 G genotypes. However, the 4 G/4 G genotype did not have significantly increased risk compared to the 4 G/5 G genotype.A total of 103 SSNHL patients and 113 healthy controls were analyzed for 4 G and 5 G PAI-1 gene alleles. A typical PAI-1 genotyping experiment is shown in Figure We further examined to see if the polymorphism of PAI-1 gene is associated with clinical outcome in patients with SSNHL Table . We wereOne of most probable mechanisms of SSNHL appears to be impaired cochlear blood circulation involving the pathogenic micro-thrombotic mechanism . There aTo investigate the potential contribution of polymorphism within the PAI-1 gene to the development of SSNHL, we recruited SSNHL patients and control subjects from Ferrara and Modena, Italy, from a white, homogeneous population. In this population, we found the frequencies of 5 G allele  had no significant difference from those of 4 G allele  either in patients or in controls. However, the frequency of the 5 G/5 G genotype was two times lower in the SSNHL group (15.5%) compared to that in the control group 30.2%). This 5 G/5 G genotype showed 2–3 times lower risk effect than 4 G/4 G and 4 G/5 G. In this study, we found the 4 G/4 G genotype had no significant risk ratio in developing SSNHL. However, the 5 G/5 G genotype appeared to have a protective effect against developing SSNHL. Our findings are supported by several previous reports that the patients with 5 G/5 G genotype are known to have lower plasma levels of PAI-1 compared to those with 4 G/4 G ,24, and .2%. ThisIt has been known that there is no effective treatment of SSNHL other than systemic corticosteroids which is generally used but has limitation to some patients . Our stuThis study suggests that the individuals with the 5 G/5 G genotype of PAI-1 have less risk of developing SSNHL, and the 5 G/5 G genotype may function as a prognostic factor in recovery from SSNHL. This result may be of clinical significance in diagnosis, treatment, and prognosis for SSNHL patients and may provide a new therapeutic strategy for SSNHL.SSNHL, Sudden sensorineural hearing loss; PAI-1, Plasminogen activator inhibitor-1; tPA, Tissue plasminogen activator; uPA, Urokinase-type plasminogen activator.Authors declare that there is no financial or non-financial competing interests in relation to this manuscript.SC, SP, MB, AM and TY conceived of the study, and participated in its design and coordination and helped to draft the manuscript. HC, CY, DC and JK involved in drafting the manuscript or revising it critically for important intellectual content and performed the statistical analysis. IK and CC carried out the molecular genetic studies, participated in drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6815/12/5/prepub


Beauveria bassiana tested for virulence against F. occidentalis in laboratory bioassays, we found strain SZ-26 as the most potent, causing 96% mortality in adults at 1×107 mL−1conidia after 4 days. The effect of the strain SZ-26 on survival, longevity and fecundity of the predatory mite Neoseiulus (Amblyseius) barkeri Hughes were studied under laboratory conditions. The bioassay results showed that the corrected mortalities were less than 4 and 8% at 10 days following inoculation of the adult and the larvae of the predator, respectively, with 1×107 conidia mL−1 of SZ-26. Furthermore, no fungal hyphae were found in dead predators. The oviposition and postoviposition durations, longevity, and fecundity displayed no significant differences after inoculation with SZ-26 using first-instar larvae of F. occidentalis as prey in comparison with untreated predator. In contrast, the preoviposition durations were significantly longer. Observations with a scanning electron microscope, revealed that many conidia were attached to the cuticles of F. occidentalis at 2 h after treatment with germ tubes oriented toward cuticle at 24 h, penetration of the insect cuticle at 36 h, and finally, fungal colonization of the whole insect body at 60 h. In contrast, we never observed penetration of the predator's cuticle and conidia were shed gradually from the body, further demonstrating that B. bassiana strain SZ-26 show high toxicity against F. occidentalis but no pathogenicity to predatory mite.Among 28 isolates of  Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), is regarded as an important economic pest of a wide range of agricultural and horticultural crops worldwide F. occidentalis has developed a high level of resistance to many chemical pesticides Neoseiulus (Amblyseius) barkeri (Hughes) (Acarina: Phytoseiidae) and the entomopathogenic fungus Beauveria bassiana  Vuillemin have been shown to be potential biological control agents of F. occidentalisWestern flower thrips, N. barkeri has been successfully employed for reducing populations of F. occidentalis in crops, such as strawberries and cucumbers B.bassiana against F. occidentalis results in high rates of mortality in laboratory screenings and greenhouse conditions F. occidentalis, it is suggested