S. pneumoniae. Search for effective vaccine continues and some serotype independent pneumococcal proteins are considered as candidates for the design of new vaccines, especially a mucosal vaccine, since pneumococci enter the body through mucosal surfaces. Selection of the appropriate adjuvant is important for mucosal vaccines, and lactic acid bacteria (LAB) with immunostimulant properties are promissory candidates. In this work, we assessed the adjuvant effect of a probiotic strain, Lactobacillus casei (L. casei), when nasally administered with a pneumococcal antigen  for the prevention of pneumococcal infection. Adjuvanticity of both live (LcV) and heat-killed (LcM) was evaluated and humoral and cellular antigen-specific immune response was assessed in mucosal and systemic compartments. The potential mechanisms induced by nasal immunization were discussed.At present, available pneumococcal vaccines have failed to eradicate infections caused by Nasal immunization of young mice with PppA+LcV and PppA+LcM induced anti-PppA IgA and IgG antibodies in mucosal and systemic compartments and levels of these specific antibodies remained high even at day 45 after the 3rd Immunization (3rd I). These results were correlated with IL-4 induction by the mixture of antigen plus LcV and LcM. Also, PppA+Lc (V and M) induced stimulation of Th1 and Th17 cells involved in the defence against pneumococci. The protection against pneumococcal respiratory challenge at day 30 after the 3rd I showed that PppA+LcV and PppA+LcM immunizations significantly reduced pathogen counts in nasal lavages while prventing their passage into lung and blood. Survival of mice immunized with the co-application of PppA plus LcV and LcM was significantly higher than in mice immunized with PppA alone and control mice when intraperitoneal challenge was performed. No significant differences between the treatments involving LcV and LcM were found.Live and heat-killed L. casei enhanced the antigen-specific immune response when administered nasally with a pneumococcal antigen. Considering the potential risk associated with live bacteria, the design of a nasal vaccine based on pneumococcal antigens and heat-killed L. casei emerges as a safe and effective strategy for the prevention of pneumococcal infections and opens new possibilities of application of dead LAB as adjuvants in vaccine formulations against other pathogens. Streptococcus pneumoniae strains and some problems associated with vaccines have made pneumococcal diseases a major health problem worldwide. At present, there are two types of pneumococcal vaccines in use: capsular polysaccharide pneumococcal vaccines (PPV) and protein-polysaccharide conjugate pneumococcal vaccines (PCV). PPV are not effective in elderly people, immunocompromised individuals or children under 2 years of age, while PCV are expensive for application in developing countries as global vaccination strategies. In addition, recent researches have shown that serotypes eradicated by PCV are being replaced by non-vaccine pneumococcal serotypes [E. coli lymphotoxin (LT), which are highly immunogenic as well as the most effective experimental adjuvants known today. However, they are also very toxic and not acceptable for human use [S. pneumoniae in mouse models [S. pneumoniae  [Lactobacillus casei expressing PspC was able to reduce nasal colonization by S. pneumoniae in a mouse model [Lactobacillus casei CRL 431 (L. casei), a probiotic strain, was able to improve the immune response of the host against respiratory pathogens in a mouse model. The effect of L. casei was associated with the stimulation of the innate and adaptative immune response [S. pneumoniae [S. pneumoniae.Pneumococcal infections are one the most common diseases in both developed and developing countries ,2. Despierotypes -5. Thus,erotypes ,7 but atuman use . In thisuman use , some ree models -14. In a and 23) , was abl and 23) ,17. A rese model . Howeverse model -21. In tresponse ,20-22. Iresponse , so theiresponse  and inveeumoniae -14. In cS. pneumoniae enters the host primarily through the respiratory mucosa, vaccination strategies by the nasal route are of great interest, especially if we take into account the fact that most vaccines delivered parenterally are not completely effective to induce mucosal immunity [Lactobacillus casei as a nasal adjuvant could be able to trigger protective immune responses against pneumococcal infection. On the basis of the above considerations, the aim of this work was to assess the mucosal and systemic immune response induced by nasal immunization with a pneumococcal antigen (PppA) associated to live and heat-killed Lactobacillus casei and to analyze the possible immune mechanisms involved in its protective effect.On the other hand, since immunity . Moreoveimmunity -31. Sincimmunity , we hypoMucosal IgA and IgG antibodies are important for the defence against respiratory infections because, while IgA prevents pathogen attachment to epithelial cells, thus reducing colonization, IgG promotes phagocytosis and prevents local dissemination of the pathogen in the alveoli as well as its passage into blood . At the p < 0.05). LcV, LcM and PppA alone showed a variable pattern of Th1 cytokine induction throughout the period assessed , nasal immunization with PppA+LcV and PppA+LcM increased IL-2 and INF-γ levels  compared with basal values. PppA+LcV induced higher IL-2 levels than PppA+LcM on days 73 in NL and on days 28 and 42 in BAL, while INF-γ levels on day 58 in BAL also were higher for PppA+LcV compared to LcM+PppA  on day 28 in BAL, while no significant differences in this cytokine were observed for other periods in mucosal fluids neither in serum. The regulatory IL-10 cytokine showed a variable behaviour, depending on the experimental group studied. However, PppA+LcV and PppA+LcM induced higher values of IL-10 in both mucosal and systemic compartments during almost all periods evaluated. This fact would be important to ensure a balanced immune response against a pneumococcal infection. Finally, IL-17A, which represents activation of the Th17 cells, was also evaluated  was able to exercise an important adjuvant effect on the specific cellular and humoral immune response. Thus, a good antigen-specific immune response was reached with the mixtures: pneumococcal Ag+LcV and pneumococcal Ag+LcM.On the other hand, during almost all periods evaluated, nasal immunization with the different treatments induced high levels of IL-4 in both mucosal and systemic compartments compared with basal levels Figure  and 3C. d Figure  and 4C. L. casei was able to prevent pneumococcal colonization in lung and pathogen dissemination in blood, showing negative hemocultures  immunization. PppA+LcV and PppA+LcM immunizations were able to reduce pathogen counts in NL significantly and prevented their passage into lung and blood. Finally, immunization with PppA alone induced a lower reduction in the number of S. pneumoniae in NL and lung compared with the other experimental groups, except for the control. In addition, this antigen alone did not prevent pathogen passage into blood.A pneumococcal respiratory infection model was used to assess nasal and lung colonization as well as dissemination of the pathogen into blood. Nasal immunization with the pneumococcal antigen associated with live (LcV) and heat-killed (LcM) es Table . Also, cS. pneumoniae serotype 14 four weeks after the third immunization.In order to determine whether the induction of specific humoral and cellular immune response induced by nasal immunization with PppA+LcV and PppA+LcM was correlated with increased resistance to pneumococcal infection, young mice were intraperitoneally challenged with S. pneumoniae serotype 14 was used because of its high potential for invasive disease and also because it is the prevalent one in our country.Animals that received the PppA protein alone or PBS were also evaluated. In both systemic and respiratory challenge assays, p < 0.01 Cox's Mantel Test), in which 100% of the mice died after day 4 post challenge. PppA+LcM showed a survival percentage of 50% with respect to PBS group but no significant differences were found compared with PppA+LcV. Mice that received PppA alone showed a survival percentage of 20%, with no significant differences with control. The animals in this group died between days 4 and 6 post challenge. These results demonstrate that both LcV and LcM are effective as adjuvants when they are associated with a pneumococcal antigen because PppA+LcV as well as PppA+LcM immunizations increased resistance to the systemic challenge with S. pneumoniae serotype 14.Young mice immunized with PppA+LcV showed a survival percentage of 60% on day 21 after challenge Figure , which wS. pneumoniae. A new conjugate vaccine PCV13 includes 6 additional serotypes with respect PCV7 and provides a broader spectrum of protection than the vaccines mentioned above [S. pneumoniae infection by nasal immunization with PppA when live (LcV) and heat-killed (LcM) Lactobacillus casei 431 was used as a mucosal adjuvant. The role of PppA in the pathogenesis of pneumococcal infection is unknown but it presents homology with a nonheme iron-containing ferritin protein from Listeria innocua and other bactoferritins.The licensed pneumoccocal vaccines , formulated as vaccines depending on serotypes, did not enable the eradication of infections caused by ed above . Howevered above , which ced above  to ensured above ,20-22. OLactococcus lactis [S. pneumoniae [L. fermentum isolates from the pharynx of BALB/c mice was able to activate macrophages in the respiratory tract and reduced nasal and lung colonization of S. pneumoniae. Also, in previous investigations, we demonstrated that the nasal administration of Lactococcus lactis (LL) enhances the protective immune response against a pneumococcal challenge in a mouse model [L. casei Shirota activated the cellular immune response in the respiratory tract and protected against Influenza Virus infection. Recently, it was reported that both live and dead Lactobacillus casei 431, the ones used in this study, improved protection against pneumococcal infection in malnourished mice [L. casei nasally administered when associated with a pneumococcal antigen. In this sense, considering that the humoral immune response is extremely important in the defence against S. pneumoniae, specific anti-PppA antibodies were evaluated. Thus, nasal immunization of PppA alone induced low levels of specific IgA and IgG antibodies while the PppA+LcV and PppA+LcM mixtures induced the highest values of antigen-specific antibodies. In a previous report it was demonstrated that passive immunization of naïve mice with serum from animals immunized with recombinant Lactococcus lactis-PppA increased the survival percentage against a pneumococcal challenge [S. pneumoniae. Moreover, it was demonstrated that nasal administration of L. casei live and killed heat increased the anti-pneumococcal IgA and IgG antibodies in the local and systemic compartments after a pneumococcal respiratory infection [L. casei increased the number and activation of alveolar macrophages and neutrophils in BAL. The phagocytosis process involved in the primary depuration of pneumococci would be facilitated by the specific antibodies. Overall, the immunostimulatory effect of the probiotic strain contributes to the strengthening of the protective immune response after vaccination. On the other hand, the nasopharynx-associated lymphoid tissue (NALT) contains all the immune cells required for the induction and regulation of the mucosal immune response to antigens delivered into the nasal cavity [L. casei would activate the immune system and enhance the response against a specific pneumococcal antigen. At present, studies are being carried out in the nasopharynx to analyze