Childhood asthma and allergies are particularly prevalent diseases. Our objective is to identify respiratory and allergic phenotypes from birth to 6 years of age, and to explore their environmental determinants, especially those related to the home environment. Data on respiratory and allergic health outcomes and domestic environmental exposure were collected for 935 mother–infant pairs from a longitudinal mother–child cohort based on mothers, included before 19 weeks of gestation in Brittany between 2002 and 2006. Information was obtained by self-administered questionnaires completed by parents at inclusion, delivery, and when the child was 2 and 6 years old. Kml3D clustering was used to describe profiles of children who shared similar trajectories of symptoms as phenotypes. Association with environmental determinants was estimated by polytomous logistic regression. Five phenotypes were identified: a reference group characterized by low symptom levels (31.1%), a transient cough phenotype (36.5%), an eczema/cough phenotype (12.3%), a wheeze/cough phenotype (11.8%), and finally a mixed phenotype (8.0%). The wheeze/cough profile was associated with postnatal exposure to glues used in renovation activities (aOR 2.3 [1.2–4.7]), and the mixed phenotype with postnatal exposure to paint (aOR 2.1 [1–4.5]). The phenotypes observed showed some consistencies with those seen in previous studies. Some exposures associated with respiratory/allergic phenotypes observed in this study are avoidable. If confirmed by further research including interventional trials, home-based environmental counseling could be a possible prevention target for primary care professionals. Asthma is the most frequent chronic disease in childhood and represents a major burden for patients, their families, and society.2The prevalence of childhood asthma and allergies varies greatly worldwide:3 To date, only three studies have collected longitudinal data for multiple 12-month profiles of respiratory and allergic symptoms.6 Rancière et al.4 simultaneously observed profiles of wheezing, dry night cough, eczema, and rhinitis symptoms from ages 0 to 4 in 2522 children from the PARIS (Pollution and Asthma Risk: an Infant Study) birth cohort. Two transient (rhinitis or wheeze) and two persistent (cough/rhinitis or dermatitis) phenotypes were identified, with a low symptoms reference group. Panico et al.6 analyzed wheezing and proxies for atopy (eczema and/or hay fever ever) in 11,632 children from the Millennium Cohort Study at ages 3, 5, and 7 and obtained a 4-trajectory clustering: a group with low symptom levels, another with low wheeze and high atopic symptoms, a third with high levels of both wheeze and atopic symptoms, and finally a group with high levels of wheeze only. Belgrave et al.5 studied wheeze, eczema, and rhinitis at ages 1, 3, 5, 7, and 11 in 9801 children from the ALSPAC  and MAAS (Manchester Asthma and Allergy Study) birth cohorts and identified eight developmental profiles: no disease, atopic march, persistent eczema and wheeze, persistent eczema with later-onset rhinitis, persistent wheeze with later-onset rhinitis, transient wheeze, eczema only, and rhinitis only.Asthma and allergic disorders commonly appear in infancy, but their pathogenesis and interplay remain incompletely understood.7 Worldwide variations in prevalence rates of asthma and allergies call attention to the potential role of the environment, as genetic differences alone cannot explain this heterogeneity.1 The literature has identified numerous avoidable determinants for respiratory and allergic disorders in childhood, in particular, the indoor environment,10 because young children usually spend most of their time at home.12 The objective of this study was to describe the joint trajectory of asthma, eczema, and rhinitis symptoms at three time points, at ages 1, 2, and 6 years, in a population-based cohort conducted in Brittany (France) from 2002 to 2006. We explored the associations of these phenotypes with some avoidable indoor environmental factors in early life.The development and expression of asthma/allergic disorders depend on gene–environment interactions.The cluster analysis, included 935 of the 3323 eligible mother–child couples from the PELAGIE  mother–child cohort Fig. .Fig. 1Flp < 0.001), had higher education level (p < 0.001), and were less often nulliparous (p = 0.008) or a smoker at inclusion (p < 0.001) , and 69.8% had at least some post-secondary education. At inclusion, 22.0% smoked, and 14.7% drank some alcohol. Most of the children were full-term (96.8%) and born by vaginal delivery (83.2%); 51.3% were boys  questionnaire,17 that follow-up bias is likely to be small.Another limitation is attrition. The questionnaires were self-administered, sent by the postal service, and lengthy (>1 h for completion) due to the broad research scope covered by the cohort. Each of these factors may have discouraged participation and induced bias in association estimates. Because the outcomes measured in this paper do not concern behavioral variables, we believe, in accordance with Greene et al.6 Comparison appears thorny; not all studies used only the ISAAC questionnaire,5 they considered different variables for phenotype identification, and health outcomes were not measured at the same time points. As Pinart et al.18 recently underlined, efforts to standardize the reporting of allergic disease phenotypes in cohorts are needed. However, similarities are apparent, especially for phenotypes. Our wheeze/cough phenotype was exclusively associated with physician-diagnosed asthma and similar to the transient wheeze phenotype identified by Rancière et al.4 Likewise, two of the studies5 identified a group with high levels of eczema symptoms and low levels of other disorders, strongly associated with physician-diagnosed eczema.4 Similarities also exist between the persistent sensitization-associated cough/rhinitis phenotype in Rancière et al.4 and our mixed phenotype: cough and rhinitis were predominant, moderate wheeze and eczema symptoms remained, and it was associated with all the physician-diagnosed conditions tested .Three previous studies have used unsupervised techniques to study multiple longitudinal respiratory- and allergy-related symptoms and to identify phenotypes.20 Our wheeze/cough phenotype was associated with early-life exposure to glue, and Rancière et al.4 observed associations between transient wheezing and postnatal exposure to new particle board furniture. Our mixed phenotype showed associations with early exposure to alkyd/acrylic paints used for interior home redecoration. Glues, particle board furniture, and paints can emit chemical compounds that, inhaled or by direct skin contact, can induce adverse health effects. Early exposure to chemicals used for indoor renovation activity is suspected of harmful effects, including the development of asthma21 and eczema.22 We did not, however, measure these compounds, and their effects are not clearly understood.The prenatal and early postnatal period is an important window of vulnerability for developing immune and respiratory systems, which are sensitive to environmental chemicals.23 It was studied in two of the papers cited above6 and observed by Rancière et al.6 in two of their phenotypes, rhinitis and dermatitis. A lack of statistical power due to our smaller sample might explain the absence of association in our study.The causal relation between postnatal exposure to ETS and respiratory outcomes in children is well established.24 More recent focus on the specific use of bleach during pregnancy has shown no association with wheezing at age 1,25 while a cross-sectional study found 10–13-year-old children living in homes cleaned with bleach at least weekly less likely to have asthma, eczema, or sensitization to aeroallergens.26 No phenotypes were associated with exposure to pets at home, whose respiratory/allergic health effects on children are currently the subject of debate.27 Surprisingly, children from our wheeze/cough phenotype were less likely to sleep on a mattress supposed to be laden with house dust mite (HDM) than the reference group. Rancière et al.4 observed a positive association between their dermatitis phenotype and the presence of a used mattress (older than 3 years at birth). Reverse causation due to the purchase of a new mattress in symptomatic children explaining our surprising association is improbable because it concerned only 19 children. HDM are thought to be implicated in the development of allergic asthma in children at high risk,28 with a nonlinear relation: both very high and very low exposure to HDM are probably protective against asthma and HDM sensitization at age 5.29 We treated the age of the mattress as a surrogate for HDM exposure in early life, but we cannot quantify the actual exposure, nor compare our sample to a population at high risk for asthma/allergy.Exposure to bleach was not associated with any phenotype. Studies of household cleaning products and their effect on asthma/allergy in children are scarce and conflicting. Maternal exposure to a set of domestic chemicals including bleach has been associated with persistent wheezing in offspring at age 3.We do not believe that phenotypes identified represent clinical entities that clinicians should relate to. The development and expression of asthma/allergic disorders depend on gene-environment interactions. From the point of view of the primary care giver, if one focuses on potential prevention targets, to date, genetic predisposition