that B.bassiana should be applied along with the releases of predatory mites under field conditions B. bassiana and predators to control F. occidentalis is a critical issue for the implementation of IPM programs. A better understanding of the factors that minimize undesirable effects of insect pathogens on natural enemies could improve their integrated utilization against pest insects B. bassiana strain SZ-26 and N. barkeri by studying the effect on the longevity and fecundity of predatory mites when offered first-instar thrips as prey. Furthermore, there are no reports for the micromorphological observations of fungal conidial inoculation processes on this predator. Thus, we studied the mortality of larval and adult N. barkeri and F. occidentalis exposed to B. bassiana strain SZ-26 and compared the fungal infection process in the predator and thrips by scanning electron microscopy (SEM). This information will enhance our understanding of the interactions between two biological control agents and help to determine the possibility of concomitant application of both in F. occidentalis classical biocontrol programmes.Most previous research has been designed to evaluate the effects of pathogens on predators directly by exposing predators to pathogen residues or by topical application, and then studying factors such as predator mortality and behavior, or indirectly by allowing predation on fungal-infected preys, or assessing predator-prey abundance in experimental crops. B. bassiana  (N. barkeri.Of the 28 strains of bassiana  tested ap<0.001) . Strain N. barkeri were maintained below 4 and 8% at 10 days following inoculation of the adult and larvae, respectively. These mortalities for N. barkeri were significantly lower that those of F. occidentalis, whose corrected mortalities reached 100% and 66%, respectively,   . No peneB. bassiana strain SZ-26 on the predator longevity and ovipositionEffect of B. bassiana strain SZ-26, preoviposition duration of predators was significantly longer as compared to the controls. There were no differences in other life table parameters, such as oviposition, postoviposition duration, female longevity and daily fecundity compared to the controls . In this study, the sub-lethal effects on N. barkeri when directly exposed to B. bassiana strain SZ-26 conidial suspension were evaluated. we observed that N. barkeri groomed conidia from their bodies so that few conidia remained on N. barkeri 48 h after treatment with B. bassiana strain SZ-26 (unpublished). One function of grooming in arthropods is the removal of foreign bodies such as fungal or mite parasites Phytoseiulus longipes was efficient in removing most capilliconidia of the fungal pathogen N. floridana through self-grooming behavior.There has been increasing interest in evaluating the sub-lethal effects of pathogens on predators. Shaw et al. F. occidentalis to predators as food, because B. bassiana infection could make the larval thrips deficient in certain essential nutrients F. occidentalis to N. barkeri will affect life table parameters of the predator still needs to be demonstrated. From our recent results, this exposure did lengthen the preoviposition period of adult females. However, this likely reflects time spent by all treated mites grooming off conidia and not a physiological effect on females. Overall, both direct bioassay and sub-lethal effects on N. barkeri indicated that B. bassiana strain SZ-26 poses a negligible risk to N. barkeri.In order to avoid being influenced by other factors, we supplied untreated larval B. bassiana strain SZ-26 conidia penetrated F. occidentalis cuticle soon after germination. The results agree with those of Vestergaard et al. F. occidentalis. In contrast, despite being able to attach to N. bakeri, it was displayed that no pathogenicity of B. bassiana strain SZ-26 to N. bakeri. No penetration of germ tube or formation of hyphal bodies was observed on dead N. bakeri further supporting the SEM results. The pathogenicity of entomopathogenic fungi is the result of mechanism pressure and proteinases which are associated with cuticle degradation N. bakeri to avoid infection by fungi. Although many studies indicate that entomopathogenic fungi are highly pathogenic against targeted insect pests while showing no detrimental effects on predators in laboratory bioassays and field investigations N. bakeri body within 48 h, reducing the infection possibility. Moreover, although conidia could germinate, they were not observed to penetrate the N. bakeri cuticle, we speculate that the different cuticle structures or proteinase targets between F. occidentalis and N. bakeri influence the fungi pathogenicity. The proteinaceous outer integument of predatory mites probably forms an effective barrier against B. bassiana strain SZ-26. In addition, few shriveled conidia were detected on the cuticle of N. barkeri after 48 h, possibly because the germinated conidia which were remaining on N. bakeri could not be glued on the susceptible host, the few