the immune mechanisms induced at this level by the probiotic strain alone and associated with pneumococcal antigens. In previous studies, it was demonstrated that the oral administration of L. casei induced an increase in IgA cells in the bronchus-associated lymphoid tissue (BALT) in lung [et al. [L. casei beneficially regulates the balance between tumor necrosis factor-alpha and interleukin 10, allowing a more effective immune response against infection and modulating the inflammatory response, with less damage to the lung [S. pneumoniae, stimulation of T helper cells was studied on the basis of the cytokines profile induced by immunization. Our results showed that PppA+LcV and PppA+LcM immunizations were able to stimulate INF-γ release in NL, BAL and serum and that LcV and LcM alone induced a similar effect although to a lesser degree. The INF-γ Th1 cytokine enhances chemokine expression and promotes pulmonary neutrophil recruitment into the infected lung, playing an important role in the neutrophil-mediated host protective responses against pneumococcal infection. IL-2 was increased in mucosa and serum by PppA+Lc and PppA+LcM immunizations during almost all the periods assayed and this effect would partly account for the high level of specific antibodies observed on day 73 after the first immunization. Although the factors influencing the differentiation of memory cells are incompletely understood, some investigations support a role of IL-2 in memory cell differentiation [S. pneumoniae colonization from the mucosal surface of the nasopharynx. In addition, IL-17 would mediate the death of S. pneumoniae in the presence or absence of specific antibodies [PppA was able to induce protective antibodies when it was administered by the nasal route associated with a mucosal adjuvant  or when s lactis ,39 enhans lactis , the deseumoniae ,19,40,41eumoniae  demonstrhed mice . All thehallenge  and theshallenge . Thus, bl cavity . Hussell in lung ,45. In c in lung ,45, alth [et al.  demonstrthe lung . Thus, wntiation ,48. Thistibodies ,50. Thustibodies .S. pneumoniae that should be harnessed in the design of effective mucosal vaccines. The effectiveness of nasal immunization by combination of PppA with live and heat-killed L. casei was evident in challenge assays carried out on day 30, after the third immunization. Thus, PppA+LcV and PppA+LcM vaccination considerably reduced pathogen counts in NL and prevented lung and blood colonization when animals were challenged nasally. In addition, these treatments allowed the survival of 50-60% of the mice after peritoneal challenge with the pathogen. Immunization with PppA alone could fail to provide adequate protection against pneumococcal infection partly because it does not induce high specific antibody levels but mainly because it would not be able to stimulate effector and memory cells in an efficient way. In this sense, nasal immunization with PppA was not a good inducer of IL-2, IL-17 and INF-γ, which are important factors in the defence against S. pneumoniae. In contrast, vaccination with PppA+Lc (LcV and LcM) was able to induce a good specific humoral and cellular immune response in the mucosal and systemic compartments. Our results indicate that L. casei 431 can act as a good mucosal adjuvant, allowing a potent specific immune response when it is associated with a pneumococcal antigen, and that the viability of the strain is not a crucial condition for stimulating the antigen-specific immune response. A recent report [Bordetella pertussis vaccine (wP) or wP with low levels of LPS (wP low) was able to protect against a respiratory lethal challenge with S. pneumoniae. However, the use of dead lactic acid bacteria (Gram+) as a nasal adjuvant represents a novel alternative for the development of mucosal vaccines using proteic antigens. This is the first report that considers the association of a specific pneumococcal antigen with a non-viable probiotic strain as an adjuvant for the development of a nasal vaccine. In addition, it is the first study dealing with the possible mechanisms involved in long-lasting specific systemic and mucosal immunity induced after nasal vaccination. The use of dead microorganisms would allow the safe application of potential vaccines in immunocompromised hosts and elderly people without the risks associated with the use of live bacteria [Lactobacillus casei 431 as an adjuvant is not comparable with other studies [L. casei emerges as a safe and effective strategy for the prevention of pneumococcal infections and opens new possibilities for the application of dead lactic acid bacteria in the protection against other pathogens.The induction and maintenance of antigen specific T-cell responses is an essential feature in the protection against t report  demonstrbacteria . The eff studies  because Lactobacillus casei associated to pneumococcal antigen was able to provide effective protection against pneumoccocal infection in mice. The results obtained in animal models do not guarantee their effectiveness in humans; however, they enable the establishment of scientific bases for subsequent testing in humans. In this sense, the use of vaccine formulations containing dead organisms is safer and more attractive for the design of human clinical trials in the short term.In this work, we demonstrated that nasal immunization with live and heat-killed E. coli-PppA was obtained in our laboratory and the development of this strain was described in a previous report from our work group [pppA gene was amplified from the chromosomal DNA of S. pneumoniae T14 with EC1 and EC2 primers (EC1 (forward): 5'GGGGCCATGGCTTGTAGAATTGAAAAAAGAA 3', EC2 (reverse): 5'GGGGTCGACTAAACCAGGTGCTTGTCCAAGTTC 3'). The PCR product was purified from agarose gel using home-made silica beads and treated with the restriction enzymes NcoI and SalI. Then, the pppA gene was ligated into the NcoI and SalI sites of pET28b using T4 DNA ligase (Promega). A recombinant plasmid containing the pppA gene, named pET-PppA, was first recovered from E. coli DH10B cells and then transformed into BL21 (DE3) cells for expression. BL21(DE3) (pET-PppA) cells were grown with aeration until they reached an OD600 of 0.8 in LB medium supplemented with neomycin (50 μg/ml) and induced with 1 mM IPTG  (Sigma) for 2 h. Whole-cell lysates were prepared, cell debris was removed by centrifugation , and the proteins present in the supernatant were run on a 12% SDS-PAGE gel to confirm expression of the recombinant PppA (rPppA). rPppA was purified from BL21(DE3) (pET-PppA) as described below.Recombinant rk group . BrieflyLactobacillus casei CRL 431 (LcV) [9 cell/day/mouse) for its later administration by the nasal route. Heat-killed L. casei (LcM) was prepared by heating bacteria in a water bath at 80°C for 30 min and the lack of bacterial growth was confirmed using MRS agar plates.31 (LcV) ,21, obtaStreptococcus pneumoniae serotype 14, kindly provided by Dr. M. Regueira from the Laboratory of Clinical Bacteriology, National Institute of Infectious Diseases, Argentina, was used. Freshly grown colonies of S. pneumoniae were suspended in THB and incubated at 37°C until the log phase was reached [g for 10 min at 4°C and washed three times with sterile PBS. Then, the cell concentration of the pathogen was adjusted to the dose used in the respiratory (106 cells/mouse) and peritoneal (108 cells/mouse) challenge assays. reached . The patRecombinant PppA (rPppA) was purified using a His-Bind purification kit (Novagen) and visualized by electrophoresis on 12% SDS-polyacrylamide gels, as previously described . The reaad libitum. Experiments were approved by the Animal Care and Ethics committee at CERELA.Three-week-old male and female Swiss albino mice were obtained from the closed colony at CERELA and each experimental group consisted of five or six mice for each period evaluated. During all experiments, animals were supplied with balanced rodent food and water L. casei (109 CFU) as the mucosal adjuvant. LcV and LCM suspension were prepared in PBS. The immunization was carried out using a protocol that comprised 3 successive administrations that included two consecutive days each time with a 14-day interval between them . Groups that received LcV, LcM, rPppA and phosphate-buffered saline solution (PBS) were used as controls. Fourteen days after each immunization and also at days 58 and 73 after the first immunization, samples of nasal and bronchoalveolar lavages  and serum were collected. For nasal lavage collection, a catheter attached to a syringe was inserted into the nostril and a total of 0.3 ml of PBS was injected. This procedure was carried out twice and the outflow was collected. Then, NL was centrifuged at 8,000 × g for 10 min to remove cell debris and clarified NL samples were collected. BAL samples were obtained according to the technique previously described [g (at 4°C), the supernatants were collected. All fluids were stored at 70°C until analysis.Young mice were nasally immunized with 30 microlitres of a mixture of 5 ug of rPppA plus live (LcV) and heat-killed (LcM) escribed ,18. Brierd immunization, control and immunized mice were challenged intranasally with the pathogen by dripping 25 μl of an inoculum containing 106CFU (log phase) in PBS into each nostril and allowing it to be inhaled. Mice were killed at 3 days after challenge with S. pneumoniae and their lungs were excised, weighed and homogenized in 5 ml sterile peptone water. Also, nasal lavages were collected as described above. Homogenates and nasal lavages were diluted appropriately, plated in duplicate on Columbia sheep blood agar and incubated for 18 h at 37°C; then, the number of colonies was counted. S. pneumoniae was identified by standard techniques [Experimental respiratory infection was induced as previouslydescribed. Thirty days after the 3chniques ,20 and tS. pneumoniae serotype 14 was grown until cells reached their exponential phase under the conditions described above, and each mouse was infected intraperitoneally with 100 ul of PBS containing 108 cells of the pathogen. Each experimental group consisted of 14 mice and survival was closely monitored for 21 days.Intraperitoneal challenge experiments were carried out 30 days after the third immunization of the animals. 2O2) and the reaction was stopped by the addition of H2SO4 1 M. Readings were carried out at 493 nm  and samples were considered negative for the presence of specific antibodies when OD493 < 0.1.Serum and BAL antibodies against PppA protein were determined by ELISA, according to previous experiments ,37. BrieCytokine concentrations in NL, BAL and serum were measured by mouse Th1/Th2 ELISA Ready SET Go! Kit , including interleukin (IL)-2 and interferon (IFN)-γ as Th1-type, IL-4 and IL-10 as Th2-type cytokines. The IL-17A as a Th17-type cytokine was also measured using the ELISA kit from e-Bioscience (BD Biosciences). The sensitivity of assays for each cytokine was as follows: 4 pg/ml for IL-2, IFN-γ and tumour necrosis factor (TNF)-α, and 2 pg/ml for IL -4 and IL-10 and IL-17 4 pg/ml.post hoc test using the StatGraphics software . Differences were considered significant at P < 0.05.Experiments were performed in triplicate and results were expressed as mean ± standard deviation (SD). After verification of a normal distribution of data, significant differences between means were determined by analysis of variance (ANOVA) with Fisher's least significant difference (LSD) Lactobacillus casei: LcM, live Lactobacillus casei: LcV, pneumococcal protective protein A: PppA.NL: nasal lavages; BAL: Bronchoalveolar lavages, heat killed EV and MM contributed in designing the study and performed the experimental assays and analysis of results. MM performed the statistical analysis, prepared and wrote the manuscript. All authors read and approved the final manuscript.