to respiratory/allergic diseases is inaccessible to intervention. This paper aimed to describing respiratory/allergic symptoms in the first 6 years of life. It was quite correctly in line with the currently admitted concept of allergic march. We generated hypotheses on the potentially harmful effect on respiratory and allergic health in children aged 0–6 of some chemical exposures that are directly related to parental behavior (renovation and redecoration) and thus avoidable. If confirmed in observational studies and interventional home-based trials, environmental counseling by primary care givers, because of their close relationship to families and potential knowledge of domestic behavior, could be particularly useful in prevention strategies for respiratory and allergic diseases in childhood.31 is a population-based mother–child cohort that enrolled 3421 women in pregnancy (before 19 weeks’ gestation) in Brittany between 2002 and 2006. Data were collected at birth and when the child was 2 and 6 years old. We studied a subgroup of 935 mother–child pairs for whom respiratory and allergic data were available at both follow ups .32 assesses four respiratory/allergic symptoms: wheezing, eczema, rhinitis, and cough. Wheezing was measured at ages 1, 2 (2 years questionnaire), and 6 (6 years questionnaire). At age 1 and 2, wheezing was defined as at least one episode of whistling in the chest or an asthma attack. Children not reported to wheeze but with physician-diagnosed asthma were also considered wheezers. Wheezing or whistling in the child’s chest and/or a wheezy sound there during or after exercise during the last 12 months defined wheezing at age 6. Eczema was defined at ages 1, 2, and 6 by a positive answer to the question: “Has your child ever had an itchy rash which was coming and going in the last twelve months?” Rhinitis symptoms were assessed for the first 2 and the sixth years of life and defined by positive answers to ISAAC questions: “Has your child ever had a problem with sneezing, or a runny, or a blocked nose when he/she DID NOT have a cold or the flu?” and “In the past 12 months, has this nose problem been accompanied by itchy-watery eyes?” The PELAGIE questionnaire did not include any specific item to assess child’s cough at the 2-year follow-up. Cough during the first 2 years of life was defined by a positive answer to the question: “Since birth, has your child ever had bronchitis or bronchiolitis?” At age 6, cough was the occurrence of dry night cough in the past 12 months.The ISAAC questionnaireTo confirm phenotypes, we assessed parent-reported comorbidities such as otitis media at age 2 or ever awakening due to breathlessness or respiratory discomfort and hay fever or other allergic rhinitis at age 6. They were also described against physician-diagnosed asthma, eczema, and food allergies as at the 6-year follow-up we also asked the parents whether a physician had ever diagnosed those disorders.We assessed indoor environmental factors known or suspected to be involved with respiratory and allergic symptoms.At age 2, we assessed: ETS, defined as maternal and/or paternal consumption of at least one cigarette per day at home since birth (yes/no); whether child spends time in rooms cleaned with bleach (yes/no); renovation activities in the dwelling since birth that used glues or either alkyd or acrylic paints (yes/no); usual home heating, defined by individual heating by at least one of the following: gas/fuel oil/wood or chimneys (yes/no).At age 2, we assessed: pets at home, that is, at least one of the following: cat/dog/rodent/bird/farm animal (yes/no) since birth; daily airing of the child’s bedroom (yes/no); and HDM exposure (yes/no), which was evaluated indirectly by the age of the child’s mattress considered old if purchased at least 3 years earlier.33 designed to study the joint longitudinal evolution of several variables over time. This method applies the k-means algorithm on longitudinal data . Moreover, several outcomes/symptom trajectories are considered simultaneously to compute interindividual distances. Each individual is represented by a matrix with his/her levels of different symptoms (lines) at different time points (columns). The multidimensional Euclidean metrics was used to calculate interindividual distances (Euclidean norm of the distance matrices). As the k-means algorithm requires predefining a number of clusters, we ran the k-means algorithm 20 times to obtain, respectively 3, 4, and 5 clusters. These numbers of clusters, based on an a priori choice were reasonable regarding our sample size and expected interpretable clinical phenotypes.6 The choice of the optimal clustering was based on the Davies Bouldin cluster validity index.34 For each clustering (different numbers of clusters), a proximity indicator reflecting the average within-cluster distance/between-cluster distance is computed. The optimal clustering minimizes this proximity indicator. The resulting clusters were then interpreted as clinical phenotypes based on the mean symptom trajectories observed in each cluster and our medical expertize. The complete cluster analysis was performed with the R software package KmL3D.33Phenotype identification relied on the joint temporal evolution of wheezing, cough, eczema, and rhinitis symptoms. We used the KmL3D unsupervised clustering method2: yes/no) and age at inclusion, parity, maternal smoking habits at inclusion (at least one cigarette per day: yes/no), family history of allergy , child’s gender, vaginal delivery (yes/no), and exclusive breastfeeding for at least 3 months (yes/no). A sensitivity analysis, included preterm children and those with a birth weight <10th centile expected for gestational age. The prevalence of these two characteristics was low and thus not appropriate for use in the adjusted regression model. Logistic regression analyses were performed with SAS 9.4, and results were expressed as odds ratios and their 95% confidence intervals (95% CI). The level of statistical significance was 0.05. Our data were analyzed in 2017.Associations between phenotypes and covariates were estimated with univariate polytomous logistic regression models. We used single random imputation by the mean or median when missing values for covariates did not exceed 5.0%. The final regression model included the lifestyle factors associated with phenotypes in the univariate analysis and all the environmental factors, regardless of the strength of their association with phenotypes. Multivariate regression models were adjusted for maternal body mass index (BMI ≥ 30 kg/mFurther information on research design is available in the Reporting SummarySupplementary Information


Cotton is an important economic crop in worldwide. It produces fiber for the textile industry and provides cottonseeds with high-quality protein and oil. However, the presence of gossypol limits the utilization of cottonseed. Two pairs of cotton near isogenic lines (NILs) with different pigment glands, i.e., Coker 312 vs Coker 312 W and CCRI12 vs CCRI12W, exhibit different gossypol contents. The glandless traits of Coker 312 W and CCRI12W are controlled by recessive and dominant genes, respectively. However, knowledge regarding the genomic variations in the NILs is limited. Therefore, the NILs genomes were resequenced and the sequencing depths were greater than 34×. Compared with the TM-1 genome, numerous SNPs, Indels, SVs, and CNVs were discovered. KEGG pathway analysis revealed that genes with SNPs and Indels from the recessive NILs and genes with Indels from the dominant NILs shared only one enriched pathway, i.e., the sesquiterpenoid and triterpenoid biosynthesis pathway, which is relevant to gossypol biosynthesis. Expression analysis revealed that key genes with variations that participate in the gossypol biosynthesis and pigment gland formation pathways had different expression patterns among the dominant, recessive glandless and glanded plants. The expression levels in the glanded organs were higher than those in their NILs. Altogether, our results provide deeper insight into cotton NILs with different pigment glands. Gossypium spp.) is a leading economic crop worldwide. In addition to producing nature fiber for the textile industry, cotton also provides a large quantity of cottonseeds containing high-quality protein and oil1. It is estimated that every kilogram fiber yield is associated with 1.65 kg cottonseeds, which contain approximately 21% oil and 23% protein2. However, the nutrient-rich cottonseeds cannot be used directly due to the presence of gossypol stored mainly in the pigment glands, which are toxic to human beings and monogastric animals3. Therefore, low-gossypol cotton cultivar breeding was always regarded as an important task. Two approaches may serve this purpose, i.e., either reducing the gossypol content directly or eliminating the pigment glands to reduce the gossypol content indirectly. Recently, some gossypol biosynthesis genes have been characterized and used to reduce the gossypol content in cottonseeds5. On the other side, as one of the most important secondary metabolites, gossypol plays important roles in cotton’s growth and development8, and is used as a medicine with anti-bacterial10, anti-cancer13 and male-contraceptive properties15.Cotton  such as glanded and glandless NILs is limited.The next-generation sequencing technology has become more advanced in recent years. The genomes of gl2 and gl3. Similarly, CCRI12W is a glandless NIL derived from CCRI12 by backcrossing, but the glandless