Lawsonia intracellularis, has a wide range of susceptible host species. Recently, L. intracellularis has been recognized as an etiologic agent of an emerging enteric disease in foals called equine proliferative enteropathy (EPE). The presence of L. intracellularis in nonruminant wildlife has raised questions regarding the role of these species in EPE transmission.Proliferative enteropathy is a global enteric disease of particular importance in pigs. The causative bacterium, L. intracellularis in wild rodents and feral cats from eight farms with confirmed EPE. Serum (42) and fecal (40) samples from resident foals and fecal samples (131), intestinal mucosa tissues (14), and mesenteric lymph nodes (14) from wild and feral animals were collected for the evaluation of the farm status and the molecular detection of L. intracellularis following the diagnosis of EPE in index cases. Fresh feces from wild rodents and feral cats were collected from the ground while walking the premises or after trapping the animals using live traps. A total of 3 brown rats, 7 house mice, 1 striped field mouse, 2 grey red-backed voles, and 3 feral cats showed evidence of prior exposure to L. intracellularis.This study investigated exposure to L. intracellularis transmission and infection in wild rodents and feral cats and provide possible evidence of interspecies transmission. The exposure of wild rodents and feral cats provides potential evidence for the spillover of L. intracellularis to wildlife species and raises the question of spillback to horses. Additionally, these animals may represent an indicator of environmental exposure or may be actively involved in the transmission of L. intracellularis to foals by acting as potential reservoir/amplifier hosts. This study is the first to demonstrate the magnitude of L. intracellularis shedding in the feces of wild rodents and feral cats and to indicate the significant infection risk that wild rodents and feral cats pose to naïve horses in South Korea.Our data add to increasing evidence demonstrating the potential for The online version of this article (doi:10.1186/s12917-017-1155-8) contains supplementary material, which is available to authorized users. Lawsonia intracellularis is the etiologic agent of porcine proliferative enteropathy (PPE). PPE is considered a disease of particular importance in the pig because it usually affects growing pigs and has a large impact on performance . Nowith PPE % [L. intL. intracellularis at farms A, C, E, F, G, and H, suggesting that the bacterium had been introduced into the surrounding environments of these particular farms. The presence of L. intracellularis in wild rodents and feral cats living in close proximity to the farm led us to assume that this pathogen might be shed into the natural environment and spread as previously described [Collectively, three rodent species and 1 feral cat species were positive for escribed . The traL. intracellularis on farms with documented EPE cases in South Korea. Based on our results, a number of free-living rodents and feral cats can be considered host or reservoir species of L. intracellularis. Furthermore, we identified a new L. intracellularis-infected host: the grey red-backed vole. This species may be an important vector/reservoir of L. intracellularis in the Far East region, including South Korea, because this species ranges across northern Eurasia, including the Korean Peninsula, and is frequently found in South Korea.From the perspective of herd prevalence in wild animals, the proportion of positive feces from the wild animals captured in this study ranged from 0 to 25% for each farm. Differences in habitat, the number of trapping events between farms, and the total numbers of each captured animal species between the farms might be at the source of this discrepancy. Additionally, the results of this study highlight the variety of animal species that are potentially involved in the shedding of L. intracellularis infection among wild rodents and feral cats around horse farms can spread through the oro-fecal route similar to domestic pigs on farms with intensive breeding [The parallel finding of the causative agent in the intestinal mucosal membrane and the corresponding mesenteric lymph nodes in one of the brown rats and two of the house mice was suggestive of the earlier findings in domestic pigs . Our obsbreeding .L. intracellularis on pig or horse farms, with the prevalence of PCR-positive animals varying substantially between farms from 4 to 83% [The importance of EPE transmission by vectors is unknown. Following experimental inoculation, histological lesions develop in laboratory mice, rats and hamsters but not in sparrows or chickens , 31–34. 4 to 83% , 17, 31.L. intracellularis in wild rodents and feral cats captured on farms with EPE was higher than previously reported, possibly due to differences in regions or sampling methods; for instance, surveys reporting prevalence rates of 1.0 and 0%, respectively, have used PCR analysis of tissue or fecal samples, respectively [Evidence of exposure to ectively , 35.L. intracellularis [L. intracellularis DNA detection in wild rodents and feral cats were not significantly different, although the research design in terms of farm selection was different between our study and the study by Truong et al. This difference might explain why the previous study by Troung et al. investigated animals captured on pig farms regardless of consideration of farm selection based on PPE outbreak by L. intracellularis infection, which was in contrast to our method. Additionally, Lee et al. reported an extraordinarily high nationwide prevalence of PPE-positive pig farms (100%) in South Korea [A random sampling-based study previously performed in South Korea reported that 15.7 and 12.5% of tissue samples from wild rodents and stray cats, respectively, tested PCR-positive for llularis . Becauseth Korea .L. intracellularis–specific IgG by serology and the molecular identification of L. intracellularis DNA in feces by PCR [L. intracellularis have been hampered by the lack of established and validated serological assays [L. intracellularis in the feces of animals generally does not provide any conclusive evidence regarding the biological state of the organism or its origin. The direct link between wild animals and EPE still needs to be proven by either characterization of the detected bacterial isolates or experimental challenges using isolates from free-living hosts. To overcome these difficulties, good diagnostic methods and increased knowledge of epidemiology and immunity are required for the L. intracellularis bacterium.Regardless of the affected species, the antemortem diagnosis of proliferative enteropathy is based on the detection of s by PCR , 37. Posl assays . Difficul assays . Among aL. intracellularis-specific antibodies in cat or rodent species. Although IPMA could be available in our laboratory in this study and actually performed for sera from some animals, the serological data for cats and rodents is not shown in this paper because the assay was not fully evaluated and still not widely accepted for sera from cats and rodents. Another limitation was the study design, which was based on voluntary enrollment instead of randomized selection. Similar limitations were reported by Pusterla et al., [L. intracellularis in wild and feral animals.A major limitation of this study was the lack of widely-accepted serological assay to evaluate  et al.,  in the UL. intracellularis to susceptible weanlings remains speculative. However, foals most likely become exposed to L. intracellularis after the ingestion of feed or water contaminated by L. intracellularis-containing feces from domestic or free-living animals. Potential reservoir hosts must be abundant on the premises, have unlimited access to feeding and drinking areas of the susceptible weanlings, and be able to maintain the agent indefinitely within their populations. From the study results, brown rats, house mice, striped field mice, grey red-backed voles, and feral cats would be considered prime reservoir host candidates in South Korea. At the very least, these wild animal species may play a role in the circulation of L. intracellularis in the natural habitats around EPE-affected horse farms in the Far East, including South Korea. Additional domestic or free-living animals might have played a role in the transmission of L. intracellularis on the horse farms enrolled in this study. Therefore, the role of clinically and subclinically infected foals in the feco-oral transmission of L. intracellularis requires further investigation.The mode of transmission of L. intracellularis in foals remains speculative worldwide, and only limited information on epidemiological findings in wild and feral animals from farms with EPE has been reported in the United States in an attempt to determine their role in the spread of L. intracellularis. This study is the first report to describe the identification of five wild mammalian hosts that are potentially associated with the shedding of L. intracellularis on farms with a known EPE status in South Korea. Additionally, this study is the first to demonstrate the magnitude of L. intracellularis shedding in the feces of wild rodents and feral cats and to indicate the significant infection risk that wild rodents and feral cats pose to naïve horses in South Korea. These results also demonstrate the importance of understanding the role of wildlife species in the development of management strategies for EPE in weanling foals in South Korea. The findings emphasize the need to enforce biosecurity measures to prevent wild animals, including wild rodents and feral cats, from entering horse stables.In summary, the infection source of


Noise-induced hearing loss (NIHL) is an increasingly growing problem in young children. This is attributed to recreational noise being the most common cause of this problem. In young children, hearing problems can delay language development and reduce academic achievements. South Africa, in particular, has limited information and protective measures regarding the conservation of hearing in school-aged children.The main aim of the study was to determine the perception of primary school teachers regarding NIHL. The study also aimed to determine if any hearing conservation programmes are being implemented in schools and the need for training of primary school teachers regarding NIHL.A survey was conducted. In order to cover the population of interest, the sampled schools in Pretoria were clustered into urban, semi-urban and rural areas.The majority of the teachers included in this study are aware of NIHL and its effects. They, however, lack the necessary resources and knowledge to effectively use this information. Most (67.5%) of the teachers indicated that they have never been exposed to children with NIHL in a school setting. It was also found that the majority (84%) of the schools included in the study do not implement hearing screening and conservation programmes.Although the sample size was limited, the results correlate with other research in this field indicating a need for planning and implementation of hearing conservation programmes in schools, including training of teachers in order for these programmes to be effective. Noise-induced hearing loss (NIHL) is a hearing impairment caused by excessive or prolonged exposure to loud sounds. The sound results in changes in the mechanism and metabolism of the inner ear as the outer hair cells (OHCs) of the cochlea are over stimulated. The endolymphatic fluids of the cochlea are also affected in the sense that the high energy transfer causes metabolic stress, which in turn results in swelling and degeneration of the balance and hearing nerve terminals that attach to the inner hair cells (IHCs). Degeneration of the hair cells of the cochlea and damage to the auditory nerve result in either temporary or permanent noise-induced threshold shifts (NITS) Edwards, .NIHL may be sudden or progressive. With the latter, it develops gradually and is more rapid in the first decade of exposure with continuous exposure being more damaging than intermittent exposure. Sudden NIHL is caused by acoustic trauma, which results from sudden exposure to high intensity sound levels that cause instant damage to the hearing mechanism, for example, explosion or gun fire at close range .A TTS is defined as a temporary increase in hearing thresholds after exposure to loud sounds. It can be caused by continuous noise and impulse noise. Continuous noise can be harmful when exposed to noise of 85 dB or more for more than 8 h and impulse noise of 140 dB or more. With continuous noise, for every 5 dB increase from 80 dB, exposure time halves, for example, 80 dB = 16 h, 85 dB = 8 h [South African National Standards (SANS), what is too loud, and how loud is too loud.These experiences tend not to cause concern, partly, because there appears to be a full recovery. However, it is widely recognised that repeated episodes of TTS can result in permanent changes within the cochlea and hearing loss. If the OHCs in the cochlear do not recover within the hours mentioned above, then the hearing loss becomes permanent, which is known as permanent threshold shifts (PTSs)  connections in a network of auditory neurons at a more central  brain level and that these cells continue to fire spontaneously . Chronic tinnitus can be as damaging to quality of life