Treacher-Collins syndrome (TCS) is a rare dominant autosomal anomaly resulting from malformation or disruption of the development of the first and second branchial arches. It is characterized by micrognathia, malar hypoplasia, and malformations of the eyes and ears. The prenatal diagnosis using two-dimensional ultrasonography (2DUS) is characterized by identification of facial malformations together with polyhydramnios. Three-dimensional ultrasonography (3DUS) has the capacity to spatially display these facial malformations, thus making it easy for the parents to understand them. We present a case of TCS diagnosed in the 33rd week using 3DUS, with postnatal confirmation using cranial computed tomography and anatomopathological analysis. Treacher-Collins syndrome (TCS) is a rare dominant autosomal craniofacial disorder 1 : 50,000) characterized by symmetrical bilateral malformations that frequently include hypoplasia of the mandibular-zygomatic complex, palpebral fissures, coloboma of the lower eyelid, and absence of medial eyelashes at the defect, malformation of the middle and external ears, and conductive hearing loss  : 50,000. TCS forDiagnosing TCS prenatally is very important because the tongue may obstruct the airways and lead to the newborn's death. A prenatal diagnosis of this syndrome may be suspected from two-dimensional ultrasonography (2DUS), through identifying the presence of polyhydramnios, micrognathia, palpebral clefts, and low-set ears , 4. ThreWe present a case of TCS diagnosed in the 33rd week by means of 3DUS, with postnatal confirmation of the anomalies by means of computed tomography (CT) on the face. We emphasize the differential diagnoses with other types of craniofacial dysostosis, along with the importance of 3DUS for enabling the parents to have a better understanding of the facial malformations.The patient was a 24-year-old primigravida without comorbidities and with adequate levels of prenatal care. She was referred to the Department of Gynecology and Obstetrics, School of Medical Sciences, Santa Casa de Misericórdia de São Paulo (FCMSCSP), because of a suspicion of severe facial malformation in the fetus, seen on obstetric level I ultrasonography at another clinic. A 2DUS examination performed in the 33rd week showed microphthalmia, a transverse facial cleft occupying the lower half of the face and bilateral auricular hypoplasia. A “golf ball” was also viewed in evaluating the four heart chambers, which was confirmed by means of fetal echocardiography. However, there were no other associated cardiac malformations. In order to view the face of the fetus better, 3DUS was performed using the SonoAce X8 device , equipped with a volumetric convex transducer (3–7 MHz). The 3D image in rendering mode made it possible to clearly view the transverse facial cleft, microphthalmia, and low-set ears . In the The patient underwent cesarean delivery at a gestational age of 35 weeks and 3 days because of fetal hemodynamic centralization, giving birth to a live female infant weighing 2,355 g and presenting Apgar 7 and 8. Due to respiratory discomfort, the newborn was provided with orotracheal intubation early on. The physical examination conducted after the birth showed micrognathia, hypoplasia of the maxilla, anophthalmia, bilateral auricular appendages, and severe microtia . CranialExtubation was not possible because of the severe facial deformity, which affected the respiratory canals, and therefore, the newborn was subjected to tracheostomy and gastrostomy. Eleven days after birth, the newborn progressed to death due to bronchopneumonia. The autopsy confirmed the physical and imaging findings.Facial dysmorphism, particularly when associated with disorders of the first and second branchial arches, generally results from a combination of migration and inadequate formation of facial mesenchyme . SeveralIn our case, the diagnosis of TCS was made in the 33rd week of pregnancy. Late diagnosis is generally due to late-starting prenatal followup at public healthcare services in developing countries like Brazil. Moreover, since Brazilian law does not permit termination of pregnancy in cases of fetal malformation, this contributes towards late diagnosis of severe fetal malformations, thus putting the pregnant women's lives at risk. Prenatally, TCS diagnosed by means of 2DUS is well established through findings of severe facial abnormalities, generally in association with polyhydramnios, which is an indirect sign of upper airway obstruction. Therefore, adequate prenatal diagnosis enables followup for the pregnant woman at a tertiary-level service, so as to provide better support for the newborn. With regard to 3DUS, there are only two reports in the literature of prenatal diagnosing of TCS, and both of these cases were also diagnosed at a late stage , 8. BothIn summary, we presented here a case of TCS diagnosed in the third trimester by means of 3DUS, which was confirmed postnatally by means of CT on the face and anatomopathological analysis. We believe that 3DUS should be used in cases of severe fetal abnormalities, so that the parents can understand the malformations better.


Tandem repeats (TRs) are often present in proteins with crucial functions, responsible for resistance, pathogenicity and associated with infectious or neurodegenerative diseases. This motivates numerous studies of TRs and their evolution, requiring accurate multiple sequence alignment. TRs may be lost or inserted at any position of a TR region by replication slippage or recombination, but current methods assume fixed unit boundaries, and yet are of high complexity. We present a new global graph-based alignment method that does not restrict TR unit indels by unit boundaries. TR indels are modeled separately and penalized using the phylogeny-aware alignment algorithm. This ensures enhanced accuracy of reconstructed alignments, disentangling TRs and measuring indel events and rates in a biologically meaningful way. Our method detects not only duplication events but also all changes in TR regions owing to recombination, strand slippage and other events inserting or deleting TR units. We evaluate our method by simulation incorporating TR evolution, by either sampling TRs from a profile hidden Markov model or by mimicking strand slippage with duplications. The new method is illustrated on a family of type III effectors, a pathogenicity determinant in agriculturally important bacteria Ralstonia solanacearum. We show that TR indel rate variation contributes to the diversification of this protein family. Today accurate multiple sequence alignment (MSA) is frequently needed in genomics and molecular biology studies. In an alignment defined by the evolution of sequence residues (rather than by its molecular structure), characters in the same column are assumed to be homologous, indicating that they have evolved from a common ancestral character. Recent successful additions to the alignment tools arsenal are the phylogeny-aware algorithms that reduce alignment errors producing biologically more meaningful alignments . Graph-bA TR is a consecutive repetition of sequence segments with a similar character pattern. In coding regions, TR mutations have direct effects on the protein product, and even non-coding TRs mutations can seriously impact genetic fitness . A numbeSupplementary Figure S10). Despite this, to ease the modeling and computation, current methods typically assume fixed unit boundaries but are still of high complexity . BoIn addition, most current alignment methods for sequences with rearrangements and repeats ,19 inferWe describe and implement a model of TR unit evolution, capitalizing on the advantages of a recent graph-based phylogeny-aware MSA algorithm . In our ProGraphMSA+TR inherits all other advantages of our previous graph-based alignment method , includiWe evaluate the new algorithm in a simulation framework incorporating TR evolution, by either sampling TR units from a profile hidden Markov model (HMM) or by mimicking strand replication slippage with duplications.The new TR indel method has been implemented within ProGraphMSA , a frameTR unit indels were modeled by paths in the graph that bypass whole repeat units. This is achieved by adding edges to the leaf sequences to allow for skipping one or multiple repeat units. The edge penalties are adjusted to account for unit indel costs. Although theoretically, an arbitrary cost model is possible, we used affine gap penalties. Also for TR unit indels, the graph-based approach is able to differentiate insertions from deletions and thus penalizes them correctly.To annotate the TR regions in a sequence with additional edges, ProGraphMSA+TR requires an MSA of homologous TR units (TR-MSA) for each type of TR in the given sequence. For each sequence to be aligned, ProGraphMSA+TR transparently calls TR detector programs T-REKS  or TRUSTIntuitively, our method should offer the best performance in reconstructing the evolution of TRs in homologs of moderate divergence and while the TR unit indel rate is low enough to produce non-overlapping events. Our approach allows only for TR unit indel events and does not enforce the alignment to be consistent in terms of TR-level homologies, i.e. two TR-level homologous characters in a sequence can be aligned to two non-homologous characters in the second sequence. Although such consistency could be achieved by column-wise alignment [e.g. profile-profile alignment ] of the To evaluate ProGraphMSA+TR, we designed a simulation algorithm, which evolved an ancestral sequence containing TRs into a set of extant sequences. The mutation process was modeled using the WAG substitution model  with chaSimulated TRs had average unit lengths between 10 and 100 amino acids. In the core TR region, we additionally simulated insertions and deletions of whole TR units with different insertion/deletion rates and different length distributions (one or multiple units at once). Most importantly two distinct modes were used for simulating TR unit indel events.‘The profile method’ sampled all inserted TR units  from a profile HMM a. As uni‘The duplication method’ mimicked strand slippage mutations  by dupliAll simulation parameters were identical for the two methods and the four TR types, except for the ancestral unit counts and the length distributions of TR unit indels . AlthougThe comparative performance of different methods was evaluated on simulated data generated with different TR unit types, different mutation and indel rates, and for two distinct simulation modes of TR unit indels . To assespecificity (or accuracy).To evaluate the inferred MSAs in terms of correctly aligned residue pairs, we used two common alignment-quality statistics fD and fM. The ‘developer score’ (fD) is the fraction of correct pairs relative to the total number of pairs in the reference alignment, i.e. it is a measure of ‘sensitivity’ . The ‘modeler score’ (fM) is computed relative to the number of pairs in the reconstructed MSA and thus provides a measure of For profile-based simulation of protein alignments with TRs, the definition of fD and fM is straightforward as TR unit insertions do not create ambiguous pairings. However, in the presence of TR unit duplication events, a global MSA becomes ambiguous: each copy of a unit can be treated either as homologous or as an insertion, i.e. every character of the corresponding TR region in the ancestral sequence has two (or more) possible pairings in the alignment, but only one can be realized owing to the nature of global alignments.Consider a scenario in which an ancestral TR unit was duplicated independently in disjoint clades . Each chThe quality of the inferred MSAs was assessed separately for TR regions and their flanking sequences. supplementary material .As the observed performance was similar for all types of TR units, full results are shown only for LRR-containing MSAs  and 7. TSupplementary Figure S10). Interestingly, the awareness of TRs helps to improve the alignment of both the TR region and the flanking regions, even if not always significantly.For programs that do not account for TR presence , we observed similar fD and fM values, which decreased, as expected, with increasing TR unit indel rates. In contrast, accounting for the presence of TRs significantly improved the performance: In general, we can observe that ProGraphMSA+TR outperformed ProGraphMSA—both in terms of sensitivity and specificity. Moreover, with true TRs (as known from simulation) ProGraphMSA+TR had better sensitivity and specificity compared with using the predicted TR units . Remarkably, the net performance advantage was always in favor of modeling TRs, as ProGraphMSA+TR achieved the best balance of combining sufficiently high specificity with clear gains in sensitivity.Overall, the simulation results for duplication and profile methods were