trait is mainly controlled by one dominant gene named GoPGF or Coker 312 is an outstanding glanded upland cotton cultivar with a high content of gossypol, while Coker 312 W, which has a low gossypol content, is a recessive glandless near isogenic line (NIL) derived from Coker 312 by backcrossing, in which the glandless trait of Coker 312 W is controlled by two the recessive genes gl2 and gl3. Coker 312 W was derived from a cross between Coker 312 and Z5629, and the glandless hybrid was selected from every generation of this cross for 16 generations. Hai-1 is a glandless cultivar, and the glandless trait is controlled by the dominant gene, gl2 and gl3, while CCRI12W is controlled by Two pairs of NILs, i.e., Coker 312 vs Coker 312 W and CCRI12 vs CCRI12W were used in the experiments. Z5629 is a glandless cultivar. and the glandless trait is controlled by a pair of recessive genes, i.e., Slits of cottonseeds were examined under a stereo microscope  attached to a desktop computer. The images were captured by a stereo microscope.The cottonseed samples were dried at 30 °C to constant weight for 2–3 days, and then ground with a grinder to a powder. Standard gossypol solutions were prepared by dissolving 0.01 g HPLC-grade gossypol in 10 mL of acetone. Standard gossypol solution of 0.010, 0.050, 0.100, 0.200, 0.500, 0.800, 1.000 and 2.000 mL were added into 10 mL volumetric flasks and adjusted to the calibration using acetone to prepare the different levels of gossypol solutions. Similarly, 0.1 g of sample was suspended into 2 mL acetone. In addition, the sample solutions were maintained in an ultrasonic bath for 45 min. Subsequently, the suspensions were filtered through quantitative filter paper followed by filtration with a 0.45 μm syringe filter . The sediment was washed three times with acetone. Then, the extraction was adjusted to 10 mL with acetone.3PO4 . The injection volume was 10 μL, and the flow rate was 1.0 mL/min. The UV detector was set at 238 nm, and the temperature was set at 25 °C. The samples were measured in triplicates.The HPLC analysis was performed using an Agilent 1100 , equipped with an auto-sampler and UV detection. A C18 column   was deployed as the stationary phase. The mobile phase consisted of Methanol/0.2% H30. The DNA samples were randomly sheared to a size of ~350 bp. The DNA fragments were end-repaired, and an ‘A’ base was added to the end of the double strand break DNA. Then, DNA adaptors  were ligated to the above products. An agarose gel was used to separate these products and excised for purification. The modified and purified DNA fragments were enriched by PCR amplification with paired-end PCR primers . Qubit 2.0  was used to measure the concentration of the library. The libraries were diluted to 1 ng/μl, and the insert size of the libraries was detected by an Agilent Bioanalyzer 2100 . To ensure the quality of the libraries, quantitative RT-PCR (qRT-PCR) was used to accurately quantify the effective concentration of the libraries (>2 nM). Then, the libraries were sequenced on an Illumina HiSeq 2000 platform .Genomic DNAs were extracted from the fresh leaves of the cotton plants using a standard cetyltrimethyl ammonium bromide (CTAB) protocol26 using Burrows-Wheeler Aligner (BWA)31. The compared results were calculated to remove the duplicated reads using SAMTOOLS32.The paired-end reads were processed to remove adaptors and low quality paired reads for quality control and filtration. The procedure to remove the low quality reads were as follows: reads with adaptor; reads containing more than 10% ‘N’s; reads with more than 50% bases having low quality value (Phred score ≤ 5). The effective reads were aligned against the reference genome sequence of the upland standard line TM-133 was used to detect the structural variations (SVs) based on the alignment results and the insert size. CALL in CNVnator34 was used to detect the copy number variations (CNVs). Annotations of all the genetic variants were performed by ANNOVAR35. To visualize the genetic variants, Circos plots were generated by Circos36.MPILEUP in SAMTOOLS was used to identify the single nucleotide polymorphisms (SNPs) and insertions/deletions (Indels). In order to reduce the detection error rate of SNPs, the SNPs with the supported reads number less than four or quality value less than twenty were filtered. CRESTTM 1st Strand cDNA Synthesis Kit . The primers used for qRT-PCR were designed with Primer 5.0 software. All primers are listed in Supplementary Dataset −ΔΔCt method.RNA samples were extracted from the samples using a Total RNA Extraction Kit . The first strand cDNA was synthesized using a PrimeScript37. GO items with corrected P-values less than 0.05 were considered significantly enriched by different genes.Gene Ontology (GO) enrichment analysis of genes containing differential SNPs and Indels was implemented by the GOseq R package38. Pathways with corrected P-value less than 0.05 indicated that different genes were significantly enriched in those pathways.Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of genes containing differential SNPs and Indels was performed by KOBAS software to test the statistical enrichment of different genesAll the sequence data for two pairs of near isogenic lines have been deposited in the NCBI Sequence Read Archive under accession number PRJNA542238.As the cottonseeds were crosscut, significant differences in the pigment glands were observed. The cottonseeds of Coker 312 and CCRI12 exhibited clearly visible pigment glands, while Coker 312 W and CCRI12W had no pigment glands -δ-C synthase gene that is also significant for gossypol biosynthesis. Previously, CAD1-A had been reported to be highly expressed in glanded cottonseed, while the expression level in glandless cottonseed was meager50. Similarly, we observed that CAD1-A had a high expression in glanded cotyledon and a low expression in glandless cotyledon, but the roots of both glanded and glandless plants exhibit significantly high expression. Moreover, another critical gene participating in the gossypol biosynthesis pathway, i.e., CYP706B1, has good potential for the manipulation of the gossypol content in cottonseed51. This gene was also significantly highly expressed in root of glanded and glandless plants. Therefore, these three genes related to gossypol biosynthesis all had an extremely high expression level in the root of the glanded and glandless cotton plants, implying that the root might have a strong ability to synthesize gossypol and that the glandless cotton root could also effectively synthesize gossypol even though pigment glands are not located all over the plant. The only dominant pigment gland formation gene, i.e., GoPGF, has been characterized by Madan et al.22. We also investigated its expression patterns in four organs of the glanded plant and found that the expression of GoPGF in all organs of the glanded plant was higher than that in the glandless plant. GoPGF expression in the root of glanded plants was higher than that in the leaf and lower than that in the stem, which is similar to previous reports. However, we found that its specific expression patterns were quite different between the glanded and glandless plants. The relatively similar expression levels in the four organs in the glandless plants and the considerably different expression levels in the glanded plants may be due to the existence of pigment glands including the density and size of pigment glands. Therefore, the dominant gene in pigment gland formation exhibited a highly different expression pattern between the glanded and glandless cotton plants.As gene expression can be affected by the position of SNPs and indels53. Sunilkumar et al. reported that they had successfully used RNAi to disrupt gossypol biosynthesis in cottonseed tissue by interfering with the δ-cadinene synthase gene expression. The present work based on the whole genomes of Coker 312, Coker 312 W, CCRI12 and CCRI12W provided lots of critical information for discovering genes controlling the biosynthesis of gossypol and the formation of pigment glands, which could be used as the candidate genes for genetic engineering breeding. With CRISPR/Cas9 system, we may use the discovered genomic information to develop the low-gossypol transgenic line and glandless transgenic line by knockout the critical genes in gossypol biosynthesis pathway, such as CYP706B1, and pigment gland formation pathway, such as Low gossypol cotton cultivar breeding was always regarded as an essential task for breeders, especially for the development of glanded plants with glandless cottonseed. Therefore, research investigating pigment glands is also urgent. Nowadays, as genetic engineering technology developed, RNAi and CRISPR/Cas9 system were applied to research on the outstanding cotton variety breedingNumerous DNA variations were identified in dominant and recessive glandless cotton NILs through re-sequencing technology. The comprehensive analysis of the variations revealed the different genes enriched in the ontologies and pathways related to the biosynthesis of secondary metabolites, which could provide a deeper insight into these four genomes. The expression profile of several genes related to gossypol biosynthesis and pigment gland formation revealed their significantly different expression pattern, which could be helpful for attaining a deep understanding of the mechanism of gossypol biosynthesis and pigment gland formation. Altogether, the large amount of sequenced data and related comprehensive analysis lay a foundation for uncovering the genes controlling pigment gland formation, which could be useful for cotton breeders developing new outstanding cotton germplasm with glandless seeds and glanded plants.Supplementary InformationSupplementary Dataset