shriveled conidia probably lost their viability. These observations and speculations may aid in explaining why N. bakeri in not infected by B. bassiana strain SZ-26. The results also enhance our understanding of the interactions between pathogen and predators. To better understand the interactions, the defense mechanisms of predators need to be further explored.From our SEM observations, No specific permissions were required for these locations/activities.None of the species used in this study are endangered or protected.The origin and source of the twenty-eight fungal isolates are shown in N. barkeri and the prey Tyrophagus putrescentiae were obtained from colonies maintained in the laboratory of insect natural enemies, Institute of Plant Protection, Chinese Academy of Agricultural Sciences. N. barkeri are reared in sterilized wheat bran-T. putrescentiae mixture and fed on T. putrescentiae in plastic boxes (15 cm×15 cm×10 cm) with lips and a circular moist sponge (10 cm diameter) at the edge of boxes for preventing escape. A hole (12 cm diameter) was cut in the lid and covered with fine mesh to allow for ventilation. Culture boxes were kept at 25±1°C, 60–70% RH and L16:D8 photoperiod in a climate controlled chamber. Cotton silk was placed on the surface of the leaves for oviposition, eggs were collected and transferred to a new plastic box using a fine paintbrush after 6 hours and allowing the emergent larvae to develop in synchrony. The newly emerged larvae and adults were obtained for experimental use.F. occidentalis was maintained as described by Liang et al Phaseolus vulgaris L.) in 0.5 L tube-shaped glass jars with snap-on lids. A hole (10 cm diameter) was cut in the lid and covered with fine mesh to allow for ventilation. Rearing jars were kept at 26±2°C, 60–70% RH and L13:D11 photoperiod in a climate controlled chamber. Thrips at similar stages of development were obtained by incubating adults on fresh, healthy plants for oviposition, removing the thrips after 3 days and allowing the different stage of thrips to develop in synchrony. The first instars and adults were obtained for experimental use.A colony of western flower thrips, F. occidentalis survival was evaluated by treating thrips with concentrations of 1×107 mL−1 conidia, which is the concentration commonly used for spray application for control of western flower thrips in greenhouses in China F. occidentalis adults were collected from the laboratory rearing colony and dipped for 5 s in the conidial suspension. Adults were allowed to dry on filter paper and transferred to Petri dishes (diameter 7 cm) lined with bean leaves and covered with plastic film which were pricked for ventilation. The Petri dishes were stored in a climate cntrolled chamber . The effects against the F. occidentalis adults were scored at day 5 after treatment. The presence of fungal mycelia was used as an indication of mycosis. Each replicate consisted of 20 adults; treatments were randomized and the experiment was replicated 3 times using different insect lots over time.The effect of the fungal isolates on adult F. occidentalis and N. barkeri using the same conditions listed above. The first instar larval and newly eclosed adult stages of F. occidentalis were inoculated by immersion for 5 s in 2 ml conidia suspension of B. bassiana strain SZ-26 and using a fine paintbrush carefully transferred to petri dish (3.5 cm diameter) lined with freshly excised bean leaf, which was placed on the surface of the water—saturated filter paper, the root vein of leaf was wrapped by moist cotton wool to slow leaf desiccation. The dish was then sealed with polyvinyl chloride (PVC) cling film and incubated at 25±1°C, 60–70% RH and L16:D8 photoperiod in a climate controlled chamber. The status of individuals was determined 10 days after treatment. Mortality was recorded daily. Each stage of thrips consisted of 8 replicates with 20 insects per replicate. The presence of fungal mycelia was used as an indication of mycosis. Controls consisted of thrips treated with 0.05% Tween-80 in sterile H2O. Bioassays for adult and larval N. barkeri were repeated as described above. Ample T. putrescentiae immatures were needed to supply as food, the dead N. barkeri were picked and placed on SDA at 26±1°C under continuous darkness, then examined under optical microscope for the presence of B. bassiana strain SZ-26 conidia or hyphal bodies. Each replicate consisted of 20 adult N. barkeri, treatments were randomized and the experiment was replicated 8 times using different insect lots over time.Based on the screening of the 28 new strains as reported above, strain SZ-26 was re-evaluated against B. bassiana strain SZ-26 and placed individually in each chamber and about 20 first-instar larval F. occidentalis were supplied as food. A male was added to each chamber for 1 d to allow mating and then the male was removed. The successfully mated females started to lay eggs, the daily fecundity of each was recorded until the females died, predators were transferred