similar, also when comparing data sets with different types of TR units. The differences in absolute values of fD and fM for the two simulation methods appear to be due to different lengths and divergences of simulated TR regions. Indeed, on average, the profile simulation method increased the number of TR units during evolution for longer trees and high TR unit indel rates, whereas the duplication simulation method decreased the total length of the TR region. Only in the case of low TR unit indel rates or short trees for data simulated with the profile method, we observed a significant but still small decrease in specificity or sensitivity of ProGraphMSA+TR compared with ProGraphMSA. Particularly, owing to the high divergence between TR units sampled from the profile HMM, the lower quality of TR predictions led to slightly lower specificity compared with ProGraphMSA without TR information. We similarly observe that the TR detection performs worse for short repeat units . Methods based on global alignment usually provide better power to infer indels, especially if they are able to discriminate between insertions and deletions in ancestral states during phylogenetically guided alignment . Hence, these methods do not facilitate accurate inference of TR unit indel events. Conversely, ProGraphMSA+TR explicitly marks possible TR unit indels in its reconstructed ancestral sequences. Combined with an appropriate cost model our approach facilitates accurate inference of TR unit indels.However, TR-unaware MSA algorithms tend to merge TR unit indels if their cost model does not account for frequent and long indels. In addition, TR unit indels can be split if gaps of the correct unit length are not favored . Consequently, the number of detected TR unit indel events was significantly lower compared with when the true TR information was used.We assessed the robustness of TR unit indel estimation by comparing the number of inferred TR indel events with the true numbers as known from the simulation. Overall, we observed a strong correlation of estimated and true indel counts. For example, We note that the results were affected by the choice of input parameters in ProGraphMSA+TR, namely, the TR unit indel rate and its extension probability. These parameters should be provided by the user; yet, they are rarely known. We suggest default values ; howeverThe input TR unit indel rate is the indel rate per TR site. To obtain a comparable parameter value from ProGraphMSA+TR, the inferred TR unit indel rate has to be divided by the estimated length of the TR region.Ralstonia solanacearum. These proteins are a family of type III effectors—the major pathogenicity determinant of the R. solanacearum species complex  to create a specific repertoire contributing to pathogenesis on different plant hosts.The TR indel process acting on the GALA family appears to be a characteristic force creating and maintaining the allelic variability.We presented a fast and accurate method for global alignment of multiple sequences with TRs. In contrast to local alignment methods, our method is able to detect insertions and deletions of TR units which are not restricted to TR unit boundaries. The method is implemented in ProGraphMSA+TR, which is built on a graph-based alignment framework , and thuIn this study, ProGraphMSA+TR has been shown to improve the quality of alignments by incorporating TR information, which can be obtained using one of many available TR detection methods, or a combination of different TR detectors and a subsequent statistical scoring of putative TR units . More acIn addition to improved alignment quality, the new method allows for an accurate inference of TR unit indel events, which facilitates the estimation of TR indel rates. This is enabled by placing indels in a phylogenetically meaningful fashion  as well R. solanacearum. Such studies may provide additional insight for detecting selective advantages from phylogenetic patterns of TR indel rates and evolutionary constraints on TR unit numbers.Here, we demonstrated the utility of ProGraphMSA+TR to study the evolution and diversification of paralogous TR regions on real data, the GALA proteins with LRRs that act as type III effectors in phytopathogenic bacteria Given the high abundance of TRs in genomic sequences , modelinFinally, our models for simulating sequences with TRs and their evolution allow for the most realistic to date simulation scenarios of such sequences. The implementation of the simulation algorithm is available from the project website (later in the text) and should be a useful standalone tool for other studies of TRs and their evolution.Project name: ProGraphMSA+TRhttp://sf.net/projects/prographmsa/Project home page: Operating system: Platform independentProgramming language: C++Other requirements: Eigen 3.1, TCLAP 1.1 or higherLicense: GNU GPLv3http://www.cbrg.ethz.ch/services/ProGraphMSAWeb server: Both the implementation of TR-aware sequence alignment and the simulation algorithm are available from the project website.Supplementary Data are available at NAR Online.Swiss National Science Foundation [ref. 31003A-127325 to M.A.] and the Germaine de Staël program of the Swiss Academy of Engineering Sciences [ref. 2011-15]; ETH Zürich (to A.M.S. and M.A.).Funding for open access charge: Conflict of interest statement. None declared.


Zbed4 mRNA and protein of mouse retina.ZBED4, a protein in cones and Müller cells of human retina, may play important functions as a transcriptional activator of genes expressed in those cells or as a co-activator/repressor of their nuclear hormone receptors. To begin investigating these potential roles of ZBED4, we studied the developmental expression and localization of both the Zbed4 mRNA in retina and other mouse tissues, and western blots showed the nuclear and cytoplasmic expression of Zbed4 at different developmental times. Antibodies against Zbed4 and specific retinal cell markers were used for retinal immunohistochemistry.northern blots showed the presence of Zbed4 mRNA was present at different levels in all the mouse tissues analyzed. The Zbed4 protein was barely detectable at embryonic day (E)14.5 but was clearly seen at E16 at both retinal outer and vitreal borders and throughout the retina by E18 and postnatal day 0 (P0). Thereafter, Zbed4 expression was more restricted to the inner retina. While ZBED4 is localized in cones and endfeet of Müller cells of human retina, in adult mouse retina Zbed4 is only detected in Müller cell endfeet and processes. The same localization of Zbed4 was observed in rat retina. In early development, Zbed4 is mainly present in the nuclear fraction of the mouse retina, and in adulthood it becomes more enriched in the cytoplasmic fraction.Zbed4 in the mouse retina suggest a possible involvement of this protein in retinal morphogenesis and Müller cell function.The patterns of spatial and temporal expression of  This protein has 82% amino acid identity with human ZBED4 and is highly conserved across species. The Zbed4 gene maps to mouse chromosome 15 in a region that is syntenic to human chromosome 22q13.3, where the human ZBED4 gene maps. In this paper we report the localization and developmental expression of Zbed4 in mouse retina. Interestingly, Zbed4 is not detected in cone cells of the mouse, in contrast with its localization in cones of human retina. Moreover, Zbed4 is abundant throughout the embryonic retina but is present only in Müller cells of the adult mouse retina.Recently we reported the isolation and characterization of a novel protein, ZBED4, which belongs to the BED subclass of zinc finger proteins and is expressed in cone photoreceptors and Müller cells (mainly detected in their endfeet) of human retina . ZBED4 h spacer) ,7. Simil2 inhalation at the appropriate postnatal day (P). Wistar rats were obtained from Charles River . Eyes of mice and rats were quickly enucleated after death, and retinas were rapidly dissected and frozen on dry ice or fixed in 4% paraformaldehyde. All experiments were conducted in accordance with the Animal Care and Use Committee of the University of California, Los Angeles and the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research.C57Bl/6J mice were obtained from colonies bred from stock originating at the Jackson Laboratories . Mice were reared under dim cyclic light and sacrificed by CO+ RNA was obtained using the Oligotex mRNA purification kit . RNA quality was determined with a bioanalyzer  and quantification was performed using a NanoDrop spectrophotometer  [Total RNA was extracted from mouse retinas using Trizol . Poly A3CO2K, 3.5 mM (CH3CO2)2Mg, 3.75 μg/ml BSA (BSA), 0.2 mM deoxyribonucleotide triphosphate (dNTP), and 0.2 μM appropriate PCR primers following the same program profile as described before [DNA-free total RNA was reverse transcribed and the first-strand cDNA was amplified by PCR using the Advantage 2 polymerase mix , 40 mM Tricine–KOH (pH 9.2), 15 mM CH+ RNA obtained from adult mouse retinal tissue were electrophoresed on a 1.2% denaturing formaldehyde agarose gel and transferred to a Hybond N+ membrane  [Zbed4 cDNA fragment containing part of the 3′ coding region and of the 3′ UTR (3461–4191 bp) was labeled with 32P-dCTP and Klenow enzyme and used to hybridize a multiple mouse tissue  and the retinal northern blots.Two micrograms of polyAA polyclonal antibody was produced in rabbit against a peptide from mouse Zbed4 containing amino acids 8–25 . After affinity purification, the specificity of the antibody for Zbed4 was determined in a similar way to that described for the human ZBED4 antibody .Nuclear and cytoplasmic protein extracts from mouse retinal tissues at different developmental ages were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents . Quantification of total protein was performed with the Micro BCA Protein Assay Kit (Thermo Scientific). Protein extracts (50 µg) were separated by sodium dodecyl sulfate PAGE on 7.5% gels  and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h in TBS-T solution  containing 3% BSA and incubated 2 h at room temperature with the rabbit primary antibody against mouse Zbed4 at a dilution of 1:1,000, followed by 1 h incubation with secondary anti-rabbit IgG antibodies labeled with alkaline phosphatase . Western blots were visualized with the alkaline phosphatase chemiluminescent DuoLux kit (Vector Laboratories).After dissection and fixation overnight in 4% paraformaldehyde, mouse and rat retinas were embedded in optical cutting temperature compound . Frozen sections (8 μm) were cut on a Leica CM1850 cryostat . Immunostaining was performed as described previously [Zbed4 mRNAs are recorded in GenBank: a 5.3-kb mRNA (GenBank BC052174) and a 5.4-kb mRNA (GenBank NM_181412). Alignment of the 5.3-kb mRNA with the Zbed4 genomic sequence shows that it is derived from two exons, one short noncoding and one long exon that contains part of the 5′ UTR and the complete 3′ UTR. The 5.4-kb mRNA includes an additional 92-bp noncoding exon upstream of the first exon of the 5.3-kb Zbed4 mRNA. Both mRNAs have the same coding region and 3′ UTR. A 731-bp probe (3461–4191 bp of the 5.3-kb mRNA) encompassing the 3′ coding region and part of the 3′ UTR of the mouse sequence were amplified and used to hybridize a mouse multitissue northern blot (Ambion) along with a membrane containing mouse retinal mRNA. A major transcript approximately 5.3–5.4-kb long (consistent with the length of mouse Zbed4 mRNAs) was detected in all tissues examined: heart, brain, liver, spleen, embryo, lung, thymus, testis, ovary, and retina  is commonly used to normalize the amount of nuclear protein loaded in each lane of a gel ,9. HowevTo investigate the expression of Zbed4 at different times during embryonic and postnatal development, immunostaining was performed on mouse retinal sections at embryonic day (E)14.5, E16, E18, P0 (with the first 24 h after birth defined as P0), P5, P8, P11, P15, P21, and P30, using the Zbed4 antibody. As seen in We previously showed that ZBED4 is mostly present in the nucleus and inner segment of cones and is detected in the endfeet of Müller cells of human retina . In the To establish the association of Zbed4 in developing mouse retina with newly differentiating cell types, we performed a series of double-labeling experiments using antibodies to several specific cell markers. Despite the general similarities between human and mouse retinas, there are notable differences between the two species in the number, ratio, and distribution of photoreceptor cells and in their morphological characteristics and physiologic responses. The human retina has a larger proportion of cone photoreceptor cells than the mouse retina and it contains the macula, a specialized area that is not present in the mouse. In addition, mice are dichromats, whereas humans are trichromats. Therefore, spatial and temporal differences in gene expression patterns and protein function are expected between humans and mice.Zbed4 mRNA, which encodes a 1,168-amino acid protein, has the same structural features of its human ortholog [In the present study we found that although the mouse It is well known that before birth in the mouse, during E16 to P0, ganglion cells are present in the innermost area of the retina , whereas the outer retina consists primarily of undifferentiated neuroblastic cells ,11. SincAt P0 and later on during postnatal development, we found that Zbed4 co-localizes with glutamine synthetase, an established marker for glial Müller cells . Zbed4 iZbed4 mRNA, as we have previously shown [It may be speculated that the different cell distribution of Zbed4 in human and mouse retinas is related to the different structural features of the gene in these species. In addition, we have previously mentioned that there is only 82% homology between human and mouse Zbed4. Since zinc finger proteins of the BED class not only bind to DNA but also to RNA or proteins , contribly shown .Interestingly, expression of Zbed4 in the nuclear fraction of mouse retina follows the opposite pattern to that in the cytoplasmic fraction during postnatal development , decreas