Glaucoma is a neurodegenerative disorder characterized by the progressive functional impairment and degeneration of the retinal ganglion cells (RGCs) and their axons, and is the leading cause of irreversible blindness worldwide. Current management of glaucoma is based on reduction of high intraocular pressure (IOP), one of its most consistent risk factors, but the disease proceeds in almost half of the patients despite such treatments. Several experimental models of glaucoma have been developed in rodents, most of which present shortcomings such as high surgical invasiveness, slow learning curves, damage to the transparency of the optic media which prevents adequate functional assessment, and variable results. Here we describe a novel and simple method to induce ocular hypertension in pigmented rats, based on low-temperature cauterization of the whole circumference of the limbal vascular plexus, a major component of aqueous humor drainage and easily accessible for surgical procedures. This simple, low-cost and efficient method produced a reproducible subacute ocular hypertension with full clinical recovery, followed by a steady loss of retinal ganglion cells and optic axons, accompanied by functional changes detected both by electrophysiological and behavioral methods. A recent study estimates that approximately 76.0 million and 111.8 million people will be affected by glaucoma in 2020 and 2040, respectively3. Current therapeutic approaches are based on either clinical or surgical procedures to reduce high levels of intraocular pressure (IOP), one of the most consistent risk factors for the development and progression of glaucoma. However, approximately 45% of patients do not respond to these approaches and experience disease progression4. Therefore, understanding the complex, multifactorial pathophysiological features of glaucomatous neurodegeneration is required for the development of effective therapies to prevent or slow down the degenerative process, either independent of or in addition to lowering the IOP.Glaucoma, the leading cause of irreversible blindness worldwide, comprises a heterogeneous group of neurodegenerative disorders defined by the progressive functional impairment and death of the retinal ganglion cells (RGCs) and their axons8. Among several distinct rodent models of glaucoma, those based on either acute or chronic ocular hypertension (OHT) most closely resemble disease in humans. Spontaneous OHT has been well characterized in mouse lines such as the DBA/2 J and DBA/2NNia strains, often used as models of age-related glaucoma9. However, these mice develop the disease relatively late in life, which demands a significant time commitment for the experiments7. There is also considerable variability in the course of both the rise of IOP and glaucomatous damage among animals of the same age10 from distinct laboratory facilities. In addition, a recent longitudinal study of 118 DBA mice described problems related to the measurement of the IOP, functional evaluation and in vivo retinal imaging, due to complex anterior segment pathology11.Modeling glaucoma in laboratory animals is a well-established strategy to both identify disease mechanisms and develop new neuroprotective approaches. In particular, rodents are low-cost, easy to handle, have a short life-span, can be genetically manipulated and the anatomy and physiology of their ocular structures are relatively similar to humansExperimentally induced, unilateral OHT carries advantages over genetic models of glaucoma, such as better control over the optic nerve and retinal pathology, as well as the availability of the fellow eye as an internal control. In addition, the bigger size of the eye globe and related anatomic structures in rats, as compared with mice, facilitates surgically induced OHT. Here we describe a novel and simple method to induce ocular hypertension in pigmented rats, based on low-temperature cauterization of the whole circumference of the limbal plexus, a vascular component of the post trabecular pathway of aqueous humor drainage, immediately upstream to the episcleral veins. Full-circle limbal plexus cautery (LPC) induced subacute OHT, with progressive glaucomatous neurodegeneration that extends beyond the period of elevated IOP. This method is easy to perform, low-cost, efficient, and leads to the loss of ganglion cells within a relatively short time after a single surgical procedure.In pigmented rats, access to the limbal plexus from the surface of the globe is noninvasive Fig. . FollowiCauterization of the limbal plexus induced transient ocular hypertension Fig. , charact13  is a cytoskeletal protein widely used as a marker of reactive changes in astrocytes and Müller glial cellsAs a preliminary assessment of optic nerve damage following LPC, we examined RGC axons in toluidine blue-stained semithin sections, at approximately 1 mm from the posterior pole of the eye Fig. . Photomiin vivo functional evaluation of the retina in most operated animals. Pattern electroretinogram (PERG) has been traditionally used both clinically and experimentally as an effective and noninvasive test to study RGC function15. As early as D3, when IOP was still elevated but only a mild and localized RGC loss was observed in the retina, there was a robust lowering of the amplitude of the steady-state PERG in experimental eyes compared with their respective controls To examine how LPC-induced OHT affects rat visual behavior, the optomotor response (OMR) was examined through observation of the animal’s ability to track a moving patterned visual stimulus  at various spatial frequencies. The visual threshold at distinct intervals following OHT showed a profile similar to that of electrophysiological tests, with reduced visuomotor acuity at D3, then a return to baseline levels at D7 and D14, and a significant decrease at D30  intracameral injection of viscoelastic substances or microbead particles (pre-trabecular)19 is invasive, with high risk of corneal damage or inflammation, as well as intraocular infections; moreover, some injected substances may reduce the transparency of the optic media, which precludes functional analysis; (2) laser scarring of the TM and/or photocoagulation of the limbal vasculature and episcleral veins (trabecular)21 require expensive equipment, are difficult to apply to pigmented animals due to high and variable absorption of laser energy, and is often accompanied by damage to the TM, corneal inflammation and ulceration, which impair functional assessment22; (3) Morrison’s procedure of injection of hypertonic saline into an episcleral vein (post-trabecular)23 requires extensive training, and leads to highly variable IOP24; (4) Shareef and Sharma’s episcleral vein cauterization (EVC)25 is also invasive, requires a slow learning curve to access the deep episcleral veins behind the rectus muscles, may lead to venous congestion because the targeted deep venous plexus actually comprises the vortex veins, which drain the entire globe including the choroid27, and leads to highly variable period of OHT, ranging from 2 weeks to 6–7 months as described by differing authors29.Widely used experimental models of glaucoma have been classified as pre-trabecular, trabecular or post-trabecular, using the trabecular meshwork (TM) as an anatomic reference30. The subacute elevation of the IOP was followed by clinical recovery in about 90% of the rats and enabled the functional evaluation of the retinas in vivo as early as the third day after surgery in roughly three-quarters of the operated animals. The LPC procedure has several advantages over other methods: (1) it is technically easy and noninvasive, with a quick learning curve; (2) it does not require expensive equipment; (3) it allows both in vivo electrophysiological and behavioral examination; (4) the reproducibility and success in generating glaucoma reduces the numbers of animals necessary for experiments, thus complying with increasing ethical concerns regarding the use of animals in basic research. Notwithstanding, structural damage in the ganglion cell layer was detected with relatively short latency, with loss at two weeks of about one third, and at one month of about half of the retinal ganglion cells, as well as extensive axon loss at the optic nerve. Therefore, it allows for studies of glaucomatous neurodegeneration, following either short- or mid-term experimental designs.The currently described method of limbal plexus cauterization (LPC) leads to a post-trabecular type of ocular hypertension, followed by retinal and optic nerve degeneration. This model is akin to human glaucoma secondary to increased drainage pressure of the aqueous distally to the trabecular meshwork, as seen in cases of Grave’s orbitopathy, cavernous sinus thrombosis, retrobulbar tumors and arterio-venous shunts31, many such patients undergo progressive visual loss despite adequate control of IOP with either topical hypotensive drugs or surgical approaches33. Gradual worsening of visual function and further structural loss in these cases most likely reflects secondary degeneration dissociated from ocular hypertension. Thus, the present subacute OHT model may contribute to a better understanding of the glaucomatous neurodegeneration to the benefit of patients34. The LPC-induced