as the effects of hearing loss . Parents and clinicians should be attentive. Measures of speech discrimination often more accurately reveal a hearing problem than the simple audiogram or hearing screening tests. Hearing loss can affect language development in young children. This cascading effect results in reduced academic achievements if information is being missed at school. For adolescents, communication difficulties can lead to social isolation. There have been reports of suicide resulting from social isolation. If hearing aid use is required, the adolescent may also have problems with the cosmetic appearance of the device or the stigma attached to wearing hearing aids. This may result in the adolescent not using the aids, or they may choose to become socially isolated or only socialise with small selected groups. This ultimately will have an influence on their quality of life Many research studies have been conducted internationally to investigate the prevalence and the prevention of NIHL in children; however, there is little research available on NIHL in South Africa. Considering the specific risks that are often unavoidable in South Africa, for example, exposure to excessively loud music in public transport vehicles, which is commonly a mode of transport in order to get to and from school as well as exposure at social gatherings and religious services, highlights the need for research specifically in the South African context. Current international research focuses more on setting standards and guidelines for PLD use. In South Africa, research studies regarding the prevalence of NIHL in youths are extremely limited. A study conducted in 2005 in Gauteng, South Africa, on teachers’ attitudes about hearing loss indicated that teachers do not have adequate knowledge, skills and training for effective implementation of hearing conservation programmes Pottas,  states tThe lack of research in South Africa highlights the need to determine the knowledge and perceptions of primary school teachers regarding NIHL, and hearing conservation is vital in order to identify possible solutions for effective implementation of hearing conservation programmes in schools. It may also create awareness that teachers and audiologists should work together for identification and management of learners with NIHL and also in promoting NIHL prevention programmes at schools. Healthy behaviours are more easily established during childhood compared with adulthood. It would, therefore, be better for children to grow up in an educational environment whereby health promotion is emphasised in order to change listening habits and attitudes. This can only be achieved if teachers are aware of NIHL as well as its effects.A descriptive research design was used for the purpose of this study, that is, a quantitative study whereby researchers seek to obtain insight and answer questions that the researcher has about the population of interest  in Pretoria, regarding NIHL. The study also aimed to determine what and if any hearing conservation programmes are being implemented in schools and the need for training and education of primary school teachers regarding NIHL and hearing conservation.A combination of purposive sampling (a form of non-probability sampling) and multistage sampling  methods was used. The purpose of using the two methods was to increase the feasibility and efficacy in order to accomplish certain goals of the research design. From a population of 283 public primary schools in Pretoria, a number of 164 schools were the representative sample. A 5% confidence interval and 95% confidence level was used to calculate the representative sample, which is most commonly used in quantitative research. To cover the population of interest, the sampled schools were clustered into urban , semi-urban (Ga-Rankuwa) and rural areas  with each cluster consisting of 55 schools. The selection criteria included qualified teachers, educating students in Grade 5–7. As most of the literature regarding NIHL in youths has focused on teenagers, targeting an age group before adolescence was deemed appropriate. All teachers that complied with the inclusion criteria were given the opportunity to participate in the study.Semi-structured questionnaires were used. These are the kind of questionnaires that include both close- and open-ended questions. The questionnaires were adapted from two studies. The first study was that conducted by Taljaard et. al.  about PLReliability is the consistency with which a measuring instrument yields a certain result when the entity being measured has not changed  was used to analyse the different variables. The Statistical Packages for Social Sciences software was used to store and analyse the data.Ethical approval was granted by Sefako Makgatho Health Sciences University Research Ethics Committee , Department of Basic Education and principals from the participating schools. Participants had to give written consent before completing the questionnaires and voluntary participation was emphasised. Researchers ensured confidentiality by having the participants complete the questionnaires anonymously. The semi-structured questionnaires were sent to the participating schools together with consent forms via emails, with some being hand delivered. Those that were hand delivered allowed clarification of questions and were collected immediately after being completed and some were collected a few days after completion. Those sent through emails were emailed back to the researchers.Out of the 164 targeted schools, only 59 were able to take part in the study. The return rate was 5% (59/283), with 19 schools from the urban area, 20 from the semi-urban area and 20 from the rural area. De Vos et al.  stated tFollowing statistical analysis, insignificant differences were found between teachers in urban, semi-urban and rural settings. This could be due to the fact that only seven universities in South Africa provide teacher training and they are in urban settings. The results are therefore mostly reported by combining the responses from all the 219 participants.n = 70) of the participants were able to describe the behaviours of a hearing impaired learner. According to Peel  of the participants indicated that they had experience in teaching learners with hearing loss, 74% (n = 162) had no experience within this area.In this study, only 26% (n = 173) of the participants indicated that audiologists, teachers and parents are responsible for identifying hearing loss in learners. Intensity of sounds was indicated as the most common risk condition for NIHL, followed by the frequency of the sound and the duration of exposure to the sounds. Non-occupational noise was identified as the least common risk condition. The majority of the participants indicated that all of the following are effects of hearing loss in order of most to least importance: academic failures, cognitive difficulties, language and speech difficulties, and social problems. These results suggest that the majority of the teachers have an idea of the impact of hearing loss in children; however, their knowledge in this area is limited as social problems, language and speech problems were indicated as the least common impact of hearing loss, and it is clear that they do not understand the cascading impact of hearing loss on a child in school. According to Doyle and Dye  of the participants were of the opinion that hearing loss can be reversed, 86% (n = 188) said NIHL can happen to anyone at any age and 10% (n = 22) thought that hearing loss can be reversed and can happen to anyone at any age. Only one participant (0.5%) thought that hearing loss only happens to old people. The results suggest that most of the teachers are aware that NIHL can affect any one at any age; however, a small percentage of teachers are of the opinion that NIHL can be reversed. This emphasises the need for training teachers to work with hearing impaired learners. The teachers’ awareness of the characteristics of hearing loss will improve as they gain more experience and training in working with hearing impaired children  agree that loud sounds or music can damage hearing; however, 4% (n = 3) of the participants were not aware of this fact. Supporting these results is a study conducted by Richburg and Goldberg  of the participants in all clusters know the importance of early identification and treatment of hearing loss. This is supported by the results of the study conducted by Hayes  of the participants reported that they have hearing screening programmes, 6% (n = 5) from the semi-urban area and 16% (n = 10) in the rural areas. This suggests that the urban and the rural areas have more schools with hearing screening programmes than the semi-urban areas. However, overall only 13% (n = 29) of the schools from all clusters do have hearing screening programmes in place. It is also not clear if these hearing screening programmes are sensitive to identifying NIHL.From the urban areas, 19% (n = 18) of the schools indicated that these programmes have been functional for more than 3 years. National prevention programmes have been described in the United States , United Kingdom (Do not lose the music) and Australia , , WHO, ; howevern = 206) of the teachers reported that they do not have the necessary equipment required to run these programmes optimally. The professionals in charge of running the hearing conservation programmes were as follows: From the urban areas, 9% (n = 7) of the participants indicated audiologists, 3% (n = 2) of them revealed speech therapists and 12% (n = 9) of them indicated nurses. From the semi-urban areas, 1% (n = 1) of the participants indicated audiologists and 2% (n = 2) revealed speech therapists. From the rural areas, 8% (n = 5) of the participants indicated audiologists and 13% (n = 8) revealed nurses. This suggests that the majority  of the school programmes are run by nurses in the urban and rural areas, with audiologists being the second most common professionals across all clusters. Under normal circumstances, hearing screening programmes should be managed by educational audiologists as they are the ones who can train other professionals to effectively take part in the programmes habilitation  and collaboration with teachers and school personnel in order to develop and implement individualised education plans (IEPs) for each child identified with a hearing loss. Informational and affective counselling and guidance for children, parents and teachers is a key responsibility for educational audiologists. The process of selecting, fitting and determining effectiveness of amplification is also an essential duty of educational audiologists, including participation in measurement of classroom acoustics and instructions for teachers with assistive technology. Clearly, educational audiologists have the skills necessary to collaborate with teachers in order to ensure the academic success of children with hearing loss. With the guidance and support of educational audiologists, teachers can be provided with the necessary tools and education to make the appropriate adaptations for children with hearing loss and implement successful hearing conservation programmes  of the teachers from all clusters have not received any prior training. An even higher percentage  of the participants indicated that they would like to be trained. These results agree with those obtained by Pottas in 2005. The study indicated that teachers do not have adequate skills and lack training for effective implementation of the hearing conversation programmes. Although teachers are educated in the methodology of providing academic education to all students, they may lack detailed information regarding the specific needs of children with hearing loss in the typical classroom. However, with the knowledge and confidence to assist children with hearing loss in the classroom, teachers can promote academic success in this population as well as support hearing conservation programmes.The results obtained suggest that the majority ; the second most common method was presentations  with posters  being the least common method. In a study conducted by Thompson, Pakulski, Price and Kleinfelder  an educational orientation to identify children with a hearing loss that might have negative academic consequences or (2) medically oriented programmes designed for the early identification of existing health conditions that necessitate medical referral. Therefore, a preventive approach for the purposes of identifying early signs of NIHL has not been a focus for school-based hearing screening programmes  training and educating teachers about NIHL and (2) planning and implementation of hearing screening and hearing conservation programmes with an educational audiologist overseeing the programme.The study sample was obtained from different clusters, that is, urban areas, semi-urban areas and rural areas, representing all sectors of Gauteng. Furthermore, although the results of the study cannot be generalised, this study serves as an initial study that can be used to investigate hearing conservation programmes in other provinces and educational sectors. Additionally, the majority (85%) of the participants were able to complete the questionnaires appropriately, suggesting that the questionnaire was clear and the participants understood what the questionnaire required and can, therefore, be successfully used in future research. The anonymous aspect of this survey could be representative of the truthful responses of the teachers as there could be no consequences from their answers.There were insufficient participants in order to generalise the results of this study to the larger population. The limited participants could be due to method of questionnaire delivery and limited time frame of the research project. Nulty  reported