Cortical cross-modal re-organization, or recruitment of auditory cortical areas for visual processing, has been well-documented in deafness. However, the degree of sensory deprivation necessary to induce such cortical plasticity remains unclear. We recorded visual evoked potentials (VEP) using high-density electroencephalography in nine persons with adult-onset mild-moderate hearing loss and eight normal hearing control subjects. Behavioral auditory performance was quantified using a clinical measure of speech perception-in-noise. Relative to normal hearing controls, adults with hearing loss showed significantly larger P1, N1, and P2 VEP amplitudes, decreased N1 latency, and a novel positive component (P2’) following the P2 VEP. Current source density reconstruction of VEPs revealed a shift toward ventral stream processing including activation of auditory temporal cortex in hearing-impaired adults. The hearing loss group showed worse than normal speech perception performance in noise, which was strongly correlated with a decrease in the N1 VEP latency. Overall, our findings provide the first evidence that visual cross-modal re-organization not only begins in the early stages of hearing impairment, but may also be an important factor in determining behavioral outcomes for listeners with hearing loss, a finding which demands further investigation. A basic tenet of neuroplasticity is that central pathways will re-organize following long-term sensory deprivation. There is ample evidence from animal and human studies of cross-modal re-organization of the cortex that occurs in both blindness t(15) = −1.69, ρ >0.05). All participants reported no issues with visual acuity and no neurological impairment.Seventeen adults between the ages of 37 to 68 years participated in this study. The study was approved by the University of Colorado at Boulder Institutional Review Board, and all participants provided written consent. Subjects were recruited via advertisements in the community, and hearing was tested for all subjects using standard clinical audiometric procedures prior to speech-in-noise and EEG measurements. Eight of the subjects  revealed clinically normal hearing thresholds  for frequencies ranging from 250 Hz to 8000 Hz. Nine of the subjects demonstrated hearing loss . On average, this group showed normal hearing from 250 Hz through 1000 Hz and a mild-to-moderate sensorineural hearing loss bilaterally from 2000 Hz to 8000 Hz. Average audiograms for the two groups are shown in Speech perception in background noise was measured using the QuickSIN™ test Participants were shown a high contrast sinusoidal concentric grating that morphs into a radially modulated grating or circle-star pattern Participants were fitted with a 128-channel EEG electrode recording net  and seated in a comfortable reclining chair in an electro-magnetically shielded sound booth. All stimuli were presented via E-Prime® 2.0, stimulus software compatible with Net Station 4 . The sampling rate for the EEG recordings was 1 kHz, with a band-pass filter set at 0.1–200 Hz.Data were band-pass filtered offline at 1–30 Hz and segmented according to the EEG activity surrounding the stimulus presentation (epochs), with 100 ms pre-stimulus and 495 ms post-stimulus time. EEG recordings were corrected to the pre-stimulus baseline, and eye-blink artifact recorded at designated eye channels was removed if greater than +/−100 µV, unless adjusted for individual subjects. Bad channels were removed from the recording and replaced with interpolated data from the remaining channels via a spline interpolation algorithm. Remaining data were averaged and re-referenced using common average reference. Individual waveform averages were averaged together for each of the two groups  to compute a grand-averaged waveform. Amplitudes and latencies for individual participants were recorded for all three obligatory visual evoked potential (VEP) peaks . The P1 peak component was observed as the first positive-going peak occurring approximately within a latency window of 90 to 130 ms, the N1 component was observed as the second peak or first negative-going peak occurring approximately between 135 ms to 200 ms, and the P2 component was observed as the third peak or second positive-going peak occurring approximately within 200 to 300 ms. If a peak component occurred outside of the described latency ranges, it was still marked and included according to the order of appearance . P1 amplitudes were defined as the onset to peak value, N1 amplitudes as the peak of the N1 component to peak of the P2 component, and P2 amplitudes as peak of the P2 component to offset value. Latencies were chosen at the highest amplitude of the peak.First we created a two-dimensional voltage map using Net Station 4 , which allowed us to examine regions of interest (ROI) around the occipital midline EEG data for individual participants were exported from Net Station and imported into EEGLAB Peak components for the VEP MGFP waveforms were selected separately for current density reconstruction (CDR) via sLORETA, with no a-priori restrictions placed on the model. The selected head model was standardized using the boundary element method (BEM) Waveforms for the two groups across the whole head (128 channels) are shown in F = 6.265, ρ <0.05). N1 amplitude was larger for the HL group  = 9.865, ρ <0.01). N1 latency was decreased for the HL group  = 7.684, ρ <0.01). Finally, P2 amplitudes were increased for the HL group  = 8.983, ρ <0.01). Overall, this trend of decreased latencies and increased amplitudes for obligatory VEP components for HL listeners is consistent with previous results, which showed evidence of cross-modal recruitment in deaf subjects P1 amplitude was larger for the HL group  in the HL group see . We labeCortical source localization, or current density reconstruction (CDR), was performed in order to visualize anatomical regions of possible cross-modal re-organization in the HL group. The sLORETA algorithm provided by CURRY® Scan 7 Neuroimaging Suite was applied to the three VEP peak components . The actAs expected, for the NH group, the visual stimuli elicited all three VEP components and activated visual processing regions, including multiple cerebellar areas, which have been shown to respond to visual motion U = 10.5, Z = −2.46, ρ <0.05). This difference in auditory performance in background noise between normal hearing listeners and listeners with mild-to-moderate hearing loss is consistent with Killion et al. Speech perception-in-noise acuity was measured for both groups using the QuickSIN™ clinical test r = −0.701, ρ = 0.001). That is, a shorter N1 latency was associated with higher scores  on the QuickSIN™ test. Overall, our results reflecting differences in speech-in-noise perception between the NH and HL groups as a function of visual evoked potentials are consistent with previous studies in deaf subjects showing cross-modal re-organization in subjects with poor speech perception r = −0.446, ρ <0.05; left ear, r = −0.540, ρ <0.05). That is, as the degree of hearing loss increased, there was a corresponding decrease in N1 latency.The N1 VEP component has been suggested as a marker of cross-modal re-organization in deafness We sought to examine whether cross-modal recruitment is evident in early stages of hearing decline or whether cross-modal plasticity is limited to the near-total sensory deprivation that accompanies profound deafness. We recorded high-density EEG in response to a visual stimulus in adults with normal hearing in the low frequencies and a mild-to-moderate hearing loss in the high frequencies. A group of age-matched normal hearing adults served as the control group. All participants were administered the QuickSIN, a test of speech-in-noise perception which is used to document clinical outcomes in patients with hearing loss.Relative to normal hearing controls, adults with mild-to-moderate hearing loss showed: (i) increased amplitude of the P1, N1 and P2 VEP components, (ii) presence of an additional positive VEP component (P2’) occurring after the P2, (iii) decreased latency of the N1 VEP, (iv) cortical re-organization as evidenced by increased activation of auditory temporal areas elicited by visual stimulation, (iv) poorer speech perception scores in noise, (v) a significant negative correlation between the degree of hearing loss and N1 VEP latency, and (vi) a strong negative correlation between speech-in-noise perception and N1 VEP latency. Overall, this pattern of results in our listeners with mild hearing loss is consistent with previous findings in deaf subjects suggesting visual cross-modal recruitment in deafness Consistent with our findings, significantly increased amplitudes of N1 and P2 VEPs with hearing loss are well-documented in deafness Current source density reconstructions (CDR) were compared between NH and HL listeners. As expected, for the visual stimulus, NH listeners showed cerebellar/occipital activation for the P1, N1 and P2 VEP components . ResponsThe HL group showed occipital/cerebellar activation comparable to the NH group, as was evident in the CDR for the P1 response. However, higher-order processing as reflected by the CDR for the N1 and P2 components showed clear evidence of cortical re-organization. Cortical activation for these components showed an emphasis in ventral stream processing within temporal cortex, including temporal gyri , which are typically associated with auditory cortical processing In a recent study, Campbell and Sharma Overall, the CDR findings are strongly suggestive of cortical re-organization facilitating cross-modal recruitment for visual processing in adults with mild-moderate hearing loss. The shift of activation to temporal cortex represents activation of the ventral visual stream, which is typically responsive to visual object form or shape changes, and is located within temporal cortex in proximity to auditory areas. The ventral stream has been implicated in the processing of facial and mouth movements Hearing loss is most consistently associated with poor outcomes in recognizing speech in background noise, a skill essential for everyday listening Overall, the VEP and behavioral results that we describe are strongly indicative of visual cross-modal re-organization in adults with mild-moderate hearing loss. This is a new finding as previous reports of cross-modal plasticity have been confined to adults with deafness, which was congenital or pre-lingual, and/or in cochlear-implanted adults Our study provides new evidence of cross-modal cortical re-organization in adult-onset mild-moderate hearing loss. Increased amplitudes of P1, N1 and P2 VEP components, decreased N1 latency, a novel P2’ component and current source density reconstructions reflecting a ventral shift in activation were observed for adults with mild to moderate hearing loss relative to normal hearing controls. Furthermore, we observed a strong negative correlation between cross-modal re-organization (as reflected by decreased N1 latency) and speech perception in noise. Future studies are needed to outline the detailed trajectory of cross-modal changes as hearing declines from a mild hearing loss to deafness. Prospective longitudinal studies will provide important information concerning the timeline of cross-modal re-organization according to severity of hearing loss, including a quantification of the degree or severity of re-organization. In addition, such studies may indicate the effect of clinical interventions, such as amplification or cochlear implantation, in reversing cross-modal re-organization.