into new chambers and supplied daily with first instars as food. The excised leaves were changed every 4–5 days and the predators were transferred into new chambers. The oviposition period and female longevity were also estimated. Controls were set up only with untreated females. For treatment and control, a total of 30 synchronized female predators were tested.The experimental units were designed with two pieces of uniform organic glass (6 cm×5 cm×4 mm), the water—saturated filter paper was placed on one piece, the freshly excised leaf of kidney beans was upside down on the surface of the filter paper, a hole (2.5 cm diameter) was punched in another piece and pressed on the leaf. A chamber was formed between two pieces of organic glass which served as the experimental platform. The newly molted female adults were inoculated by immersion for 5 s in 2 ml conidial suspension of T. putrescentiae immatures were supplied to predators as food. After 1, 2, 12, 24, 36, 48, 60 and 72 h, the SZ-26 -treated samples were fixed in 70% ethyl alcohol for 24 h, than dehydrated in a ascending series of ethyl alcohol , left to air dry for a few seconds and mounted on SEM stubs with double-sided carbon tape. Dried samples were sputtered with gold and observed with the SEM under Quanta 200 FEG at high-vacuum mode.For SEM observation, the predatory mites and thrips were collected and inoculated by immersion for 5 s in 2 ml conidial suspension of the SZ-26 strain, then transferred into the chamber (10/species in each chamber). Ample All statistical analyses were carried out using SPSS software


Mycoplasma hyopneumoniae is a prevalent swine respiratory pathogen that is a major cause of economic loss to pig producers. Control is achieved by a combination of antimicrobials, vaccination and management practices, but current vaccines offer only partial control and there is a need for improved preventative strategies. A major barrier to advances in understanding the pathogenesis of M. hyopneumoniae and in developing new vaccines is the lack of tools to genetically manipulate the organism. We describe the development and optimisation of the first successful plasmid-based system for the genetic manipulation of M. hyopneumoniae. Our artificial plasmids contain the origin of replication (oriC) of M. hyopneumoniae along with tetM, conferring resistance to tetracycline. With these plasmids, we have successfully transformed M. hyopneumoniae strain 232 by electroporation, generating tetracycline resistant organisms. The persistence of extrachromosomal plasmid and maintenance of plasmid DNA over serial passages shows that these artificial plasmids are capable of self-replication in M. hyopneumoniae. In addition to demonstrating the amenability of M. hyopneumoniae to genetic manipulation and in optimising the conditions necessary for successful transformation, we have used this system to determine the minimum functional oriC of M. hyopneumoniae. In doing so, we have developed a plasmid with a small oriC that is stably maintained over multiple passages that may be useful in generating targeted gene disruptions. In conclusion, we have generated a set of plasmids that will be valuable in studies of M. hyopneumoniae pathogenesis and provide a major step forward in the study of this important swine pathogen. Mycoplasma are characterised by their lack of a cell wall, small genome size and utility in synthetic biology. They are responsible for a number of important diseases in humans and other animals. Among the most important species is Mycoplasma hyopneumoniae that is the cause of enzootic pneumonia, a chronic swine respiratory disease of global economic importance to pig producers  revealed two DnaA boxes (each with two bp mismatches with the Escherichia coli consensus sequence 5′-TTATCCACA-3′) upstream and downstream of the dnaA gene within AT-rich regions . The tetM gene and promoter from Enterococcus faecalis were amplified from plasmid pIVT-1  [M. pulmonis [GenBank: NC_002771.1] [M. conjunctivae [GenBank: NC_012806.1] [M. ovipneumoniae [GenBank: AFHO00000000.1]   , M. pulm12806.1]  and M. oin gene 6 bp and t studies . oriC reJ872396]  into theHindIII. DNA was separated by electrophoresis on 0.8% agarose and blotted onto Hybond-N + membrane  using standard methods [bla ampicillin resistance gene of pGEM-T or the tetM gene were generated from PCR-amplified DNA  and 1 μg was digested to completion with  methods . DNA wasM. hyopneumoniae, we constructed a simple plasmid containing the predicted oriC region of M. hyopneumoniae strain 232 and tetM conferring resistance to tetracycline. A large 2.1 kbp region of the M. hyopneumoniae strain 232 genome encompassing the entire dnaA gene and upstream and downstream AT-rich regions was selected. Homologous DNA sequences of other mycoplasmas demonstrating a similar gene arrangement have shown success in functioning as an oriC in artificial self-replicating plasmids [tetM gene has been used as a selection marker for mycoplasmas [M. hyopneumoniae