Parus humilis) has evolved a distinctly long and curved beak from other parids. An integration of morphometrics, phylogenetics, transcriptomics and embryology allows us to address the evolutionary and developmental mechanisms of the adaptive beak structure observed in the Ground Tit.Beak morphology exhibits considerable adaptive plasticity in birds, which results in highly varied or specialized forms in response to variations in ecology and life history. As the only parid species endemic to the Qinghai-Tibet Plateau, the Ground Tit (Parus major). We detected that 623 genes were significantly differentially expressed in embryonic upper beaks between the two species, 17 of which were functionally annotated to correlate with bone development and morphogenesis, although genes related to bone development were not found to undergo accelerated evolution in the Ground Tit. RT-qPCR validation confirmed differential expression of five out of eight genes that were selected from the 17 genes. Subsequent functional assays in chicken embryos demonstrated that two of these genes, FGF13 and ITGB3, may affect beak morphology by modulating levels of osteoblasts and osteoclasts.A morphometric approach quantified that the Ground Tit has a comparatively longer and more decurved upper beaks than other parids. We estimated that the ancestor of the Ground Tit likely had a short straight upper beak similar to most current recognized parid species using an ancestral state reconstruction. This morphological specialization is considered an adaptation to its ground-oriented behavior on the high plateau. To identify genetic mechanisms behind this adaptive change, a comparative transcriptomic analysis was applied between the Ground Tit and its closely related species, the Great Tit  contains supplementary material, which is available to authorized users. The avian beak is a highly evolvable structure. The malleability of beak morphology permits birds to adapt, sometimes rapidly, to diverse ecological niches . VariousBMP4 has been found to play a role in regulating both the width and depth of the prenasal cartilage (pnc) in the frontal nasal mass (FNM) of both Darwin’s finches and chickens [CALM1, IHH, DKK3, TGFBR2 and CTNNB1, is responsible for regulating the length and width of the pnc and premaxillary bone (pmx) in birds [ALX1 and HMGA2 are associated with beak shape [Studies of the genetic mechanisms underlying variation in beak morphology have identified multiple genes responsible for the development of beak morphology in birds. For example, chickens , 7. The in birds –10. Two ak shape  and beakak shape , respectak shape –15.Parus humilis) was considered to be a member of the family Corvidae based on its phenotypic similarities with ground jays of the genus Podoces  [Traditionally, the Ground Tit (ed beak) , 17. Howed beak) , 19. Theed beak) . In resped beak) , 22, theed beak) .Parus major). They are closely related species with a divergence time of approximately 7.7-9.9 million years [To explore the evolutionary and developmental mechanisms of the adaptive beak structure observed in the Ground Tit, we firstly employed linear and geometric analyses to quantify variations in beak morphology between the Ground Tit and other parids. We then identified potential candidate genes contributing to beak development in the Ground Tit by comparative transcription between the Ground Tit and the Great Tit  and linear discriminant analysis (LDA) showed that beak size in the Ground Tit is significantly different from that in all other parids  and centroid size , indicating that evolution of beak shape was also phylogenetically independent. Multivariate regression detected no significant allometry between shape and size (P = 0.064) even though 49.04% of total interspecific variation in shape was predicted by size variation. Procrustes ANOVA on Procrustes coordinates showed significant interspecific difference in beak shape . Discriminant function analysis (DFA) and canonical variate analysis (CVA) clearly distinguished Ground Tits from all other parids , Atlantic Canary (Serinus canaria) and American Crow (Corvus brachyrhynchos) were identified for phylogenomic analysis. After removing genes with errors and saturated mutations, 1873 genes were retained for subsequent analyses. For all 1873 genes, the mean dN/dS ratio of the Ground Tit was higher than that of other lineages, but these differences were not statistically significant , we defined 623 genes to be significantly differentially expressed, including 148 up-regulated and 475 down-regulated genes in the Ground Tit relative to the Great Tit  to be validated for their observed expression levels and patterns in embryonic upper beaks at stages 28/29 using reverse transcription quantitative PCR (RT-qPCR). RT-qPCR validation confirmed five out of the eight genes showing significant differences in expression in the upper beaks of Ground Tits relative to that in Great Tits, including FGF13 in FGF signaling pathway, FRZB and WIF1 in canonical Wnt signaling pathway, and ITGB3 and NFATC1 in OCD signaling pathway  were assayed by in situ hybridization on sections of upper beaks at stages HH28/29. In situ hybridization supported transcriptome and RT-qPCR results that Ground Tits expressed higher level of FGF13 but lower levels of WIF1 and ITGB3 than Great Tits  and recombinant ITGB3 protein (rITGB3) showed an elevated proportion between upper and lower beak length relative to control embryos Fig. . These iGeometric analysis separated more species pairs and detected differences among all species  in the FGF pathway. Previous reports have highlighted roles of FGFs in inducing neural crest cells to promote skeletal outgrowth of the beak , 32, 33.ITGB3, an adhesion receptor highly expressed in osteoclasts, is required for the interaction of osteoclasts with bone substrates during bone resorption , 38. ITGFGF13 and ITGB3 might affect the beak morphology by regulating levels of osteoblasts and osteoclasts, respectively. Although FGF13 and ITGB3 were found to affect both upper and lower beak parts in chicken embryos, we infer that they could affect the upper beak more than the lower beak in embryos of Ground Tits and Great Tits according to their higher expression in upper beaks , depth (at the nares) and width (at the nares) of the upper beak Fig.  that detCollect Statistics on Tree Set option in Morpho J [Regression option [We also compared the shape of the upper beak in the Ground Tit with that in other parids using geometric morphometric analysis. A total of 352 lateral images of upper beaks of the skin specimens were used in the geometric analysis  [P-values below 0.05 were used as the threshold for statistical significance. Wilcoxon rank sum tests were used to test whether the mean dN/dS value of the Ground Tit differs significantly from those of other lineages.Genomic coding sequences from five avian species, including the Ground Tit , Great Ton years  in BEASTon years . Based oon years . To reduon years . The likon years . P-valuete (FDR) . FDR-adjAt early stages of embryonic development, initiation, migration and differentiation of neural crest cells contribute to avian beak morphogenesis , 64. Beahold (TranGen) at 4 °C. Eight stage-matched Ground Tit and 9 Great Tit embryos were dissected for RNA extraction. Three matched embryos of each species were fixed in 4% paraformaldehyde (PFA) solution for in situ hybridization. The field collection of embryos was performed with the permission of the State Forestry Administration of China and conformed to the National Wildlife Conservation Law of China.We collected embryos during two breeding seasons (April 2013 to July 2013 and April 2014 to July 2014) in Nagqu, Tibet (Ground Tit) and Zuojia, Jilin (Great Tit) , which produced a total of 21.91 GB raw reads. We used Cutadapt to remove adapters and reads with >5% unidentified nucleotides [http://hannonlab.cshl.edu/fastx_toolkit) to remove reads with length of <90 and reads with <90% bases that had Phred quality score > 20, as well as used Trimmomatic to trim and pair reads using default setting [Total RNA was extracted from four embryonic upper beaks from each species using TRIzol reagent (Invitrogen). The quality of extracted RNA samples was examined using a Nanodrop spectrophotometer and an Agilent 2100 Bioanalzyer. Sequencing libraries (non-strand-specific) were constructed for two qualified RNA samples from each species according to the manufacturer’s protocol (Illumina). RNA sequencing was performed based on 100 bp paired-end reads using an Illumina HiSeq 2000 in the leotides , used FA setting . The res setting .P-value less than 0.001 [We quantified gene expression based on two data types; the simple read counts calculated by HTSeq-count , and thean 0.001 . To decran 0.001  to enrican 0.001 , 81.P-values and fold changes . cDNA, primers and TransStart SuperMix (TranGen) were mixed in a 20 μl reaction to amplify each gene. RT-qPCR was run in triplicate on a Roche LightCycler 96 using a program: 94 °C for 30 s, 40 cycles at 94 °C for 5 s, 60 °C for 15 s and 72 °C for 10 s. The relative expression (∆Cq) of all genes was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is inversely proportional to the real expression levels. We examined significance in relative expression using Student’s t-test. Expression differences were calculated using the -∆∆Cq method [FGF13, WIF1 and ITGB3 and hybridization signals were visualized using confocal epifluorescence.To verify expression levels and patterns of genes identified by transcriptome analyses, we performed RT-qPCR for eight candidates in upper beaks of embryos because of limitation of embryo numbers (four replicates per species). Seven of them were selected from pathways related to bone development with the highest es Table . FRZB, aq method . In situq method  on parafWIF1, FGF13 and ITGB3, can affect beak morphological traits. Eggs were incubated at 38 °C and 60-70% humidity. At HH30, 10 μl of recombinant mouse ITGB3 protein (200 ng/μl) (R&D Systems) (n = 15), recombinant mouse WIF1 protein (250 ng/μl) (R&D Systems) (n = 15), and FGF13 recombinant protein (50 ng/μl)  (n = 15), was injected into the vitelline vein using a sterilized glass needle. Prior to injection, we opened an ~2 cm oval window in minor diameter using a scalpel and removed the external membrane using fine forceps. Control embryos (n = 15) were treated with 0.1% bovine serum albumin (BSA). Surviving embryos were collected at HH38, fixed with 4% PFA and stored in 100% methanol after gradient dehydration. We collected a total of 43 survived embryos consisting of 13 controls, 12 rFGF13 treatments, 10 rITGB3 treatments, and 8 rWIF1 treatments.Chicken embryos were manipulated using a biochemical strategy to test if changes in expression of the candidate genes, Embryos were stained with 0.1% Alizarin Red, and cleared in gradient glycerol . LateralAdditional file 1:Supplementary materials. Included are 8 supplementary figures  and 18 supplementary tables (Tables S1-S18). (PDF 1215 kb)Additional file 2:The beak size data of 13 parids used in this study. (XLS 93 kb)Additional file 3:The beak shape data of 13 parids used in this study. (XLS 91 kb)Additional file 4:The description of expression abundance of the 623 DEGs. (XLS 217 kb)