The potential of such conjugates to purge latently infected cells was also demonstrated in a cell-to-cell viral inhibition assay by measuring their efficacy to inhibit the spread of HIV-1LAI from infected human peripheral blood mononuclear cells to Jurkat T cells over a period of 30 days following viral activation with 100 nM SAHA (suberoylanilide hydroxamic acid). The IgG-like half-life was not significantly different from that of the parental antibody, as shown by the mean serum concentration of one prototype in mice at 72 h. These encouraging results provide a rationale to develop further novel anti-HIV agents by coupling additional antibodies of interest with alternative HIV-inhibitors via recombinantly-produced, self-assembling, modules.We constructed novel HIV-1 fusion inhibitors that may overcome the current limitations of enfuvirtide, the first such therapeutic in this class. The three prototypes generated by the Dock-and-Lock (DNL) technology to comprise four copies of enfuvirtide tethered site-specifically to the Fc end of different humanized monoclonal antibodies potently neutralize primary isolates (both R5-tropic and X4-tropic), as well as T-cell-adapted strains of HIV-1 in vitro. All three prototypes show EC There are about 32 antiretroviral products approved for the treatment of the HIV-1/AIDS pandemic 4, they are expected to provide the therapeutic benefits of T20 with the added advantages conferred by the IgG component, one of which would be improved pharmacokinetics with a longer serum half-life to allow less frequent dosing than the twice daily currently required for T20. Moreover, depending on the targeting specificity and effector functions of the conjugated antibody, whether binding, neutralizing or not, the resulting DNL constructs could eliminate both infected cells and free virus via several known mechanisms Enfuvirtide  was the first drug in the class of HIV-1 fusion inhibitors to receive approval in 2003 for treating AIDS patients 4 of cP4/D10 and two humanized mAbs, namely, h734, a non-immunoreactive variant of h734 (anti-indium-DTPA IgG1), and hLL2 or epratuzumab (anti-CD22 IgG1), that do not bind to HIV-1, for assessing the effect of other cellular targeting on anti-HIV potency. Our results show, surprisingly, that each of the three DNL conjugates of T20, designated cP4/D10-(T20)4, h734-(T20)4, and hLL2-(T20)4, is more potent than T20 in inhibiting HIV-1 in vitro, even when the antibody has no HIV-1-specificity, and that the hLL2-(T20)4 conjugate clears from blood in mice in a prolonged time, similar to unconjugated hLL2 IgG.Among the various antibodies that bind and neutralize HIV-1, the murine anti-gp120 (V3 loop) antibody, P4/D10, is distinguished by its additional feature of inducing ADCC to eliminate infected T cells E. coli, and isolated from the cell lysates by HIS-Select without refolding and further purification. Although the DDD2-T20 was found to comprise a mixture of crosslinked congeners of varying molecular sizes, the identity of DDD2-T20 was verified by LC-MS  of cAMP-dependent protein kinase A (PKA), referred to as the DDD module, and the other bearing the anchoring domain (AD) of an interactive A-kinase anchoring protein (AKAP), referred to as the AD module. Among the distinctive features of DNL are the spontaneous formation of a dimer of the DDD module and the self-assembly of the DDD module with the AD module into a non-covalent complex, which is subsequently rendered covalent with disulfide bonds to enhance stability in vivo by LC-MS , which s4, and the unconjugated T20 to inhibit the infection of HIV-16920, a primary, nonsyncytium-inducing (NSI), CCR5-tropic, clade B isolate 4 is significantly higher in molecular weight  than either DDD2-T20  or T20 , these data are presented in molar concentrations. Both a standard 9-day neutralization assay (4 over both DDD2-T20 and T20. The h734-(T20)4 complex exhibited an EC50 of about 0.1 nM, compared to about 2 nM for DDD2-T20 , and about 10 nM for T20. Similar results showing the higher potency of h734-(T20)4 compared to DDD2-T20 and T20 were also observed on assay  and a pron assay  reveal tobserved  in a cel4 constructs to inhibit viral replication was further attested by comparing the potency of P4/D10, cP4/D10, cP4/D10-(T20)4, h734-(T20)4, and hLL2-(T20)4 for neutralization of HIV-1IIIB in Jurkat T cells , CXCR4-tropic, clade B isolate IIIB in Jurkat T cells 4 are as potent as cP4/D10-(T20)4 in neutralizing HIV-1  T cells  and HIV- T cells ; (ii) cPV-11IIIB  and HIV-IB  bng HIV-1 ; and 4 to inhibit the spread of HIV-1LAI from infected PBMCs to Jurkat T cells over a period of 30 days following activation with 100 nM SAHA (suberoylanilide hydroxamic acid). For comparison, cP4/D10, T20, and hLL2 also were included. A substantial and persistent increase in HIV replication, measured by either p24 ELISA 24 ELISA  or p24-p24 ELISA , was obs4 appears to be stable in vivo for at least 3 days, because the serum concentrations measured by the two assays, one designed to quantify only the intact hLL2-(T20)4 and the other to determine all the hLL2-containing species, were comparable at 6, 24 and 72 h. Based on these results, the bioavailability, as determined by the mean serum concentration of hLL2-(T20)4 at 72 h, was no less than half that of hLL2 (85.3±37.2 vs. 131.2±41.3 nM), with the difference not being statistically significant .As shown in The DNL method combines genetic engineering with site-specific conjugation to enable self-assembly of two modular components only with each other, resulting in a covalent structure of defined composition with retained bioactivity 4 derivative may acquire a substantially higher activity in inhibiting or neutralizing the infectivity of HIV-1 than unconjugated T20, which is a notable advantage over certain conjugates of fusion inhibitors not made by DNL e by DNL . For exaOn the other hand, newer antibodies with broader and more potent HIV-binding and neutralization activities across clades are continuously being identified or engineered Current strategies designed to enhance anti-HIV potency via simultaneously intervening multiple aspects of the HIV-1 entry process have evolved from the earlier combination of gp41-targeting T20 with either the CD4-attachment inhibitor PRO 542 4 studied are highly active against primary isolates (both R5-tropic and X4-tropic) and T-cell-adapted strains of HIV-1, showing inhibitory EC50 values in the subnanomolar range, which are 10- to 100-fold more potent than enfuvirtide (4 or h734-(T20)4 results from increased binding of the clustered T20 to the exposed gp41 or Fc receptor-mediated antiviral activities The three prototypes of IgG-(T20)uvirtide  and, remE. coli is a notable advance and should have a substantial reduction on manufacturing costs for future T20-derived therapeutics. Moreover, the generation of IgG-(T20)4 is relatively simple, which we have accomplished with several other types of IgG-based DNL conjugates, including IgG linked to interferon-α, interferon-λ1, ranpirnase, G-CSF, EPO, and human protamine. Based on the current data of in vitro potency and IgG-like half-life, we do not expect a large dose of IgG-(T20)4 would be needed either.The current production process of T20 is complex, requiring numerous synthetic steps. Thus, the feasibility to produce functional DDD2-T20 in 4 to comprise individuals failing HAART therapy, where several doses of the DNL conjugates may effectively reduce the number of infected cells and circulating virions. Another potential patient population comprises individuals on effective HAART, with the goal of targeting and deleting the few persisting, viral-antigen-exposing or producing, cells. Any potential immunogenicity of these engineered constructs should be mitigated by use of humanized or chimeric antibodies, especially when administered to immunosuppressed AIDS patients.Before entering human trials, key preclinical studies will include in vitro testing of efficacy in inhibiting enfuvirtide-resistant virus, and in vivo evaluations in appropriate murine and primate models to assess