immediate IOP peak, to about twice the control value, is also akin to an acute angle closure crisis, marked by sudden IOP rise due to either complete or near complete anatomical blockade of the iridocorneal angle35, and the LPC model may help understand mechanisms of retinal degeneration due to the latter condition.LPC all around the cornea induced immediate OHT, which peaked at 1–3 days after surgery , as well as the significant correlation between cell loss and pressure load Fig. , rule ou44, as shown in other animal models47 as well as in human patients49. Nevertheless, controversies about the actual contribution to glaucoma of neuroinflammatory responses, inclusive of other glial cell types, are still unsettled50.The early appearance and progressive increase in GFAP immunostaining of glial cell processes, support the hypothesis that neuroinflammation is an early event of the pathophysiology of glaucomaAs soon as 3 days after LPC, the steady-state PERG recordings indicated an impairment of electrophysiological responses. At that time, experimental eyes were hypertensive and the numbers of normal looking axons were only slightly reduced, but RGC numbers were similar in both eyes. Following the return of IOP to basal levels at 7 days after LPC, PERG amplitude recovered slightly but remained significantly lower than in the fellow eyes. From this time onwards, the numbers of both Brn3a-labeled RGCs and optic axons, as well as PERG amplitude declined in synchrony.54. Moreover, decreased IOP in ocular hypertensive patients and mammals is associated with variable degrees of functional recovery57. Functional improvement associated with recovery of normal IOP levels may also be related to a recently characterized axogenic mechanism that produces transient enhancement of the excitability of mouse ganglion cells after induction of OHT58.The time course of PERG changes, as well as those of both FERG and OMR, suggest an immediate functional effect directly tied to increased OHT, followed by partial recovery and progressive functional loss dissociated from the subacute insult. It has been proposed that increased IOP, even without an established cell loss, is able to impair retinal function both in humans and in animal models of glaucoma59. The optomotor response is a compensatory reflex that requires functional communication between ON-OFF direction-selective RGCs and the nucleus of the optic tract, and has been widely used as a sensitive test to study visual function in rodents60. Given the selective involvement of a minor population of RGCs, as well as their restricted axonal projections, our result suggests that retinal damage caused by subacute OHT may also show cell-type specificity62. Future studies are required to confirm this hypothesis. Notably, an impact of ocular hypertension upon blood flow dynamics at the optic nerve head has been suggested as a major mechanism of initiation of glaucomatous damage, with ensuing compromise of RGCs energy metabolism63, and oxidative stress markers have been found in the serum, retina and aqueous humor of glaucoma patients64. Notwithstanding, distinct models of ocular hypertension that led to IOP levels similar to our study failed to find vascular changes or hypoxic damage65, and retinal ischemia caused by ocular hypertension was reported only at IOP levels varying from 110 mmHg to 140 mmHg67. Given that a threshold level of IOP required to induce retinal ischemia in rats has been estimated at higher than 60 mmHg69, even considering the depressing effect of general anesthesia70, the current average peak IOP of 24.8 ± 0.9 mmHg at post operative day one  were handled following the recommendations from the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. The experimental protocols were approved by the Ethics Committee on the Use of Animals in Scientific Experimentation from The Health Sciences Center, Federal University of Rio de Janeiro (protocol 083/17). The animals were housed in controlled conditions of temperature and 12-hr light-dark cycle (lights on at 6 am and off at 6 pm), with standard food and water Rats were deeply anesthetized with an intramuscular injection of a solution containing ketamine hydrochloride (75 mg/kg) and xylazine hydrochloride (5 mg/kg). After 5 minutes, the surgical plane of anesthesia was checked by toe pinch, the right eyelids were kept separated with the aid of a curved forceps, and the globe was gently pushed forward to help expose the limbal vasculature and allow topical anesthesia of the eye surface with ophthalmic proxymetacaine hydrochloride 0.5%. The animal was placed on slight lateral decubitus, and a low-temperature ophthalmic cautery  was used to cauterize the limbal plexus in a 360° approach around the cornea, visualized through a stereo microscope  at 40x magnification. Care was taken not to compromise the periphery of the cornea. Clinical signs of a successful procedure were the presence of small circular cauterization marks on the scleral limbus, the disappearance of blood along the limbal vasculature, and mydriasis in the experimental eye. After surgery, a drop of ophthalmic prednisolone acetate (10 mg/mL) was applied, and maintained in contact with the anterior surface of the right eye during approximately 40 seconds, after which it was replaced by an ophthalmic ointment of antibiotics (oxytetracycline hydrochloride 30 mg/g and polymyxin B 10.000 U/g).® XL, Reichert, Buffalo, NY)71 applied to the surface of the eye topically anesthetized with ophthalmic proxymetacaine hydrochloride 0.5%. The tip of tonometer touched the center of the cornea perpendicularly, just enough to move the globe slightly posterior26. Measurements were done immediately before and after the surgical procedure, then every day in the morning up to 14 days after surgery. An average of 12 to 20 individual readings were taken from each eye, possible “off” readings and those that resulted in instrument-generated averages with >10% error71 were discarded. The experimental eyes were examined under a magnifier, and clinical evolution was recorded of corneal transparency, limbal revascularization, chemosis and conjunctival hyperemia, or other intercurrences such as hyphema, corneal dystrophy or scleral instability associated with uveal prolapse.Rats were lightly anesthetized , and IOP was measured using a handheld tonometer , and the active electrode  was inserted carefully so as to avoid piercing the cornea, at the temporal periphery of the transitional zone74. The reference and ground electrodes were also stainless steel needles , positioned subcutaneously into the ipsilateral temporal canthus and the left hind limb, respectively. Signals acquired with the recording electrode were differentially amplified, digitized and processed using a MEB-9400K system . The waveforms were stored and analyzed using the software Neuropack Manager v08.33 .Both the flash electroretinogram (FERG) and the pattern electroretinogram (PERG) were recorded at 3, 7, 14 and 30 days after surgery. The animals were deeply anesthetized with intramuscular ketamine and xylazine , so as to decrease the chances of artifacts through involuntary muscle movements and other sources of noise during the exam. An additional injection of one half the initial dose was usually necessary during the exam, to maintain the animals properly anesthetized throughout the ERG assessment, which lasted approximately 90 minutes. The anesthetic agents used do not affect the amplitude of the responsesThe full protocol was recorded sequentially for either eye, beginning with the right  eye, and consisted of the FERG to evaluate the function of the outer retina (photoreceptors and bipolar cells), followed by the PERG to assess the function of the RGCs. After certifying the surgical plane of anesthesia, and with the animals adapted to the environmental light , the photopic FERG was recorded using a white strobe flash , with 10 ms duration and 20 J energy, generated by a xenon lamp at 20 cm from the stimulated eye, and 20 individual responses (1 Hz) were averaged. We analyzed the b-wave amplitude and implicit time, a component that reflects the function of the On-bipolar cells related to the cone pathway. The activity of cones was isolated through the flicker ERG, recorded after the photopic FERG with flash energy of 1.2 J and frequency of 15 Hz. Twenty individual responses were averaged, and the amplitude at the frequency of the stimulus was extracted off-line using a fast Fourier transform (FFT) algorithm programmed with Matlab software . Then, the rats were dark adapted for scotopic FERG recording. We recorded a 20 J single flash response, which elicited a mixed potential from both rods and cones (a-wave amplitude and implicit time) and their related On-bipolar cells (b-wave amplitude and implicit time), with a predominance of the rod pathway. We used a short scotopic protocol of 3 minutes in completely darkness, both to study the kinetics of retinal dark adaptation and to abbreviate the exam, as a measure to prevent the occurrence of cataract due to corneal damage induced by the active needle electrode. Signals were differentially amplified, digitized at a sampling interval of 100 µs, filtered  and stored for subsequent analysis.2), presented at an LCD monitor  positioned at 20 cm from the examined eye. The band-pass filter was set to 1 Hz–100 Hz, and signals were averaged 200–300 times to stand out from noise. Six distinct spatial frequencies were presented, in the following order for all animals: 0.018; 0.585; 0.073; 0.037; 