To assess whether WMC also plays a role when listeners without hearing loss process speech in adverse listening conditions, we surveyed published and unpublished studies in which the Reading-Span test (a widely used measure of WMC) was administered in conjunction with a measure of SiN identification, using sentence material routinely used in audiological and hearing research. A meta-analysis revealed that, for young listeners with audiometrically normal hearing, individual variations in WMC are estimated to account for, on average, less than 2% of the variance in SiN identification scores. This result cautions against the  assumption that individual variations in WMC are predictive of SiN identification independently of the age and hearing status of the listener.With the advent of cognitive hearing science, increased attention has been given to individual differences in cognitive functioning and their explanatory power in accounting for inter-listener variability in the processing of speech in noise (SiN). The psychological construct that has received much interest in recent years is working memory. Empirical evidence indeed confirms the association between WM capacity (WMC) and SiN identification in older hearing-impaired listeners. However, some theoretical models propose that variations in WMC are an important predictor for variations in speech processing abilities in adverse perceptual conditions for  Figure 1). Such work reflects the emergence of the new interdisciplinary research field of Cognitive Hearing Science . WM is considered by many psychologists as a “cognitive primitive,” due to its moderate-to-very-strong associations with different aspects of hot  has received considerable attention in recent years .For example, a series of audiological research studies investigated whether individual differences in WMC, measured by the version of the RSpan test developed by and normal-hearing (NH) listeners, Furthermore, on the basis of an extensive review of behavioral studies concerned with the effects of cognitive factors on SiN perception in HI A similar assumption that the same crucial cognitive processes are at work in all listeners, independently of their age and hearing status, is made in recent models of speech/language processing e.g., . For exa1 and a measure of SiN identification to participants with audiometrically normal hearing sensitivity.To assess the claim that individual variability in WMC accounts for differences in SiN identification even in the absence of hearing loss, we surveyed studies administering the RSpan test2 and a general literature search, we were able to compile data from 19 published and unpublished studies that complied with our inclusion criteria3. Since several studies measured SiN identification against different types of background maskers or for different performance levels, a total of 41 data sets was entered into the meta-analysis . For each data set, the Pearson correlation coefficient  and associated 95 and 99% confidence intervals  are indicated, as well as the performance level at which the participants were tested, the type of masker, the sentence material4, the age range of the sample and the sample size. Within each of the three sections of Figure 2, data sets are organized by decreasing performance level . For identical performance levels, data sets are ordered by masker type, representing presumed increasing masker complexity, from “simple” notionally steady noise5 through sinusoidally or speech-envelope-modulated noise to speech babble. Interestingly, some of the studies for which the data were reanalyzed on our request (indicated by an asterisk against them) did not even report the correlation between WMC and SiN identification in NH listeners in their original publication.To ensure consistency with experimental conditions in investigations of HI listeners, only studies presenting sentence material routinely used in audiological and hearing research against spatially co-located background maskers were considered. In addition, we only examined studies in which the effect of age was controlled for, in order to avoid inflated estimates of the correlation between WMC and SiN tasks caused by the tendency for performance in both kinds of tasks to worsen with age. The effect of age was controlled for either by restricting the analysis to a narrow age range, or by statistically partialling out the effect of age when using data from participants across a wider age range. Based on a request posted on the Auditory Listr values varied widely from -0.29 to 0.64, with almost a quarter of the values being negative, indicating that sometimes low-WMC individuals showed better SiN identification than individuals with high WMC. CIs were rather large, suggesting that studies were underpowered , and, in most cases, the intervals included the value zero.Across all data sets, the observed Seemingly in contradiction with the ELU-model prediction of higher WM involvement for speech identification in increasingly adverse listening conditions, there was no obvious trend for more consistent or stronger correlations in more difficult listening conditions . In fact, there is some (descriptive) evidence of stronger associations between WMC and SiN identification in easier listening conditions [see results in section I for the same listeners in high- and low-performance-level conditions in Moreover, comparisons across different data sets obtained for similar performance levels did not show that inter-individual variability in WMC were more consistently or strongly associated with SiN identification for more complex maskers or target speech, as has previously been speculated e.g., . For exaAt the same time, the strength of the correlation varied even for studies using very similar test conditions and participant groups. For example, at a performance level of 50%-correct for IEEE sentences presented in a steady noise masker, the correlation for young NH listeners was either -0.29  or -0.01r values to Fisher’s z scale. Across all 24 data sets, the average r value was 0.12. In other words, individual variations in WMC in young people with audiometrically normal hearing are estimated to account for, on average, less than 2% of the variance in SiN identification scores.As there was a sufficiently large number of data sets from studies restricting their sample to young listeners (aged 18–40 years), a random-effects meta-analysis model was used to estimate the average correlation among these studies. This kind of analysis has the advantage not only of assuming that the true treatment effect differs from study to study, but also accounts for the fact that multiple measures can arise from the same study . The analysis was performed using the R package metafor  and a trFigure 2), and when averaged across maskers, even after partialling out the effects of age and hearing sensitivity  while stronger and significant correlations were observed for the middle-aged (40–59 years) to old–old (70–91 years) age groups . A linear regression of SiN identification scores against age, RSpan scores and their interaction showed that the slope of the linear dependence of SiN identification performance on RSpan scores indeed increased significantly with age (p ≤ 0.001). This illustrates the moderating effect of age on the relationship between WMC and SiN identification, cautioning that the statistical control of the effect of age by computing partial correlations is not necessarily appropriate.Given the considerably smaller number of data sets in each of the two other categories, involving older listeners, we did not compute a summary statistic. However, it is noteworthy that in the largest study included in the survey, using listeners from a wide age range, significant correlations between WMC and SiN identification were found for unmodulated and modulated background noises  is a reliable and strong predictor of SiN identification in young listeners with normal hearing thresholds. Recent experimental work on the perception of interrupted speech, another form of signal degradation, is consistent with this finding .It could be argued that the cognitive and speech tests used in the studies surveyed here are suboptimal or inappropriate measures of WMC and SiN processing, respectively e.g., . HoweverAnother criticism could be made regarding the fact that SiN identification was predominantly assessed for performance levels close to 50% correct, obscuring the possibility that WMC and SiN identification are linked to a greater extent than reported here at other performance levels. Indeed, according to the ELU model, a greater mismatch between sensory and mental representations, and hence a higher involvement of WM-based identification processes, is predicted as speech-to-noise ratios become less favorable. However, this does not seem to be borne out by the collected results. Alternatively, it has also been argued that WM-based restorative processes in older HI , 2012 anHence, all things considered, the results of this meta-analysis caution against the  assumption that individual variations in WM determine SiN processing in all its forms and independently of the age and hearing status of the listener.per se. First, individual differences in WMC have sometimes been shown to explain some of the variability in performance in more linguistically complex tasks, such as the comprehension of conversations  may still prove helpful in improving the prediction of aided SiN identification performance for older audiological patients. Future evidence based on new large samples, independent of those repeatedly investigated in previous studies , 2011, cvs. specialized WM system in language comprehension is not new (In conclusion, even though the question of a general  not new  and it h not new . FinallyCF collated, analyzed and plotted the data, and wrote the paper. SR analyzed the data, and revised and commented on the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MR and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Introduction: Noise pollution is one of the most important problems in industry that has an effect on the auditory system and other physiological parameters, as well as persons in noise exposure situations. While noise-induced hearing loss is preventable, once acquired, hearing loss is permanent and irreversible.Methodology: In the current study, noise in various sections of Flour Company in Lamerd estimated via the audio recorder, which revealed that the operators’ expression remained larger than the state criterion; hence, the perception experiment (audio recorder) was performed on the operators and its outcomes were examined via utilizing SPSS 16 of version. Findings: Overall, Pearson relationship r = 0.453 discovered among job reports and the performance decline between all operators by significant stage p≤0.05. Moreover, T-test applied to examine noise impact on operators included in boisterous rooms (mean more than 85 dB) also average=26. 71 and regular deviation=11.72 got (p≤0.05) that was greater than 25db (as the standard hearing threshold). Conclusion: The outcomes of audio measuring and T-test revealed that the noise corruption has an impact on the hearing of bodies operating in noisy rooms.  Environment pollution is a secondary and unwanted product of different industrial activities that has exposed the environment to further danger [2]. Noise contamination is one of the extremely significant difficulties of the industries that influence the hearing procedure and other physiological factors of the human’s body, as well as bodies in sound exposure situations [3]. This effect and its rate are different according to the personal and environmental features. Important individual characteristics are age, work experience, race, nutrition, and diseases [4]. In addition, the exposure to noise can cause social and psychological problems [5]. Although noise-induced hearing failure is preventable, already received, the hearing failure is perpetual and immutable [6]. About 30 million workers in the USA are exposed to dangerous noise level  because of which hundreds of million dollars per year have been estimated for the hearing loss due to noise pollution [9]. Statistics of the World Health Organization evaluates 4 million dollars as daily damage [10].The requirement for industry in various communities has led to constructing different production factories and industries .The position, control, and reduction in the exposure time are regarded as necessary measures. Performing proper control methods can hold the noise of workplace at a standard level [12]. Mac Donald’s consulting engineers group investigated the noise pollution in Tehran city in 1977 for the first time, based on which, the noise rate in Tehran city was reported at 55- 85 db [13], showed that noise has a meaningful impact on the operators’ ears, especially in 4000 Hz frequency; such that only 48.3% of the operators had a normal hearing.Investigating the workers’ hearing status in noisy halls of Tehran Azmayesh Factory  on noise exposure rate and permanent noise-induced hearing of workers of Shadri spinning factory in Yazd city, the results showed that noise and work records have a direct link via their hearing loss.In addition, in a research performed by Qotbi et al. : NIHL for both ears was obtained as it follows  indicated that, there is a direct relationship between age, work record, and daily work hours with a decrease in the hearing rate. Parviz Poorkhanzadeh [17] also indicated that there is a clear link among the hearing loss and the rise of noise intensity and increase of exposure time, which our research results supported. In a study by Santana and Ferrite, a direct clear link is obtained between age, hearing loss, and occupational exposure to noise [18]. A research of Hong and Kim indicated that, there is a meaningful relation between the occupational exposure to noise and the hearing loss [19]. The relation between the hearing reduction and age and work experience has been proven in a study done in Ethiopia [20], however in the research of Fariba Asghari et al. [21], no strong relationship was seen between the hearing loss and work experience and there might be various reasons according to various elements in noise effect. The maximum noise rate was in production and the sifter halls and other halls were less noisy according to the path of these two halls. The audiometric findings revealed that the mean of hearing loss in workers of two noisy units was higher than the other units and the total average, which indicated the link among the increase of environment noise intensity and reduction of hearing rate. Also, there is a clear link among age and work record of people working in the Flour Factory of Lamrard and NIHL; while the relation of its results via the other researches in this area supported this matter. Researches done by Mohammad Heydarian Moghaddam [This research is the outcome of M.A. thesis in Islamic Azad University of Bandar Abbas. AcknowledgementFinally, we deem it necessary to thank the manager and workers of Nasr-e-Lamerd Flour Factory and the experts of the Health Center in Lamerd for their sincere assistance and cooperation in doing this study.


Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion body-based protein production is often limited by the lack of techniques for the detection of correctly refolded protein. Thus, the selection of the refolding conditions is mostly achieved using trial and error approaches and is thus a time-consuming process. In this study, we use the latest developments in the differential scanning fluorimetry guided refolding approach as an analytical method to detect correctly refolded protein. We describe a systematic buffer screen that contains a 96-well primary pH-refolding screen in conjunction with a secondary additive screen. Our research demonstrates that this approach could be applied for determining refolding conditions for several proteins. In addition, it revealed which “helper” molecules, such as arginine and additives are essential. Four different proteins: HA-RBD, MDM2, IL-17A and PD-L1 were used to validate our refolding approach. Our systematic protocol evaluates the impact of the “helper” molecules, the pH, buffer system and time on the protein refolding process in a high-throughput fashion. Finally, we demonstrate that refolding time and a secondary thermal shift assay buffer screen are critical factors for improving refolding efficiency. Escherichia coli (E. coli) is a cheap and readily accessible recombinant expression host, up to 70% of eukaryotic proteins expressed in E. coli are insoluble as stated by reports from the Center for Eukaryotic Structure Genomics (CESG)5. Despite this major attrition and the availability of a large number of well-established expression systems , E. coli is still the most widely used expression platform for protein production, due to advantages such as high growth rate in inexpensive medium, rapid biomass accumulation, ease of scale-up and high productivity8. In addition, a great number of expression vectors, engineered strains and many cultivation strategies are well characterized for the high-level production of heterologous proteins in this organism. Therefore, the development of a systematic refolding method that can be robustly applied for a wide range of protein candidates to efficiently recover correctly folded and biologically active recombinant proteins from inclusion bodies is highly attractive.The demand for innovative protein-based therapeutics to address drug-resistant diseases is ever more pressing. Although the Human Genome Project completed in 2004 has provided a wealth of biological targets for exploration by structural biology and therapeutic drug design, the number of human protein structures determined by NMR spectroscopy, X-ray crystallography and cryoelectron microscopy has not increased dramatically. One of the main obstacles is the availability of large amounts of the target protein. Although 9. Although the protein found in inclusion bodies cannot be directly used for studies due to lack of biological activity, they provide a highly enriched source of target proteins with high purity. Consequently, several refolding methods have been extensively reported . However, as many proteins can only be refolded under very specific conditions, the development of systematic screening methods that can screen multiple refolding conditions in parallel is still challenging.Inclusion bodies are composed of aggregates of unfolded, partially folded and misfolded protein. They are often formed due to a lack of chaperones, failing to reach a correct conformation in the reducing environment of the cytoplasm and potentially undergoing proteolytic degradationTM Protein Refolding Kit [Molecular Dimensions Limited]) designed to identify optimal refolding conditions are available commercially. Despite this, refolding efforts are still limited by the lack of analytical assays to monitor multiple refolding experiments in parallel. More often, the refolding process is detected by surrogate assays such as turbidity or absorbance that cannot readily discriminate between properly folded and misfolded proteins. Similarly, SDS-PAGE, size exclusion chromatography (SEC) and reversed-phase HPLC assays can solve this problem but are time-consuming and are not compatible with high-throughput methods15. Recently, Biter et al. demonstrated that differential scanning fluorimetry (DSF) guided protein refolding (DGR) allows for discrimination between folded, misfolded and unfolded states of refolded protein16. However, the pH, Anion, Cation crystallization trial (PACT) screen described is optimized for screening protein crystallization conditions rather than protein refolding. The PACT screen DGR based assay evaluates the effect of pH, anions and cations on a sparse matrix basis and does not address widely used refolding agents, such as chaotropes (urea) or aggregation inhibitors (arginine).Several fractional factorial refolding kits  as an extension of the DGR approach. Our research demonstrates that DGR can be applied for determining refolding conditions of proteins that contain more specific and complex buffer compositions. To establish and validate the refolding screen, four different proteins were used: the receptor binding domain of hemagglutinin (HA-RBD), interleukin-17A (IL-17A), mouse double minute 2 homolog (MDM2) and the inhibitory receptor ligand of programmed death receptor 1 (PD-L1). We describe the development of a dedicated and systematic protocol that is able to evaluate the impact of pH, buffer system, time, redox couples as well as a range of additives on the protein refolding process. We also show that a secondary thermal shift assay (TSA) screen performed on the refolded material provides improved dialysis conditions for the removal of refolding agents.E. coli overnight culture was used to inoculate 1 L Luria–Bertani broth. The cells were grown at 37 °C with shaking (180 rpm), 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added when the OD600 reached 0.6. After induction, the temperature of the culture was lowered to 18 °C and the cells were incubated overnight. The cells were subsequently harvested by centrifugation at 5,000 g for 20 min.For protein expression experiments, 5 mL of an 4, 0.01 mg/mL DNAse and 0.1 mg/mL lysozyme were added. The solution was incubated at room temperature for 15 min. Cell debris was removed by centrifugation . The resuspension, sonication and centrifugation procedure was repeated 4 times using lysis buffer with 0.5% Triton X-100. The inclusion bodies were incubated in lysis buffer with 0.5% Triton X-100 overnight. Finally, the inclusion bodies were washed twice in wash buffer .The cell pellets were resuspended in lysis buffer  and 5 mM ethylenediaminetetraacetic acid (EDTA)). The cell suspension was sonicated for 2 min and 2 mM MgSOInclusion bodies were resuspended in either 8 M urea or 6 M guanidine-HCl. Insoluble material was removed by centrifugation at 19,000 g for 20 min.The denatured target protein was refolded by shock dilution at a ratio of 1:20. 10 μL of denatured protein solution at ~5 mg/mL was added into each well of a 96-well plate. Subsequently, 190 μL of refolding buffer from a pre-prepared master plate was transferred to the plate containing the denatured protein. Each well contained a final volume of 200 μL. After set up, the plate was incubated with gentle shaking at room temperature or 4 °C.For the 96-well refolding detection assay, a 25x dilution of the 5000x SYPRO Orange stock (Invitrogen) was prepared in water. For each well, 5 μL of this diluted SYPRO Orange and 45 μL of the refolding sample was added. The plate was covered with sealing tape and centrifuged at 600 g for 1 min to remove any air bubbles. A BioRad CFX96 RT-PCR machine was programmed with a 3 min equilibration time at 22 °C. Subsequently, the plate was heated from 22 to 90 °C with stepwise increments of 0.5 °C per 30 s, followed by the fluorescence reading optimized for SYPRO Orange at 485 nm (Excitation) and 530 nm (Emission) after each step. The 200 μL refolding sample prepared allows for the refolding experiments to be analyzed by DGR at four different time points, aiding the identification of the optimal incubation time for the refolding process.17. Briefly, samples from the up-scaled refolding step were concentrated to a maximum of 1 mg/mL and spun to remove any precipitation . SYPRO Orange (Invitrogen) was prepared by diluting 2.5 μL of the stock solution in DMSO into 500 μL of the concentrated refolded protein sample. For each well in the 96-well plate, 5 μL of protein/SYPRO Orange solution was diluted with 45 μL of the buffer screen formulation. Then the plate was centrifuged at 600 g for 1 min before the start of the assay to remove any air bubbles. The TSA analysis was performed as described above.The subsequent step uses a TSA analysis to screen for dialysis conditions to remove refolding agents. 2.5 mL of denatured protein sample (~12.5 mg) was added dropwise into 50 mL of the refolding buffer identified above, under continuous stirring. The mixture was incubated under stirring for a period of time (as identified from the time points tested in the previous screen). The optimal buffer for subsequent purification and/or concentration could then be identified using a more standard thermal shift assay approach (DSF/TSA)After identification of a suitable dialysis buffer, scale-up refolding was repeated and the refolded sample was dialyzed against the dialysis buffer identified. The dialyzed sample was then concentrated to 20 mL using an Amicon 6050 concentrator and further concentrated by a Vivaspin 20 centrifugal concentrator (Sartorius) at 2,000 RCF at 4 °C.The concentrated proteins were purified via SEC using a Superdex 200 10/300 or HiLoad 16/60 Superdex 75 column  pre-equilibrated with the identified dialysis buffer using an NGC liquid-chromatography system (Bio-Rad). Where necessary, the molecular masses of the proteins in solution were determined using static light scattering (SLS). A miniDAWN TREOS from Wyatt technology was connected in line with the NGC system. Proteins were concentrated to 2 mg/mL before injection.22.Our refolding buffer screen is composed of a “primary pH screen” Fig.  and a “s29. This screen covers a pH range from 4.0 to 10.0 (the pH of MMT buffer was 4.0–9.0). Importantly, we use 4 different buffer systems: MIB buffer, CHC buffer, MMT buffer and single buffers. These buffer systems are composed of several reagents, ensuring pH stability over a large range. The different buffer systems not only allow us to explore the effect of pH, but also the impact that different buffer compositions may have on the protein refolding process. The initial DGR screen  