biodistribution, pharmacokinetics, immunogenicity, and dose-response effects. We envision the primary target AIDS patient population for IgG-(T20)IIIB and HIV-1Lai), primary isolates (HIV-16920 and HIV-16794), all of clade B, and, were obtained from The Swedish Institute for Communicable Disease Control. The phenotypes and biological properties of HIV-16920 and HIV-16794 have been described 4 constructs are provided in Methods S1.Jurkat T cells were originally purchased from ATCC  by The Swedish Institute for Communicable Disease Control. Jurkat T-cell line-adapted strains (HIV-1IIIB and HIV-16794 (or HIV-16920) in Jurkat T cells and PHA/IL-2 activated PBMCs, respectively. Briefly, HIV-1 viruses (50 or 100 TCID50) were mixed in duplicate with serially diluted test articles in complete medium  in a final volume of 100 µL/well. After incubation for 1 h , Jurkat T cells  or PBMCs  were added (100 µL/well) and incubated overnight (16–18 h). The cells were washed twice with RPMI 1640, replenished with complete medium containing test articles (200 µL/well), and incubated further for 72 h, at which time half of the medium in each well (100 µL) was harvested for p24 ELISA, and 100 µL of fresh medium was added to replenish each well. Afterwards, cell culture supernatants were assayed for viral levels at the indicated time in a similar fashion.Virus neutralization was performed against HIV-16 cells/mL were cultured in complete medium for 48 h at 37°C in 5% CO2. Cells were then collected, washed, counted, suspended in fresh RPMI 1640 medium at 2.5×106 cells/mL, and infected overnight with HIV-1LAI (150–250 TCID50/mL). On the next day, cells were washed 3 times with complete medium, counted, dispensed (200 µL) onto 96-well cell culture plates at 150,000 cells/well, and grown in complete medium for 3 days, at which time 100 µL from each well was removed for p24 ELISA and 100 µL of fresh medium was added to replenish each well. After 7 days, all medium was harvested and cells were washed 3 times with complete medium. Jurkat T cells were then added to the wells  in complete medium, half of which were also given SAHA at 100 nM, and serially diluted hLL2-(T20)4, T20, hLL2 and cP4/D10 were added to designated wells. Every third day, after removing 100 µL from each well for p24-ELISA, 100 µL of fresh medium were added to replenish each well and this operation was repeated over a period of 4 weeks.PBMCs at 2.5×10ad libitum. Animals were injected s.c. with hLL2-(T20)4  and anesthetized with ketamine/xylazine prior to collection of blood samples at 0.5, 6, 24 and 72 h from 2, 3, 3, and 3 mice, respectively, and stored at −70°C until analyzed by ELISA. A parallel study was performed with hLL2 IgG  in the same fashion. The serum samples from mice injected with hLL2-(T20)4 were examined by two different ELISAs, one designed to quantify only the intact hLL2-(T20)4 and the other to quantify all hLL2-containing species, with or without the linked T20. For quantification of hLL2-(T20)4, plates were coated with F(ab′)2-specific, goat anti-human IgG, and the captured antibodies probed with a mouse anti-DDD2 mAb (5E3) developed in-house, followed by HRP-conjugated goat-anti-mouse IgG. For measuring all hLL2-containing species, plates were coated with AffiniPure Goat Anti-Human IgG, F(ab′)2 Fragment Specific  and the captured antibodies were probed with a rat anti-idiotype mAb to hLL2 , followed by Peroxidase AffiniPure F(ab′)2 Frag Goat anti-Rat IgG, FcR Frag Spec (Jackson ImmunoReserach). The second assay was also used for measuring the serum levels of hLL2. The animal study was approved by the Institutional Animal Care and Use Committee (IACUC) of the Center for Molecular Medicine and Immunology  in compliance with NCI guidelines.Naïve female SCID mice  purchased from Taconic Farms  were housed in Thoren units with food and water t-test was used for comparisons between mouse serum concentrations of the different agents after analyzing the data with an F-Test (Microsoft Excel 2003) for possible difference in variance. P-values less than 0.05 were considered statistically significant.Statistical comparisons between the groups of data obtained with different compounds were performed using the nonparametric Mann-Whitney U and Kruska Wallis tests. A one-way ANOVA nonparametric test was performed using GraphPad Prism version 4.0a for Macintosh, or OS 9 Apple GraphicPad Software , and was used for comparisons of HIV-1 isolation and p24 antigen positivity between the study groups. A two-tailed Figure S1LC-MS analysis of DDD2-T20. Electrospray ionization time of flight (ESI-TOF) liquid chromatography/mass spectrometry (LC-MS) was performed with a 1200-series HPLC coupled with a 6210 TOF MS . The DDD2-T20 was reduced with 50 mM tris(2-carboxyethyl)phosphine for 30 min and resolved by reversed phase HPLC (RP-HPLC), using a 20-min gradient of 30–80% acetonitrile in 0.1% aqueous formic acid with a Jupiter C4 5 µ column . For the TOF MS, the capillary and fragmentor voltages were set to 5000 and 200 V, respectively. The observed mass (11824.53 Da) closely matched (26 ppm) the calculated mass (11824.23) of the deduced amino acid sequence.(PPTX)Click here for additional data file.Figure S24 constructs.Size Exclusion HPLC analysis of IgG-(T20) The hLL2-(T20)4 and cP4/D10-(T20)4 each resolved as a single protein peak at retention times of 7.85 min and 8.55 min, respectively, which were shorter than that of the hLL2-IgG-AD2 (8.33 min) and cP4/D10-IgG-AD2 (8.86 min), respectively, and consistent with their molecular size.(PPT)Click here for additional data file.Figure S3Antibody binding measured by ELISA. (A) P4/D10 and cP4/D10 showed similar binding avidity to recombinant gp160. (B) Comparative binding to a synthetic V3 peptide representing the third variable loop of HIV-1 gp120 outer envelope protein. Replicate samples were washed with 8 M urea or saline. For both P4/D10 and cP4/D10, similar antibody titer was measured with 8 M urea and saline, giving an avidity index of 0.98, suggesting a similarly strong binding avidity for each antibody.(PPTX)Click here for additional data file.Table S1Comparative potencies of anti-HIV fusion inhibitors.(DOC)Click here for additional data file.Methods S14 constructs with DNL; antibody-binding ELISA.Methods are provided for: chimerization of P4/D10; construction of expression vectors for cP4/D10 and cP4/D10-AD2; expression and purification of chimeric P4/D10 and AD2-containing Abs; cloning, expression and purification of DDD2-T20; generation of IgG-(T20)(DOC)Click here for additional data file.


Although pulse pressure variation (PPV) is essentially proposed as a predictor of fluid responsiveness , it has n = 8), intravenous endotoxin (n = 8), hemorrhagic shock  or hemorrhagic shock with endotoxin . Hemodynamic parameters, measured by pulmonary artery and femoral arterial catheters, were assessed at baseline (TB) and at 20 (T20), 40 (T40), 60 (T60) and 80 (T80) minutes. Groups and times were compared with two-way ANOVA followed by Tukey test (P < 0.05).Fifty-one anesthetized, mechanically ventilated pigs were randomly allocated to four groups: control (P < 0.001), with no difference between groups. MPAP was significantly higher in group H+L than in HEM at T20 (P < 0.001), T40 (P < 0.001), T60 (P = 0.009) and T80 (P = 0.013). Within group H+L, MPAP was significantly above TB in all timepoints, but was highest at T20 and T40 , decreasing significantly at T60 and T80 (to 26 ± 5 mmHg). PPV increased significantly in groups HEM and H+L (both P < 0.001) from T20 to T80. There was, however, a statistical difference between HEM and H+L at T20  and T40 , which disappeared at T60, when PPV in group H+L increased further.At T20, the systolic volume index in groups HEM and H+L dropped significantly (Even though PPV was affected by the magnitude of MPAP during the peak hemodynamic effects of early endotoxemia, its ability to detect acute decreases in preload was not entirely compromised, in the conditions of the present study. Additional research should help determine possible associated factors that interfere with PPV in related conditions.