0.292; and 0.146 cycles/degree (cpd). The amplitude of the stored sinusoidal waveforms was analyzed using a custom-made FFT algorithm programmed through Matlab language .The steady-state PERG amplitude gathers the component of the wave most likely associated with the RGC bioelectrical response (NII deflection of the transient-state PERG), generating a stable and reproducible sinusoid. The stimulus consisted of contrast reversal of a checkerboard with black and white squares alternating at 15 reversals/sec, with constant average luminance , one at a time, on top of a platform in the middle of the arena delimited by 4 computer monitors arranged in a quadrangle. Before starting the exam, the rats were allowed to habituate for approximately 2 minutes on the platform. The test was done in photopic conditions, while the monitors displayed the image of a virtual cylinder with vertical alternate black and white stripes, moving around the animal in a fixed rotation speed (12 degrees/seconds) and contrast (100%). The rats were watched by one observer (RLa) through a video camera positioned above the platform. The experimenter followed the freely moving rat’s head with a crosshair superimposed on the video image, which corresponded to the center of the virtual cylinder75. The OMR is a smooth tracking movement of the animal’s head and neck in concert with the rotation direction of the gratings, of which clockwise or counter-clockwise movements test the left or the right OMR-related visual pathway, respectively. The spatial frequency of the stimulus (cycles/degrees) was incrementally changed until the tracking movement couldn’t be noticed by the experimenter anymore. The highest spatial frequency that elicited a clear OMR was considered the threshold of visual acuity of the tested eye. The Optomotry® software was set on manual/separate mode, and each animal was examined in single time points (naïve testing)59. When an animal eventually dropped off the platform during the exam, it was immediately returned to the platform and the test was resumed69.The animals were positioned in an Optomotry76. Then, the retinas were immunolabeled with an anti-Brn3a primary antibody , followed by a donkey anti-goat polyclonal IgG secondary antibody, conjugated with Alexa Fluor® 555 , at RT. DAPI  was used for nuclear staining . The retinas were flat mounted in glass slides with the vitreous side up, and coverslipped in antifade mounting medium .The rats were euthanized by inhalation of carbon dioxide, the eyeballs were carefully removed and chemically fixed by immersion in 4% paraformaldehyde (PF4%) in phosphate buffer 0.1 M overnight at 4 °C. For RGC counting, retinas were dissected so that tissue topographic orientation was preserved, using the nasal caruncle and the choroid fissure as landmarks77, Brn3a+ cells were counted by an experimenter unaware of the source of the photos, and mean cell density was estimated from these counts for each retina.To estimate the density of RGCs, flat mounts were examined under a confocal epifluorescence microscope  using a Plan-Neofluar 40x/1.3 objective. For each quadrant of the retina, 8 photos were taken, of which 2 were from central retina (~0.9 mm from optic disc), 3 from mid-retina (~2.0 mm from optic disc) and 3 from peripheral retina (~3.7 mm from optic disc), to a total of 32 photos per retina. Using the FIJI software®, 4583; Sakura Finetek USA), and frozen with liquid nitrogen. Cryostat sections (14 µm thick) from the temporal hemi-retina were mounted on gelatin-coated glass slides and immunostained by overnight incubation at 4 °C with anti-GFAP primary antibody , developed with donkey anti-mouse polyclonal IgG secondary antibody, conjugated with Alexa Fluor® 555 . Sections were examined under an Axiovert 200 M (Zeiss) microscope with a 100x/1.3 objective. For each animal, GFAP+ processes crossing the inner plexiform layer were counted in a total of 9 fields from at least 3 distinct sections Representative images were acquired under a LSM 510 Meta (Zeiss) microscope using a Plan-Neofluar 40x/1.3 objective.For immunolabeling of astrocytes, eyeballs fixed overnight with PF4% were cryoprotected by immersion in sacarose 30% overnight at 4 °C, embedded in Optimal Cutting Temperature Compound , and postfixed for 1 h in 1% osmium tetroxide containing 0.8% potassium ferrocyanide and 5 nM calcium chloride in 0.1 M cacodylate buffer (pH 7.4). Segments were rinsed in 0.1 M cacodylate buffer (pH 7.4) and distilled water, and block-stained in 1% uranyl acetate overnight, dehydrated in a graded acetone series, infiltrated with Polybed 812 resin (Polysciences) and polymerized at 60 °C for 48 h.Semithin (500 nm) cross sections were cut in an RMC ultramicrotome and collected on slides, stained with toluidine blue, mounted with Polybed 812 resin, and imaged under a Zeiss Axioskop 2 Plus microscope (Zeiss). For quantitative analyses, 4 images at 100x magnification were taken systematically from the cross sections of each nerve. Images were processed with the software ImageJ, and the number of myelinated fibers was manually counted by the same person (C.A.A.).Statistical analysis was performed using GraphPad Prism 6.0c software. Data are expressed as mean ± SEM, except for Fig. Dataset 1


Staphylococcus aureus (MRSA) is a community- and hospital-acquired pathogen that frequently causes infections but also can survive on the human body asymptomatically as a part of the normal microbiota. We devised a comparative genomic strategy to track colonizing USA300 at different body sites after an initial infection. We sampled ST8 S. aureus from subjects at the site of a first known MRSA infection. Within 60 days of this infection and again 12 months later, each subject was tested for asymptomatic colonization in the nose, throat and perirectal region. 93 S. aureus strains underwent whole genome shotgun sequencing.USA300 methicillin-resistant S. aureus from the nose, throat and perirectal sites from 15, 11 and 15 of them, respectively. Twelve months later we isolated S. aureus from 9 subjects, with 6, 3 and 3 strains from the nose, throat and perirectal area, respectively. Genome sequencing revealed that 23 patients (ages 0–66 years) carried USA300 intra-subject lineages (ISLs), defined as having an index infection isolate and closely related colonizing strains. Pairwise distance between strains in different ISLs was 48 to 162 single nucleotide polymorphisms (SNPs) across the core regions of the chromosome, whereas within the same ISL it was 0 to 26 SNPs. Strains in ISLs from the same subject differed in plasmid and prophage content, and contained deletions that removed the mecA-containing SCCmec and ACME regions. Five strains contained frameshift mutations in agr toxin-regulating genes. Persistence of an ISL was not associated with clinical or demographic subject characteristics. We inferred that colonization with the ISL occurred about 18 weeks before the first assessment of asymptomatic colonization.Among 28 subjects at the initial sampling time, we isolated mec, loss and gain of other mobile genetic elements, and mutations in the agr operon.Clonal lineages of USA300 may continue to colonize people at one or more anatomic sites up to a year after an initial infection and experience loss of the SCCThe online version of this article (10.1186/s12866-018-1336-z) contains supplementary material, which is available to authorized users. Staphylococcus aureus is one of the most common human bacterial pathogens, causing skin and soft tissue infections (SSTI), bacteremia, osteomyelitis and other disseminated infections  was used to cross-validate SNPs predicted by Parsnp and to identify frameshifts and small indels. Plasmid content was found to be variable between strains (discussed below), and therefore not useful for comparisons across the whole dataset. One strain  fell outside of the USA300 subgroup and was therefore excluded from the rest of the study, leaving a total of 89 strains. Due to these exclusions , 2 patients were not represented by any samples, and we therefore concentrated our analysis on the 29 remaining patients, 28 of whom had an USA300 initial infection isolate. From the Parsnp alignment of the 89 strains, we extracted a core region of the USA300 chromosome of 2471 kbp (86%). The 14% of the chromosome excluded because it was missing in at least one of the genomes included SCCmec, ACME, the SaP15 pathogenicity islands, and the SLT and βC prophages [A whole genome alignment of all the newly sequenced USA300 genomes was performed using Parsnp v1.2  against rophages . Other rThe concatenated common aligned portion , members of an ISL isolated all shared < 30 SNPs in their core genome regions. We performed univariate testing  to determine if subject characteristics  or time 2 (12 months)  were retained prospectively for further study and underwent whole genome sequencing. Nose, throat and perirectal body sites were cultured at time 1, and S. aureus was isolated from 16, 15 and 17 patients, respectively. Approximately 12 months after the initial visit (time 2), we isolated S. aureus from 6, 3 and 4 subjects from nose, throat and perirectal sites, respectively  was lost during the course of the study, and we treated the remaining strains as outside of an ISL. Based on the phylogeny we removed six strains from ISLs because they were obviously phylogenetically unrelated to the other strains in the ISL. Five subjects  had colonizing strains from a different ISL than the initial infection isolate, suggesting colonization with more than