contains a variety of buffer systems in combination with 100 mM NaCl. Similarly, the second part (row E–H) contains these buffers combined with 0.4 M L-arginine hydrochloride, the most commonly used aggregation inhibitor to suppress aggregation and enhance protein refoldingeen Fig.  focuses een Fig. , which fThe optimal condition found in the primary pH screen was used as a standard composition to prepare a secondary additive screen, by diluting the additives into the identified conditions of the primary screen. Conditions A1-A9 are used to analyze the effect of 3 different sugars: glucose, sucrose and lactose at three different concentrations. Conditions A10-A12 contain 3 different detergents, which can bind to protein surfaces to prevent aggregation. Conditions B1-B8 contain 2 different chaotropic agents (0.5–2 M): guanidine-HCl and urea, which are commonly used for dissolving protein inclusion bodies and may promote refolding at lower concentrations. Glycerol and PEG  are commonly used protein stabilizers and are screened in conditions B9-C12. Conditions D1-D12 compare the effect of different concentrations of arginine (0.4–1 M) and 3 amino acids . Conditions E1-E12 are included to analyze protein refolding in the presence of NaCl/KCl over a wide concentration range (50–1000 mM). This allows exploration of whether low or high ionic strength will contribute to protein refolding process. Several metal salts with different valences are contained in conditions F1–F9. EDTA was also included in this group to investigate the refolding process in the absence of divalent metals. The buffer found in the primary pH screen in triplicate was contained in F10–F12 to ensure that the results from the assay are robust and reliable. G1–G12 is used to evaluate the effect of 12 different salts. Finally, two different reducing agents (β -ME and Dithiothreitol (DTT)) in several concentrations are included in H1-H12.31. HA-RBD is located within residues 63–286 of the influenza virus H1N1 and has a molecular weight of approximately 25 kDa, containing 4 cysteine residues. A synthetic gene corresponding to A/H1N1/2009 influenza virus hemagglutinin was cloned into the pETM11 vector32 and expressed in E. coli BL21* (DE3) cells. The inclusion bodies were denatured in Tris-HCl buffered 8 M urea solution (pH 8) at a protein concentration of 5 mg/mL. The denatured inclusion bodies were first submitted to the pH refolding screen. The results showed that pH and the presence of arginine play a key role in the refolding process. Firstly, when refolding experiments were performed over a pH range in single buffers  is one of the major surface proteins of the influenza virus and plays an important role at the beginning of the viral cycleers Fig. , Row H, ers Fig. . Furtherers Fig. . Finallyers Fig. . As the This buffer was then used as a standard addition in follow-up refolding experiments using the secondary additive screen Fig. . These eOur results also showed that refolding time has a substantial impact on the refolding process Fig. . When th17, was used to identify an dialysis buffer for the removal of refolding agents. From this screen, four key factors were found to stabilize the refolded protein: 50 mM Tris buffer at pH 8, 200 mM sodium chloride and 10 mM sodium citrate . A second DSF-based screen (TSA buffer screen), similar in approach to that used in other laboratoriesate Fig. . SurprisP21, with unit-cell parameters a = 39.5 Å; b = 73.7 Å; c = 73.7 Å and α = 90°; β = 100.72°; γ = 90°  in the space group 90° Fig. 33. The sion Fig. .33. A synthetic codon optimized gene encoding human IL-17A was also cloned into a pETM11 expression vector and expressed in E. coli BL21* (DE3) cells. The inclusion bodies were denatured in a buffer containing 20 mM Tris pH 8.5, 6 M guanidine-HCl, 100 mM NaCl, 5 mM EDTA and 10 mM DTT. From the first pH refolding screen, it is clear that pH and choice of buffer system have a substantial effect on the refolding of IL-17A. Results showed that IL-17A only gave a clear melting transition signal at pH 9.5 , produced by Th17 cells, has been implicated in the pathogenesis of various autoimmune diseases such as rheumatoid arthritis9.5 Fig. . At this9.5 Fig. . In the Refolding time was also found to play an important role in the refolding of IL-17A Fig. . Three tAs a result of this analysis, 50 mM CHES at pH 9.5, 100 mM NaCl, 0.4 M arginine were selected as the refolding buffer. A subsequent TSA assay demonstrated that IL-17A was more stable in MES buffer (pH 6) than CHES Buffer (pH 9.5) Fig. . In addi34. By adding 5 mM cysteine and 0.5 mM cystine to the refolding buffer described above, we were surprised to see a clear transition curve when the protein was incubated in refolding buffer for only five min  expression vector  was expressed in E. coli BL21* (DE3). The inclusion bodies were dissolved in a buffer containing 20 mM Tris pH 8.5, 6 M Guanidine-HCl, 100 mM NaCl, 5 mM EDTA and 100 mM DTT. The refolding screen was first conducted at room temperature, however, no clear transition curves was shown by DGR. Then the refolding was further explored at 4 °C. In sharp contrast with IL-17A, MDM2 showed transition signals in nearly all conditions of the buffer systems at a lower temperature  treatmenture Fig. . Based oure Fig.  showed ture Fig. . Subsequ2 was shown to stabilize the protein. As predicted, MDM2 shows improved stability in this buffer . Correct folding of MDM2 is indicated by size-exclusion chromatography and strong binding to a known inhibitor of the MDM2:p53 protein-protein interaction  was cloned into a pET21b expression vector  and expressed in E. coli Rosetta 2(DE3) pLysS. The inclusion bodies were denatured in buffer containing 50 mM MES (pH 8.0), 10 mM Na-EDTA, 1 M urea, 6 M guanidine-HCl and 1 mM DTT. As shown in Fig. The inhibitory receptor ligand of programmed death receptor 1 (PD-L1), which plays an important role in the maintenance of peripheral immune tolerance, has become a very promising target for the treatment of melanoma and other types of cancerD of 0.5 µM.The results also showed that the refolding incubation time has an influence on the refolding process. From Fig. . We obseE. coli that are sequestered to the insoluble fraction of the cell has become a major bottle neck in the protein structure analysis and therapeutic drug development. Expression in other systems such as yeast, insect cells, mammalian cells and cell-free systems may overcome folding problems and produce biologically active proteins that contain post-translational modifications, but expression in these systems often gives lower yields of the recombinant proteins and the cost is relatively high for large scale protein production. In this context, recovering biologically active proteins from bacterial inclusion bodies is attractive because of the high protein expression yields and low cost. However, there is no universal procedure that satisfies all protein folding requirements to date. In particular, the refolding process is highly limited by the lack of reliable biophysical methods to rapidly detect the formation of correctly refolded protein. In this assay, we established a systematic refolding procedure with the help of the DSF/DGR technique that allows for the detection of the protein refolding in an efficient manner. In addition, the refolding buffer screens we designed allow for the exploration of a wide pH range, different buffer systems, time, temperature as well as a large number of chemical additives in a high throughput manner.In the post-genome era, the large proportion of proteins overexpressed in It is well known that pH plays an important role in protein solubility, underscoring the necessity of exploring a broad pH range for our generalized primary refolding screen. Based on our primary pH screen results, it is clear that we can establish an effective refolding pH for all four proteins investigated. By comparing the DGR signal of our samples, we can also conclude that the effect of the pH on protein refolding is protein-specific. MDM2 showed a very broad refolding pH range, HA-RBD and PD-L1 showed a narrow pH range while IL-17A could only be refolded at a single defined pH.Although pH is important, it is not the only variable to consider in our primary refolding screen. We also introduced four different buffer systems to the primary screen. The advantage of using different buffer systems was demonstrated by our results. In each case, differences in the type of buffer can be observed. In particular IL-17A could only be successfully refolded following our approach in the CHC or CHES buffers. These results strongly indicate that the reagents composing the buffer system can have an impact on the refolding process and are an essential parameter to screen.M of IL-17A became significantly higher. Our screen allows the examination of the effect of arginine in combination with different buffer system and is shown to be another essential parameter in successful refolding. Arginine was selected for our screen based on a literature survey that demonstrated that it is the most frequently used “helper” molecule in refolding. While we do not currently explore the use of other frequently used “helper” molecules (such as the detergent Non-Detergents Sulfobetaines (NDSB)), these could be added to the protocol with an expansion of the primary screen and an associated increase in sample/experimental time.In the presence of arginine, the fluorescence transition signals of HA-RBD and PD-L1 were significantly increased and the T39. Finally, by deconvoluting the effects of pH, ions and additives we can achieve a more efficient screening of the complex chemical space that governs protein refolding.We have also designed a secondary additive screen that consists of different types of protein stabilizers. By screening these additives the refolding yield of HA-RBD could be increased. As the combination of aggregation inhibitor with protein stabilizer has been reported since 1990s, we believe that it is a very promising combinatorial approach to increase refolding yieldOur refolding procedure also allows for detection of the refolding signal at different time points. Our data shows that this can be an essential parameter to monitor. In our study, HA-RBD had the optimal refolding efficiency when the refolding time was lower than 1 h and the protein melting transition signal disappeared after 6 h. IL-17A and MDM2 showed the opposite effect: a weak DSF signal was found at 3 h, while the transition signal increased with time and the highest refolding signal was found at 15 h. A signal for PD-L1 