It has been shown that low levels of pigmentation increase susceptibility to noise-induced hearing loss in humans. For this reason, white populations develop more pronounced noise- induced hearing loss in comparison to black populations. Similarly, blue-eyed individuals exhibit greater temporary threshold shift than brown-eyed subjects; still, no strong correlation has been verified between the lightness of hair color and susceptibility to noise-induced hearing loss. This study was performed with the purpose of investigating a possible association between hair color and the degree of hearing loss due to firing noise. Study Design: Prospective observational study. Setting: A tertiary referral center with an accredited otorhinolaryngology-head & neck surgery department. A total of 57 military recruits were divided into two groups; light-colored (blond and light brown) and dark-colored hair (dark brown and black). The two groups were matched based on history of firing noise exposure  and the level of hearing loss at 2, 3, 4, 6 and 8 kHz sound frequencies was compared between them.The results showed that the mean level of hearing loss of light-colored hair individuals (20.5±17dB) was significantly greater than that of dark-haired subjects (13.5±11dB), (P=0.023).The results indicate that hair color (blond versus black) can be used as an index for predicting susceptibility to noise-induced hearing loss in military environments. Therefore, based on the individual's hair color, upgraded hearing conservation programs are highly recommended.  Today we are all surrounded by a very noisy environment. Beside presbycosis (age-related hearing loss), the most common type of sensorineural hearing loss is related to high-noise environments. Noise-induced hearing loss is a major problem in industrialized countries and, due to the high rate of industrialization in developing countries and the lack of adequate protective equipment, this disorder is ever increasing in prevalence . StudiesExcessive noise exposure is annoying, produces stress, impairs communication, interferes with work and leisure time activities, and in high enough doses, produces permanent damage to the auditory system which leads to significant hearing loss . OccupatOne of the supposed factors predisposing a person to noise-induced hearing loss is low pigmentation and a low melanin content of the inner ear ,6. It isIt is interesting to note that the presence of melanocytes is not limited to the peripheral auditory system. Brainstem abnormalities have been diagnosed in both animals and humans with pigment disorders; moreover, studying albino rabbits has shown that they have 24% smaller neurons in the medial superior olivary nucleus in comparison with normal animals and that the branching density of the dendrites of these cells is significantly reduced ; this inIn 1980 Carter and colleagues conducted a study on 257 students in their third year of primary school, which aimed at investigating the correlation between eye color and susceptibility to noise-induced hearing loss. After performing audiometric tests it was seen that at 4 kHz sound frequency, hearing loss in the left ear of those children who had low pigmentation of the iris (blue or green) was higher than those with a highly pigmented iris (black or brown) .The main goal of this present study was to compare firing noise-induced hearing loss in military recruits with light-colored (blonde or light brown) hair with hearing loss in those with dark-colored hair. We also aimed to evaluate the exact level of hearing loss in the same two groups following exposure to gunfire noise. This was a prospective case-control study. The study population consisted of a group of military recruits in the age range of 18 to 29 years with a normal baseline audiogram, who had all taken the same two-week routine course of military education and then visited a specially designed army clinic. Among them, 57 cases including 114 ears were randomly selected for further follow up.The research material was a specially designed questionnaire consisting of questions on demographics and hair color; occupational noise history including number of firings, weapon type and training in and use of hearing protection; and non-noise risk factors including head injury, ear disease, medications and solvent exposure. Audiometry and tympanometry studies were also used in this investigation.The military recruits who had undertaken the two-week educational course were re-evaluated with audiologic tests 10 days after finishing the course. At this time point the aforementioned questionnaire was filled in by 62 cases. The subjects’ medical files were studied for the presence of a previous normal audiogram . Based on the specially designed questionnaire, those with a normal initial audiogram and who had been exposed to a similar number of firings (10 at least) with a similar weapon type were selected for the study. The selected military forces were then divided into two groups: dark-colored hair (black and dark brown) and light-colored hair (light brown and blond). Subsequently, their post-educational-course audiometries were compared. Among them, one who had blue eyes with dark hair and two with light brown-colored eyes were excluded from the study to eliminate the iris colour effect. Based on the data collected from the questionnaires, most of the study population had fired with a Klashinkov weapon, the most commonly used weapons following that were a Klashinkov machine gun, G3, Dushka, G3 machine gun and RPG; the cases other than Klashinkov shooters were also omitted from the study to eliminate sound variation bias. In total 57 cases were entered into the study. None of the studied military men had used self-protective equipment or were affected with any known disease. Ototoxic drug usage history was also negative in all cases.The required study sample size with a confidence interval of 95%,  with light-colored hair and 37 (74 ears) with dark-colored hair. They all had a normal tympanogram and acoustic reflex. The age range of the light-colored group was 18 to 29 years with a mean age of 25.5 ± 1.8 years; all such cases were right-handed shooters. The age range of the dark-colored hair group was 18 to 28 years with a mean age of 26.5 ± 1.2 years; one case in this group was a left-handed shooter. Based on the obtained results it could be concluded that firing noise-induced damage is predictable based on a consideration of the lightness of hair color; those with light hair have a greater susceptibility when compared with those with dark hair.Permanent noise-induced hearing loss is caused by either an acoustic trauma (e.g. a brief exposure to a very intense blast-like sound) or chronic long term exposure to the loud sounds associated with a noisy occupation. A long-The present study showed that the hearing threshold of the right and left ear of the studied cases are not necessarily identical and that the left ear showed a higher level of hearing loss. It is already known that left-ear hearing loss is more probable in right-handed people . AsymmetPrevious studies conducted by researchers ,20 have Regarding the responses given to the questionnaire, subjects appreciated the potential benefit of wearing hearing protectors but instructions in their proper use and education on the hazards of noise exposure were poor and the available hearing protection devices were reported to be incompatible with other gear, uncomfortable, and an impediment to communication.The obtained results indicate that a person's susceptibility to firing noise can be predicted by the lightness of their hair. In this respect those with light hair are more susceptible to firing noise-induced hearing loss in comparison with dark haired individuals. As the important role of the melanin content of the inner ear in the damage caused through high-noise environments has been proven in many studies, it is suggested that the melanin content of the inner ear of military personnel should be estimated based on the lightness of their hair, and in this way firing noise-induced hearing loss would be avoidable in susceptible cases. Based on our results we highly recommend that approaches to the upgrading of hearing conservation strategies and methods for strengthening the existing scheme for hearing conservation be used to further minimize such related risks.


Eeyore mutant mice have early onset progressive hearing impairment and show abnormal structure of the sensory epithelium from as early as 4 weeks of age. Ultrastructural and histological analyses show irregular hair cell structure and degeneration of the sensory hair bundles in the cochlea. The identification of new genes involved in hearing is central to understanding the complex genetic pathways involved in the hearing process and the loci at which these pathways are interrupted in people with a genetic hearing loss. We therefore discuss possible candidate genes within the linkage region identified in eeyore that may underlie the deafness phenotype in these mice. Eeyore provides a new model of hereditary sensorineural deafness and will be an important tool in the search for novel deafness genes.Animal models that recapitulate human disease are proving to be an invaluable tool in the identification of novel disease-associated genes. These models can improve our understanding of the complex genetic mechanisms involved in disease and provide a basis to guide therapeutic strategies to combat these conditions. We have identified a novel mouse model of non-syndromic sensorineural hearing loss with linkage to a region on chromosome 18.  Despite advances in our knowledge of genes that contribute to human disease, in many cases the causative gene remains unknown. The use of animal models in the search for causative disease genes has been revolutionary in providing insights into complex mammalian developmental and genetic pathways http://hereditaryhearingloss.org/). In addition, the same gene can cause syndromic or non-syndromic forms of deafness and some recessive forms can even be caused by two mutations in different genes that are from the same functional group Hearing loss is the most prevalent sensory impairment in man, affecting as many as 1 in 500 newborns N-ethyl-N-nitrosourea) mouse screen at the Australian Phenomics Facility (APF). The hearing loss is congenital in these mice and affected progeny show abnormal structural morphology within the cochlear sensory regions. The causative mutation is inherited as a single recessive locus on chromosome 18, between 12,368,762 and 33,968,545 Mb. To date no genes in this region have been associated with human hearing loss. Genes within this linkage region are therefore novel candidates for human hereditary hearing loss and identification of the causative mutation will provide additional insights into the genetic pathways responsible for the correct maintenance of hearing.We have identified a novel mouse model of non-syndromic, sensorineural hearing loss via a comprehensive ENU , Australia (http://tasq.uq.edu.au/).All procedures were approved by the Royal Children's Hospital Animal Ethics Committee, RCH AEEC #A585. Mice were screened for hearing loss as part of a large-scale ENU mutagenesis program at the APF, as described previously eeyore mice, as previously described Clickbox observations and Auditory Brainstem Responses (ABR) were used to screen for hearing loss in eeyore mice were identified as having a severe hearing loss by the tests described above. Affected eeyore mice were outcrossed to the BALB/c mapping strain and bred to produce affected F2 offspring. Genomic DNA from tails of hearing and deaf littermates was isolated by Proteinase K digestion and phenol/chloroform extraction to use for homozygosity mapping. DNA from 20 affected eeyore mice were analysed using Sequenome MassARRAY (AGRF). The deafness locus was mapped using methods described previously eeyore mice were analysed using SNP-specific primers designed using Primer3 TM v3.1 Terminator Cycle Sequencing Kit (Applied Biosystems) and products read using an ABI 3130×l capillary genetic analyser (Applied Biosystems). Sequencing chromatograms were compared to the published gDNA sequence using Mutation Surveyor (v2.60) software and the genotype of affected mice determined. Using the UCSC genome browser Mutant Mice were anaesthetised with isoflurane and culled by cervical dislocation according to the National Health and Medical Research Council Australian code of practice for the care and use of animals for scientific purposes . Adult mouse cochleae were dissected and processed as described eeyore littermates. Briefly, the middle ear was exposed by dissection of the bulla and removal of the tympanic membrane, taking care not to damage the malleus underneath. The malleus, incus and stapes were removed from the middle ear with care, stored in PBS and photographed with a Leica DC200 camera (Leica Microsystems Ltd).Ossicles were dissected from half heads of adult (14 week old) hearing and deaf eeyore mice and processed for H&E staining as described previously Cochleae were isolated from 8, 12 and 24 week hearing and deaf Cochleae from 12–14 week old mice were dissected, fixed and processed for SEM as previously described eeyore mice was measured by ABR thresholds. Affected mice showed a severe hearing loss at 4 weeks of age , the Ensembl Genome Browser (URL: http://www.ensembl.org), and the UCSC Genome Browser (URL: http://www.genome.ucsc.edu). This 21.6 Mb region contains 143 genes that were analysed for likelihood of causing the deafness phenotype using the program PosMed The heritability of the deafness phenotype in F2 progeny was consistent with a recessive inheritance trait, with approximately 25% of offspring affected (14/56). SNP analysis in the s3696933 , based oeeyore spans 21.6 Mb of chromosome 18 and contains 143 genes, each of which may contribute to the phenotype identified in our mouse model. Candidate genes ranked by PosMed were examined to identify their known function and likelihood of causing the deafness phenotype in eeyore mice. The top-ranked candidate genes within this region are listed in The causative locus identified in Of particular interested are candidate genes that have previously been associated with deafness such the water-selective membrane channel Aquaporin 4 (Aqp4). Expression of this gene has been reported within the cochlear sensory cell epithelium and glial cells of the auditory nerve Cadherin 2 (Cdh2 or N-cadherin), the class III phosphoinositide 3-kinase (PIK3C3) and Myosin 7b (Myo7b) are also plausible candidates within this linkage region as they all have family members that have been identified in deaf patients or mouse models of hearing loss. The cadherin family member, Cadherin 23 (Cdh23), is a major component of sensory hair cell tip links eeyore mice represent a novel model for studying progressive sensorineural recessively inherited hearing loss, common in the human population but with limited information on its molecular basis. Eeyore mutants are severely deaf at an early age which progresses to profound deafness within a number of weeks. Morphological and structural defects are detected within the cochlea indicating sensorineural deafness in these mice. The critical region localizes to a novel 21.6 Mb region on chromosome 18, containing a number of genes that could be implicated in the pathogenicity of this disorder. The underlying genetic mutation and mechanism remains to be elucidated, however this region has not previously been associated with recessively inherited non-syndromic deafness in humans. The rapid speed and reduced costs of targeted genome sequence analyses in mouse models of human disease, could potentially lead to the identification of the molecular mutation responsible for the hearing impairment in eeyore. The identification and subsequent characterisation of mouse models such as eeyore is fundamental to our understanding of the molecular mechanisms involved in normal hearing, and where this goes wrong in hearing impairment. We therefore report eeyore as a novel mouse model of NSHL that will provide new insight into the genetic mechanism underlying progressive and age-related hearing loss, and contribute to the validation and development of novel therapeutics when corresponding genetic lesions are identified within the human genome.Our