one USA300 lineage. After removing these non-conforming strains, we were left with 23 subjects with an ISL that included an initial infection isolate and at least one other body site isolateOf the 23 patients with a USA300 ISL, 15, 11, and 15  at the time 1 (enrollment) visit had an an isolate from the same ISL in the nose, throat and perirectal area, respectively. Following up one year later , 6 (21%) patients still had a strain from the same ISL in the nose, and 3 (11%) each in the throat in the perirectal area . The ranges for each body site were: nose, 0–22 (median 4.5), throat, 0–19 (median 7) and perirectal 0–26 (median 3.5). As expected, within host analysis showed that there were a significantly greater number of SNPs within ISLs between the initial infection isolate and strains from time 2 (range 2–26: median 10) compared with time 1 (range 0–12: median 3) .The intra-ISL relationships were further analyzed by measuring the number of SNPs between each strain and the initial infection isolate Fig. . There wp < 7.7 e-13) , or at the 3 body sites sampled at time 2   Fig. a. There S. aureus changes in accessory gene content mediated by prophage, genome island insertion and plasmid transfer are important mechanisms for genome evolution. However, tracking these changes is problematic using short read shotgun data. Loss or gain of a single element might affect dozens of genes at once, and plasmids and prophages undergo frequent recombination and share common sequences, making identification difficult [In ifficult . NeverthS. aureus integrase (Sa1–7) [For prophages, we used the scheme of Goerke et al., who defined 7 distinct sequence types of  (Sa1–7) . Presenc (Sa1–7) . In addi (Sa1–7) , with thStaphylococcus spp. plasmid sequences downloaded from NCBI . In the Jamrozy et al. study, in contrast to our findings, 146/154 (95%) USA300 strains from SSTIs carried the pUSA300HOUMR backbone plasmids and 5% (8/154) carried pUSA03-like plasmids. Families of smaller (< 10 kb) plasmids were present in some of our strains. The cryptic pUSA01 plasmid was found in 57 (64%) strains; pC194-like plasmids [tetK similar to the pUSA02 plasmid [To examine plasmid content we used the combination of BLAST alignments against DNA sequences from the Gram positive replicon typing scheme of Lozano et al.  and 344 plasmids  were fou plasmid   were MSSA. For subject 1, the situation was reversed. The index infection isolate was MSSA, but the colonizing strains isolated were all MRSA. The most parsimonious explanation was loss of the genes in the ancestor of the initial infection isolate shortly after colonization of the subject. Neither subject 1 nor 4 was treated with a β-lactam antibiotic for the index infection.In subject 29, who was not treated with a β-lactam antibiotic for the index infection, the nose isolate had lost its ebp) with 27 mutations; this protein is also the largest in the genome . USA300HOU_0192 encoding a non-ribosomal peptide synthase (2397 aa), had the second most changes with 7 mutations. Most mutations were only found in one strain and more than 95% were found in 7 or fewer genomes. This was a result of the sampling strategy employed in the study, where a maximum of seven strains was taken from the same subject . Mutations that occurred frequently were those found only in the reference , in deeper branches of the tree, and those causing ciprofloxacin resistance.Of the 1882 SNPs in core regions, 1494 (79%) were in genes and of these, 1101 (73%) caused non-synonymous or premature termination changes. This high percentage of mutations affecting amino acid sequences is typical when comparing closely related bacteria before purifying selection has time to operate  and has We were interested to determine if there were any patterns in terms of the function of genes that acquired SNPs only in a certain body site, as it was possible that there was tissue-dependent selection that may act over multiple genes linked by common phenotype. We found 50, 190, 187 and 246 SNPs unique to nose, perirectal area, throat and initial infection isolate, respectively. Using PANTHER  we lookemec cassette, making them MSSA. Two strains isolated from the perirectal region of subject 20 were missing the ɸSa2 prophage carrying the PVL lukSF genes. These accessory genes are postulated to be important for SSTIs caused by USA300 .agr regulatory locus that would theoretically render them less capable of causing toxin-mediated tissue damage [agrC gene. Three of six strains from subject 11, on a monophyletic branch of the ISL phylogeny carried the same frameshift in the agrA gene encoding the regulatory protein, suggesting a single mutation event. This suggested that functional products of agr were not necessary for long-term survival on patients. Also, the mutant strains were isolated at both timepoints in all three body sites, thus demonstrating that S. aureus strains had migrated among anatomic sites on this subject.We also noted that 5 strains contained frameshift mutations in the e damage , 60 .In this study we showed that g et al.  sequenceg et al. . Our fing et al. , who fouS. aureus ISLs at different body sites over time is also novel in the literature. These data have therefore enabled us to perform a number of novel analyses on clonal variation within ISLs.There have been few prior studies of longitudinal carriage of MRSA with whole genome sequencing to assess persistence of clonal carriage. To the best of our knowledge, there have been no previous such studies in which multiple body sites have been tested along with genome sequencing of an initial infecting isolate. In addition, comparison of S. aureus mutation rate to be between 1.3–3.3 × 10− 6 SNPs per genome per year [S. aureus colonization. Other studies have come to different conclusions, pointing to particular anatomic sites as potentially more important for long-term colonization than others, for example the anterior nares or the oropharynx [For example, we found that only a median of 2 SNPs separated the ISL isolates from the predicted MRCA sequence Fig. a. The smper year , 63–66, opharynx , 68. Thimec elements. The latter two are particularly significant as they are considered central to the success of the USA300 MRSA clone, although naturally occurring deleted strains are not infrequently isolated [agr mutant strains, as have others who have studied S. aureus colonization [mec + and agr + strains.We did not identify a specific gene or locus that acquired an unusual number of SNPs suggestive of positive selection. With the number of genomes in our study, we had limited power to detect parallel mutations, especially those that may have been associated with adaptation specifically to the anatomic niches of the nose, throat or perirectal region. We did, however, encounter a significant number of genetic changes associated with movements of mobile elements during the history of colonization, including phage and plasmid gain and loss and loss of ACME and SCCisolated , 22, 69.nization , 59, 70.This study had a number of limitations. Due to sampling design we were constrained to sequencing one colony at each anatomic site/time point and could not estimate the extent of within-strain variation on a single host . In thisS. aureus asymptomatic, longitudinal colonization of humans. There are many unanswered questions related to the evolution of S. aureus during colonization, transmission and disease. We do not know how long an ISL persists on individuals nor how effective various classes of systemic antibiotics are in reducing colonization. We know that individual clones persist for years in households, but there may be expansion of a reservoir with frequent transfer of strains between individuals and fomites [S. aureus. It would also be invaluable to compare the S. aureus strain to the background skin microbiome in order to start to investigate the source of selective pressure to lose or gain genetic elements such as SCCmec and the selective pressure leading to persistence or loss of S. aureus colonization. Future research that address these questions will support the development of interventions to prevent S. aureus infections in susceptible people.We have used comparative genomics to sketch out some of the features of  fomites , 27. We S. aureus ISLs. We showed colonization of USA300 after an index USA300 MRSA infection of a patient for up to a year. Persistently colonizing strains could be found in all anatomic sites tested: nose, throat, and perirectal area. We showed that colonization by USA300, including MSSA and MRSA, was less prevalent when sampled more than a year after an initial infection. During the course of colonization there were changes in plasmid and prophage content and antibiotic resistance, including loss the SCCmec cassette to create MSSA strains. Using similar genome-based approaches but with more intensive sampling, it may be possible to define evolutionary constraints giving rise to bottlenecks emerging after exposure to antimicrobials and to determine the natural history of asymptomatic colonization of people by pathogenic S. aureus strain types as well as perhaps their risk of recurrent infection.Using genome sequencing we were able to plausibly define USA300 Additional file 1:Patient metadata. Breakdown of all metadata fields collected and summary of genetic results. Includes antibiotics administered to patient during the study period. (XLSX 36 kb)Additional file2:Core and recombinant regions of USA300 TCH1516. Sheet1 = list of core blocks identified by parsnp. Sheet2 = list of recombinant blocks in the core region identified by ClonalFrame (XLSX 59 kb)Additional file 3:Table S1. agr frameshifts. Figure S1. Provenance of strains sequenced in the study. Figure S2. USA300 ISL strains isolated from each body site. Figure S3. SNP frequency by genome. Figure S4. within ISL estimates for pangenome. Figure S5. Plasmid distribution. Figure S6. Phage distribution. (PDF 1040 kb)Additional figures and tables. Additional file 4:Antibiotic resistance phenotypes. Table of phenotypic antibiotic results for the initial infection isolates. (CSV 8 kb)