strain 232 to tetracycline. Following the methods of Hannan [M. hyopneumoniae strain 232 with 10 μg of pMHO-1, cells were incubated for 3 h at 37 °C and then plated onto Friis agar containing 2 μg/mL tetracycline. Triplicate repeats were performed and after 14 days incubation, no growth was observed for pMHO-1 or “no DNA” controls. We considered that the tetM promoter sequence may be inactive in M. hyopneumoniae and/or that the tetracycline concentration may be too high to be overcome by transformed organisms. The tetM sequence under control of its own promoter, as derived from Enterococcus faecalis, is expressed at levels sufficient to confer tetracycline resistance in several mycoplasmas [spiralin gene promoter of Spiroplasma citri that has be shown to be active in several mycoplasma species [tetM and the spiralin gene promoter sequence, we produced plasmids pMHO-3 and pMHO-4 containing tetM with the putative promoter regions of the M. hyopneumoniae strain 232 P97 ciliary adhesin gene [ldh) gene respectively. In addition to altering the promoter sequences controlling tetM expression, we sought to determine whether or not a lower concentration of tetracycline would enhance the growth of transformants. For other mycoplasmas, transformed cells are typically selected using concentrations of between 2 and 4 μg/mL tetracycline [M. hyopneumoniae 232 on Friis agar, we lowered the concentration of tetracycline to 0.2 μg/mL. M. hyopneumoniae strain 232 was transformed in triplicate with plasmids pMHO-1, pMHO-2, pMHO-3 and pMHO-4 and plated onto Friis agar containing 0.2 μg/mL tetracycline. After incubation for 14 days tetracycline resistant colonies were present in bacteria transformed with all four oriC plasmids but were completely absent in the “no DNA” control, demonstrating activity of the four promoter sequences. Morphologically, colonies were identical to untransformed M. hyopneumoniae colonies, but colony growth lagged by approximately 3 days , with fewer for pMHO-3 (5.8 × 10-5/CFU) and pMHO-1 (1.7 × 10-5/CFU), and least with pMHO-4 (8.3 × 10-6/CFU). Individual colonies were picked from agar and grown in Friis medium containing 0.5 μg/mL tetracycline, after which they were sub-cultured every 2–3 days in 1 μg/mL tetracycline. After three serial passages, plasmid DNA was extracted and separated by electrophoresis. Plasmid DNA was readily identified for each transformant whereas no plasmid DNA was seen with untransformed M. hyopneumoniae 232  was reduced by 300 bp by removing excess DNA sequence lying upstream and downstream of the AT-rich intergenic regions (pOriC-ii). With both AT-rich regions and the dnaA gene still present, as expected, there was no reduction in transformation efficiency  onto Friis agar with and without 0.2 μg/mL tetracycline. After 14 days growth, colony counts were performed. We observed a rapid reduction in tetracycline resistant colonies following serial passage in medium containing no tetracycline, showing that selection is necessary to maintain resistance, and suggesting that the transformants can readily be “cured” of plasmid DNA  transformed M. hyopneumoniae at passage 19. We predicted that integration of plasmid pMHO-2 into the M. hyopneumoniae strain 232 genome would generate a 4.6 kbp HindIII digestion fragment that would be detected by a DIG-labelled probe annealing to the tetM gene. By separating a larger amount (2.5 μg) of HindIII-digested total DNA and using a DIG-labelled tetM gene probe, we were able to visualise a 7.4 kbp band consistent with plasmid DNA in three transformants and a 4.6 kbp band consistent with integrated plasmid DNA in two transformants. This would suggest that recombinase systems active in M. hyopneumoniae are capable of allowing homologous recombination to occur, and therefore the minimum oriC plasmid may be useful in directing homologous recombination to target genes. Finally, we found that many of our UK field isolates exhibited greater resistance to tetracycline than M. hyopneumoniae 232, making it unachievable to select for transformed colonies on Friis agar containing 0.2 μg/mL tetracycline (data not shown). To circumvent this problem, we replaced the tetM gene of pMHO-2 with the puromycin N-acetyltransferase (pac) gene from pMiniTn4001PsPuro [pac gene also contains the spiralin gene promoter of S. citri and confers resistance to puromycin, an aminonucleoside that has no veterinary clinical use, and therefore will be generally applicable as a genetic marker for M. hyopneumoniae. Transformation of M. hyopneumoniae strain 232 with pMHOpuro resulted in a transformation frequency of 1.6 × 10-4/CFU compared to 4.6 × 10-5 for plasmid pMHO-2 (containing the tetM gene). Thus puromycin may be a suitable selection marker for M. hyopneumoniae isolates that are resistant to tetracycline.To determine the stability of our plasmids upon serial passage over time, individual transformants were picked into Friis medium containing 0.5 μg/mL tetracycline and passaged 19 times. At passages 3, 8 and 19, total DNA