was only observed when the protein was refolded for 15 h. These results indicate that time is also an important factor in refolding efficiency and that initially poor refolding results may not be indicative of the ultimate refolding efficiency in any given buffer.Our data also confirm that the addition of redox couples can be a critical factor in successful refolding. In two cases (IL-17A and PD-L1) refolding could be detected as soon as 5 minutes incubation in the presence of the “correct” redox couple. However, in both cases successful refolding could be achieved over a longer incubation time in the absence of the redox couples. This indicates that the redox state of the solution may play a role in the rate of refolding of these proteins, but is not necessarily essential. However, we cannot exclude that the redox state is essential for the final stability of the refolded protein.2 also helped. Again, CaCl2 was not identified to be a major contribution to the refolding of MDM2, as judged from the results of the additive screen. Additionally, the optimal pH for stabilizing IL-17A was found to be at pH 6, rather than refolding pH of 9.5. Based on our results, we concluded that while the refolding buffer can refold the protein into its native state, it does not necessarily mean that the refolding buffer is optimal for long term-protein stability. Therefore, screening for a protein stabilization buffer screen is critical after refolding the protein. It is possible that a buffer condition that supports the transition from an unfolded to a folded state (eg. by stabilizing an intermediate state) may act in a destabilizing manner on a folded for exactly the same reasons. Thus, we believe that many refolding protocols could be significantly improved by assaying for buffers that stabilize the final refolding state and that the combination of DGR and TSA provides a rapid mechanism to perform these analysis.After refolding, it was necessary to remove the “helper molecules”, such as arginine, that might affect the measurement of the protein concentration or other downstream experiments. However, one problem we normally encountered was that the refolded protein is frequently prone to aggregation after the sample was dialyzed to the refolding buffer without the “helper” molecules. This is potentially due to the sample containing a sub-fraction of mis-folded protein or the samples being partially refolded. Indeed it appears from our data that buffers optimal for protein refolding are not necessarily ideal for subsequent sample stability. Hence, we suggest that the simple step of performing a TSA buffer screen to determine an ideal stabilizing buffer for the refolded protein can dramatically improve the overall yield of protein from a refolding protocol. Interestingly, we found that the best pH and additives for refolding generally have differences with the buffer that is optimal to stabilize the refolded protein. For HA-RBD, sodium citrate was shown to stabilize the refolded protein after the arginine was removed and that concentration of HA-RBD to over 0.5 mg/mL was not possible in the absence of sodium citrate. However, citrate was not observed to have a significant effect on the refolding of HA-RBD, as judged from the DGR signals. MDM2 was more stable at pH 8, rather than in the refolding buffer at pH 8.5 and 1 mM of the divalent metal CaClet al. We have further expanded their approach to include an analysis of critical factors, such as time, redox couples, buffer composition and a secondary TSA to increase the chances of a successful refolding experiment.Our data further demonstrates the utility of DGR in establishing refolding conditions as pioneered by Biter


DFNA5 pathogenic splice-site variations and a novel benign frameshift variation to further support the gain-of-function mechanism of DFNA5 related hearing impairment, targeted genes capture and next generation sequencing were performed on selected members from Family 1007208, 1007081 and a sporadic case with sensorineural hearing loss. Reverse transcriptase polymerase chain reaction was conducted on the proband from Family 1007208 to test how the splice-site variation affects the transcription in RNA level. A novel heterozygous splice-site variation c.991-3 C > A in DFNA5 was found in Family 1007208; a known hotspot heterozygous splice-site variation c.991-15_991_13delTTC was identified in Family 1007081. Both the splice-site variations were segregated with the late onset hearing loss phenotype, leading to the skipping of exon 8 at RNA level. In addition, a novel DFNA5 frameshift variation c.116_119delAAAA was found in the sporadic case, but was not segregated with the hearing impairment phenotype. In conclusion, we identified one novel and one known pathogenic DFNA5 splice-site variation in two Chinese Families, as well as a novel DFNA5 frameshift variation c.116_119delAAAA in a sporadic case, which does not the cause for the hearing loss case. Both the two pathogenic splice-site variations and the nonpathogenic frameshift variation provide further support for the specific gain-of-function mechanism of DFNA5 related hearing loss.To report two  DFNA5 is the fifth locus of ADNSHL and the related gene is DFNA5 . The numerous ADNSHL related genes variations types include nonsense, missense, splice-site, insert and deletion (InDel) and copy number variation (CNV) and so on. For DFNA5 (MIM #600994), only 6 splice-site variations have been reported to be pathogenic for hearing loss  in exon 7 has been identified in a Japanese family, which was also unlikely to be the disease-causing gene variation, as another pathogenic POU4F3 variation was identified in the family affected members11.Autosomal dominant non-syndromic hearing loss (ADNSHL) accounts for approximately 20% of non-syndromic hereditary hearing loss, following by autosomal recessive non-syndromic hearing loss. To date, 37 ADNSHL related genes have been identified and more than 60 loci have been located , we provide further evidence for the hypothesis that the mechanism of DFNA5 associated hearing loss is the gain-of-function.In this present study, by identifying a novel disease-causing For Family 1007208, a total of 22 family members, including 10 clinically affected and 12 unaffected individuals were ascertained in this study Fig. .Figure 1Three cases and eight normal members were ascertained from Family 1007081 Fig. . All of For the sporadic case, who was 12 years old when she first visited our clinic, no family history was described in this core family Fig. . She hadMFI2/DFNA5/NSUN5/TECPR1/MLLT3/CNOT1/AKAP8) were reserved. The same bioinformatic analysis was performed on Family 1007081 and the sporadic case.For 1007208, after the filtration of common and low frequency variations, there were 51 high confidence potentially harmful variants located on the coding region which were shared by two cases but not in normal control. Combining the expression information from MGI database, we select the genes that can be detected in inner ear. Then 7 variants located on 7 genes , DFNA5 is also predicted to be a candidate gene for age-related hearing loss (presbyacusis) since DFNA5 variation related hearing loss showed similar phenotypic with presbyacusis. To investigate the association of DFNA5 with presbyacusis may provide insight into the presbyacusis mechanism. In addition, DFNA5 was also proposed to be related to noise induced hearing loss in a case-control study13.The hearing impairment in all of the affected members in Family 1007208 and 1007081 involved high frequency or the whole frequency. The severity of the ten affected individual in Family 1007208 and the three patients in Family 1007081 revealed no obvious tendency of the progression with aging. Like the phenotype of the family with c.991-2 A>G, the severity and the audiogram type of the affected members in this study are also variable intrafamiliallyDFNA5 related hearing loss are concentrated in Europe and Asia, among the previous reported eleven families which have identified DFNA5 splice-site variations, only two of them originated from Europe, while all of the other nine families come from Asia  all of these splice-site variations, though different on DNA level, they all lead to identical effect on mRNA level and protein level in part of the studies; 2) it is not the haploinsufficiency mechanism like other ADNSHL related genes  DFNA5 knock-out mice had no significant hearing impairment, though mutant mice showed the additional fourth row outer hair cells14; 4) Mutant DFNA5 leads to cell death when transfected into mammalian cell lines (HEK293T and COS-1) and yeast cells15. Except for splice-site variations, no other variation types have been reported to be disease-causing for hearing impairment. In previous study, a frameshift variation c.640insC has been proved to be not pathogenic due to it was not segregated with the hearing loss phenotype10, the frameshift variation c.116_119delAAAA in this study is also not disease-causing according to the current evidence we got.To date, a total of seven variations of ns Table . All of DFNA5 was identified in a Dutch ADNSHL family in 19981, numerous studies have focused on it, both relating to hearing loss and involving in cancer. DFNA5 is expressed in the cochlear hair cells and is a regulator of apoptosis cell disassembly. The molecular mechanisms of DFNA5 related hearing loss and cancer are totally different. DFNA5 variations, which lead to hearing loss, are expected to increase apoptosis, while for DFNA5 involving in cancer, DFNA5 loses its capacity to reduce apoptosis9. The function of DFNA5 protein is unclear, which is proposed to play a role in apoptosis and in a p53-regulated response to DNA damage. Neither mice nor zebrafishes are ideal model for clinical phenotype of DFNA5 related hearing loss, though they have expression of the DFNA5 homologous gene. In a patient with DFNA5 pathogenic variation, histopathology of the inner ear showed the loss of inner and outer hair cells and severe degeneration of the stria vascularis and spiral ligament throughout the cochlea16. In human cell lines with mutant DFNA5, MAP-kinase-related activity was up-regulated, inhibition of which could partially attenuate the mutant DFNA5 induced cell death17.Since DFNA5 splice-site variation c.991-3 C > A and a known recurrent DFNA5 splice-site variation c.991-15_991_13delTTC in two independent families, which were co-segregated with the post-lingual autosomal dominant hearing impairment phenotype. No other pathogenic variations or modifier genes were identified. RT-PCR of DFNA5 expression revealed that affected cases had less than half expression than normal control, which may play a gain-of-function role in hearing impairment. In addition, we also identified a frameshift variation in DFNA5 gene, which was not segregated in the family, further supporting the gain-of-function mechanism. However, the mechanism of the hearing loss and the specific variation-induced pathological changes in vivo remain unclear, even little information is available for humans, many more experiments and variations are needed to decipher their genetic code.In conclusion, we reported a novel The study was approved by the Committee of Medical Ethics of Chinese PLA General Hospital. Written informed consents from all the participants in the family were obtained. The methods were performed in accordance with the approved guidelines.18. Caloric testing was performed on some patients to obtain data on semicircular canal function, and cVEMP was tested with tone burst stimulus type (500 Hz) and 100 dBnHL stimulus intensity. Some patients who had tinnitus were estimated by tinnitus assessment. HRCT was performed on the proband of Family 1007208 to verify whether the family members had other complications other than hearing disorders.Two hearing loss families, including a five-generation family Family 1007208) with 141 members and a four-generation family (Family 1007081) with 11 members segregating ADNSHL, and a sporadic case without family history were ascertained from the Chinese PLA Institute of Otolaryngology, Chinese PLA General Hospital . Whole exome sequencing and target region sequencing were performed on these three pedigrees. Using Nimblegen SeqCapEZ Exome v3.0 kit and Illumina HiSeq. 2000 platform, we obtained the whole exome sequencing of sample II-1, II-3 and II-4 from family 1007208. II-1 and II-4 are cases. II-3 is a normal control. Coverage of exome region was 99.10% and mean depth was 102.95X. The other pedigrees 1007081 and the sporadic case used target region capture panel containing 127 hearing loss known causative genes and Illumina HiSeq. 4000 platform, which were conducted according to the previous procedures12.After sequencing and quality assessment, we aligned the reads onto the human reference genome hg19 (NCBI build 37.1) by software BWA (version 0.7.10) and SOAP aligner/SOAP2 (version 2.21). Then we use GATK (version 3.3–0), Samtools (version 0.1.19), SOAPsnp (version 1.03) and Platypus (version 0.7.2) to indentify SNPs and Indels. To obtain rare variations, common variants and low frequency variants in the 1000 Human Genomes Project database, HapMap Project database, ExAC database, EVS database and BGI in-house databases were excluded (0.5%). All remaining variants were considered to be rare variations and were annotated using Ensembl VEP, OMIM, MGI, Gene Ontology, HGNC gene annotation database and so on. Then we reserved harmful variations located on coding region including missense, nonsense, frameshift, indel and splice-site variations  and analyzed using an ABI 3700XL Genetic Analyzer .Total RNA from three patients and one normal control of Family 1007208 were extracted from the white cells of the whole blood samples using the cell RAN kit according to the standard protocol , and RNA concentration and purity were determined on a NanoDrop spectrophotometer. Synthesis of cDNA was carried out following the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific). RT-PCR was performed by using Takara SYBR Premix Ex Taq (Tli RNaseH Plus), the two alleles were amplified separately three times using the same quantity of the same template. TaqMan 7700 Sequence Detection System was used to monitor in real time the increase in fluorescent signal. Primers for cDNA sequencing were: forward primer: 5′- GCTGCGCATGGGATATCTTC -3′; reverse primer: 5′- TTCAGGGGAGTCAAGGTTGG -3′ (product size: 509 bp).Supplementary file


Borrelia burgdorferi, the causative agent of Lyme disease. The level of OspC varies dramatically among B. burgdorferi strains when cultured in vitro, but little is known about what causes such variation. It has been proposed that the difference in endogenous plasmid contents among strains contribute to variation in OspC phenotype, as B. burgdorferi contains more than 21 endogenous linear (lp) and circular plasmids (cp), and some of which are prone to be lost. In this study, we analyzed several clones isolated from B. burgdorferi strain 297, one of the most commonly used strains for studying ospC expression. By taking advantage of recently published plasmid sequence of strain 297, we developed a multiplex PCR method specifically for rapid plasmid profiling of B. burgdorferi strain 297. We found that some commonly used 297 clones that were thought having a complete plasmid profile, actually lacked some endogenous plasmids. Importantly, the result showed that the difference in plasmid profiles did not contribute to the ospC expression variation among the clones. Furthermore, we found that B. burgdorferi clones expressed different levels of BosR, which in turn led to different levels of RpoS and subsequently, resulted in OspC level variation among B. burgdorferi strains.Outer surface protein C (OspC) is the most studied major virulence factor of  Borrelia burgdorferi, is a spirochete that has a dual-membrane cell envelope. Unlike other Gram-negative bacteria that have lipopolysaccharide (LPS) on the outer membrane, B. burgdorferi outer membrane lacks LPS and contains numerous lipoproteins. These outer surface lipoproteins are differentially expressed and play key roles in host adaptation during its enzootic cycle between ticks and mammals  complex includes B. burgdorferi sensu stricto (s.s.), B. afzelii, and B. garinii. B. afzelii, and B. garinii are found outside of the United States. B. burgdorferi s.s. are highly heterogeneous with significant variation in virulence and endogenous plasmid content. B. burgdorferi s.s. strain B31, which was isolated from a tick collected on Shelter Island  sample of a Lyme borreliosis patient , low pH (pH 6.8–7.0), and high cell density , ospC expression in strain 297 can be easily achieved by directly cultivating the spirochete at 35 or 37°C without temperature shift. The level of OspC in 297 strain can be readily visualized as one of the most dominant bands in the protein profile when separated on Coomassie stained gel. This advantage has contributed in part, to the discovery of the first regulatory pathway in B. burgdorferi, the Rrp2-RpoN-RpoS pathway. In this pathway, the two-component system Hk2-Rrp2 activates the σN–σS (RpoN-RpoS) sigma factor cascade, leading to the production of σS (RpoS), which in turn, controls expression of many virulence genes including ospC and many other genes  medium with 2.5% agar]  by centrifugation at 8,000 g for 10 min and washed two times with PBS (pH 7.4) at 4°C. Pellets were suspended in SDS buffer containing 50 mM Tris-HCl (pH 8.0), 2% sodium dodecyl sulfate (SDS), and 10 mM dithiothreitol (DTT). Cell lysates (5 × 107 cells per lane) were separated by 12% SDS-PAGE and stained with Coomassie blue or transferred to nitrocellulose membranes . Membranes were blotted with monoclonal antibodies against FlaB, RpoS, and BosR . General guidelines for primer design were as follows: (a) Primers should be at least 20 nucleotides but not longer than 28 nucleotides, to minimize the possibility of primer-dimer formation and unspecific binding; (b) CG nucleotides should be distributed equally on both ends; (c) All PCR products should differ in size by at least 20 bp in order to allow efficient separation by gel electrophoresis. See primer sequences in Tables Multi-alignment among all 19 endogenous plasmid sequences of B. burgdorferi (as DNA template), 2.5 μl of 10 × Ex Taq Buffer, 2.5 μl of 10 × dNTP Mixture, 1 μl of Ex Taq DNA polymerase, and 2 μl of 12.5 × primer mix. Given that each primer pair has different amplification efficiency, the concentrations for each primer pair used in the primer mix were adjusted based on the PCR result. For the primer mix to amplify linear plasmids (mixLp), 222 μl of 12.5 × mixlp primer stock includes12 μl of 50 μM primer stock for lp25, lp54, lp28-4, 15 μl for lp38, and 10 μl for lp28-1, lp28-3, lp28-5, lp28-6, lp17, lp36. For the primer mix to amplify circular plasmids (mixCp), 210 μl of 12.5 × mixcp primer stock includes 15 μl of 50 μM primer stock for cp32-3, 10 μl for cp26, 10 μl for cp32-1, cp32-4, cp32-5, cp32-6, cp32-9, cp32-11, cp32-12. The PCR reaction conditions were as the following: (1) 1 cycle of initial denaturation at 94°C for 5 mins; (2) 40 cycles of amplification including denaturation at 94°C for 30 S, annealing at 54°C for 1 min, and extension at 68°C for 1 min; (3) 1 cycle of final extension at 68°C for 5 min. PCR products were separated using 3% metaphor agarose gel  in TBE buffer  by running 100 V for 1 h and then 80 V for 2 h.PCR was performed by using TaKaRa Ex Taq kit . Twenty five microliters of optimized PCR reaction includes 100 ng of boiled whole cell lysate of B. burgdorferi strain 297 contains at least 10 linear and 10 circular plasmids ranging from 17 to 54 Kb. The published plasmid sequences for strain 297 include 19 plasmids . Since the sequence of lp25 of strain 297 is not available, the primer pair for lp25 of strain 297 was designed based on the bptA gene sequence from lp25 of strain B31  the difference in plasmid contents may contribute to the difference in OspC production between AH130 and PL133.Given that certain gene(s) on endogenous plasmids can affect OspC levels of ospC expression, and absence of lp28-5 leads to high OspC level. Given that the parental clone 297 contains mixed clones with OspC high or low phenotypes, one cannot draw conclusion from parental 297 that the presence of lp28-5 can also have high OspC level. Thus, we investigated whether absence of lp28-5 leads to high OspC level by screening 297 clones isolated from semi-solid agar plate that lack lp28-5, and identified three such clones   Figure . While t) Figure . This daospC expression variation among strain 297 clones, we examined the upstream regulators that govern ospC expression. OspC has a RpoS-type promoter which is controlled by RpoS .In addition, rpoS expression in B. burgdorferi, but the mechanism remains unknown. Recently, it was reported that BosR undergoes autoregulation .XX, YY, XY, YL conceived and designed the experiments; XX, JD, and TL performed the experiments; XX and TC analyzed the data; XX, YL, and XY wrote the paper. XX and YY contributed equally to this work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.