was extracted and Southern hybridisation performed with a DIG-labelled probed specific for the 01PsPuro  to generM. hyopneumoniae has been a major obstacle to progress in understanding the survival and pathogenesis of the organism. Using simple artificial self-replicating plasmids containing the oriC of M. hyopneumoniae and an antimicrobial resistance gene, we have shown for the first time that the organism is susceptible to transformation. We found that the transformation conditions optimised by Hedreyda et al. [Mycoplasmas are suitable for M. hyopneumoniae. However, other critical factors include titration of the tetracycline concentration to a level that can readily be overcome by the TetM resistance protein, the use of high voltage for electroporation and the use of a large excess of plasmid DNA. Additionally, we found that the choice of promoter affected the expression of the tetM gene, and as is the case with other mycoplasmas, the spiralin gene promoter of Spiroplasma citri is a suitable choice for M. hyopneumoniae. Our oriC system allowed us to assess the function of putative M. hyopneumoniae promoter sequences, and confirmed the identity of two promoter sequences that were active in controlling tetM expression. The P97 promoter sequence was recently predicted and we have verified that this is functional in our plasmid system [ldh promoter, and again demonstrated its activity in our plasmid system. These M. hyopneumoniae-specific promoters will be valuable in the generation of further tools for the genetic manipulation of the organism. Using our system, we verified that the origin of replication of M. hyopneumoniae lies in the region of dnaA and includes two short AT-rich regions. It has been suspected that the oriC of M. hyopneumoniae lies in this region, but unusually within the AT-rich regions there are only two DnaA boxes that resemble the E. coli consensus sequence, whereas for other mycoplasma species, there are typically multiple DnaA boxes [dnaA gene, but these are not required for a functional oriC. We hypothesised that the oriC regions of mycoplasmas showing a high degree of nucleotide sequence similarity to M. hyopneumoniae would function in M. hyopneumoniae, and enable stable plasmid maintenance without integration by homologous recombination. However, it appears that oriCs are very specific to their host species and none of the oriC regions from four closely related Mycoplasma species functioned in M. hyopneumoniae.The inability to genetically manipulate a et al.  and usedd system . Using td system , we alsoaA boxes . AdditioM. hyopneumoniae sequence that functioned in an oriC plasmid (pOriC-iv/v) was approximately 700 bp in length and included both AT-rich regions. This plasmid was maintained extrachromosomally over at least 19 passages with selection pressure, equivalent to over 8 weeks post-transformation. However, when selection pressure was removed, tetracycline resistance was rapidly lost within 5 passages. Having demonstrated that plasmids carrying sequences with a high degree of homology to regions of the M. hyopneumoniae genome can undergo integration by homologous recombination, it is likely that this plasmid will be useful in generating targeted disruptions in genes of interest. By incorporating sequences encoding mycoplasma genes of interest, it is likely that we will be able to direct homologous recombination and cause disruption of these genes, as has been shown with other mycoplasmas [tetM, we demonstrated that pac conferring resistance to puromycin can be expressed in M. hyopneumoniae strain 232 from our oriC plasmids when driven by the spiralin gene promoter sequence of S. citri. While M. hyopneumoniae strain 232 is exquisitely sensitive to tetracycline, as demonstrated in this study, many of our recent field isolates of M. hyopneumoniae are relatively resistant (data not shown). However, these strains are susceptible to puromycin, therefore pac may be a more suitable selection marker for generating transformants in other M. hyopneumoniae isolates.The minimum oplasmas ,12. In aM. hyopneumoniae is indeed susceptible to genetic manipulation by transformation with self-replicating plasmids. In doing so, we have developed a system that will undoubtedly be useful in genetic studies of M. hyopneumoniae and probably other mycoplasma species. Additionally, we anticipate that our findings will be a key step towards the development of a transposon-mediated system to generate random mutants of M. hyopneumoniae, which will take us a step closer to unraveling the pathogenesis of this poorly understood pathogen.In summary, we have shown that The authors declare that they have no competing interests.Experiments were conceived by GAM, ANR & BSC. Experiments were performed by GAM, ASD & DM. BSC, JTB, ANR, PL, BWW, AWT and DJM assisted with experimental design. The manuscript was written by GAM & ANR and was approved by all authors.BRaDP1T consortium. List of researchers collaborating in the BRaDP1T consortium.Click here for file