Khawia rossittensis . The process of spermatid formation is accompanied by the presence of ultrastructural characters not described in traditional models of spermiogenesis, e.g., apical electron-dense material, the two striated roots situated unusually opposite each other, branching of typical striated roots, an intercentriolar body comprising five electron-dense and four electron-lucent layers, rotation of both free flagella and flagellar buds to the median cytoplasmic process at 90°, and a complete proximodistal fusion. The synchronous rotation of both flagellar buds and growing free flagella is an evolutionarily linked pattern favouring the hypothesis that the Caryophyllidea are not ancestral but are secondarily derived from polyzoic forms. Electron tomography analysis has revealed a unique feature of two helicoidal tubular structures in the central electron-dense core of the axoneme of mature spermatozoon. These data provide new insights into the architecture of the 9 + ‘1’ axoneme, which is shared by male gametes of all trepaxonematan Platyhelminthes.Monozoic caryophyllidean cestodes, intestinal parasites of cyprinid fishes, represent a group of tapeworms with an unclear evolutionary history. As spermatology may provide phylogenetically important data, the spermiogenesis and ultrastructure of the mature spermatozoon have been investigated using an integrative approach combining transmission electron microscopy, cytochemistry and electron tomography in  This group apparently played a key role in the evolution of cestodes2 and represents a basal or nearly basal group of tapeworms; however, phylogenetic relationships within the order Caryophyllidea and its evolutionary interrelationships with the most relative group of tapeworms remain unsolved4.Caryophyllidean tapeworms are monozoic, i.e., without external and internal proglottidation, and contain only a single set of reproductive organs; there are 42 genera and 122 valid species distributed in four families7. Although the database of existing spermatological characters of cestodes has been considerably extended in the last decade8, substantial gaps in the knowledge on the spermatology of the most basal tapeworm groups persist9. Of the 12 species of the order Caryophyllidea that have been the subject of ultrastructural spermatological studies, 6 species belong to the family Lytocestidae, namely, Atractolytocestus huronensis10, Caryophyllaeides fennica11, Khawia armeniaca12, Khawia sinensis13, Lytocestus indicus14, Monobothrioides chalmersius15.Sperm characters provide especially important datasets useful for phylogeny16) has been analysed using electron tomography in a recent pilot study of the lytocestid caryophyllidean tapeworm Caryophyllaeides fennica11. The results showed for the first time that two tubular structures are present in the central axonemal electron-dense core in Trepaxonemata. The helicoidal nature of the centre of the complex axonemal core has been reported using electron tomography analysis in one-axoneme spermatozoa of the evolutionarily more derived tapeworm Nippotaenia mogurndae17. These interesting findings call for further detailed electron tomography studies of other caryophyllidean cestodes and cestode groups to elucidate the basic sperm structure of type 9 + ‘1’ in the Trepaxonemata.The basic sperm structure of the trepaxonematan type 9 + ‘1’ . This character also appears in other cestode groups, e.g., Amphilinidea26, Spathebothriidea28, Diphyllobothriidea32, Bothriocephalidea38, and Proteocephalidea39.Electron-dense material in the apical region of the zone of differentiation in the early stages of spermiogenesis has been observed in lytocestid tapeworm K. rossittensis in the present study has been reported in the lytocestid Caryophyllaeides fennica11, the caryophyllaeid Caryophyllaeus laticeps25, and the diphyllobothriidean Diphyllobothrium latum29. These spermatology findings may support the hypothesis based on molecular analysis that Caryophyllidea and Diphyllobothriidea are sister groups40. Another remarkable and unusual feature of Eucestoda is the branching of typical striated roots, where one typical root is branched into two or three arms. However, further observations are needed to elucidate the occurrence and/or importance of this unusual architecture of striated roots in cestodes.The atypical arrangement of the two striated roots in opposite direction during the early stages of spermiogenesis as described in 6. Its composition may be closely related to the evolution of cestodes. The remarkable variability of this cell structure has been recorded in caryophyllidean cestodes of the family Lytocestidae. Here, the ICB is either absent, namely, in Monobothrioides chalmersius10 and Atractolytocestus huronensis13, or present and may consist of a variable number of electron dense layers, i.e., one in K. armeniaca12, three in K. sinensis13 and L. indicus14, or five in K. rossittensis (present study). Whereas the ICB of most caryophyllideans contains three electron-dense layers25, five electron-dense layers have been detected in Archigetes sieboldi41 and in K. rossittensis (present study). The latter pattern has also been reported in some diphyllobothriideans30 and trypanorhynchs42.The intercentriolar body (ICB), a phylogenetically important character, is considered to be a plesiomorphic feature of the Eucestoda43. Subsequently, this feature was observed in some lytocestids13, caryophyllaeids41, capingentids23 and some evolutionarily higher non-cyclophillidean cestodes with one axoneme spermatozoa, i.e., tetraphyllideans44, tetrabothriideans45, or nippotaeniids17. The flagellar bud may represent a strongly reduced flagellum developed during the spermiogenesis of cestodes with two-axoneme spermatozoa, and consequently, the rotation of both free flagella and flagellar buds may indicate a derived stage of spermiogenesis in the Caryophyllidea17.The rotation of both free flagella and flagellar buds to the median cytoplasmic process at 90° has not been described in traditional models of the spermiogenesis of the Caryophyllidea46. An investigation of glycogen using a cytochemical method by Thiéry18 revealed the presence of glycogen granules in the cytoplasm of prenuclear, nuclear, and postnuclear regions in the mature spermatozoa of K. rossittensis. Similar results were reported in two other caryophyllideans, C. laticeps25 and C. fennica11. It should be noted, however, that intraaxonemal glycogen has not been detected in the mature spermatozoa of any caryophyllideans or Platyhelminthes with the 9 + ‘1’ trepaxonematan structure.Glycogen is a branched polymer of glucose residues that presumably serves as an energy reserve for the motility of spermatozoa and other activities essential to the fertilization of ovaK. rossittensis. A similar structure was described in the lytocestid C. fennica for the first time15. Virtual tomogram slices of both lytocestid cestodes showed the two central tubular structures, which may resemble two free parallel central singlet microtubules of the 9 + 2 axoneme of cilia and flagella, as described by Fawcett and Porter47 for the first time. On the other hand, the helical appearance of central doublet microtubules in cestodes  are urgently needed for the effective use of these new characters for the clarification of systematic and phylogenetic questions within the Platyhelminthes.Khawia rossittensis  were obtained from the intestine of prussian carp Carassius gibelio   captured from the Tisa River, Southeastern Slovakia. The fish were dissected using the standard methods described by Ergens and Lom50. All applicable institutional, national and international guidelines for the care and use of animals were followed and approved by the Animal Care and Use Committee of the Institute of Parasitology, Slovak Academy of Sciences, Košice (Slovakia). Tapeworms were cooled in a 0.9% NaCl solution and immediately processed for ultrastructural studies in the following way.Live adult specimens of 4 in 0.1 M cacodylate buffer for 2 hours at 4 °C, dehydrated in a graded alcohol series and embedded in Spurr epoxy resin. Semithin sections were cut with glass knives on an LKB Bromma 8880 ultramicrotome, stained with methylene blue and examined under a light microscope for the localization of the testes and vas deferens. Ultrathin sections were cut with a diamond knife on a Leica Ultracut UCT ultramicrotome, placed on copper grids and double stained with uranyl acetate and lead citrate. The grids were examined on a JEOL 1010 transmission electron microscope operating at 80 kV.The worms were cut into small pieces, fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 10 days at 4 °C, washed with 0.1 M sodium cacodylate buffer, postfixed in 1% OsO18, the periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) technique, using the following protocol: ultrathin sections were placed on gold grids, treated in 1% PA (20–25 minutes), washed with distilled water, processed in 1% TSC (40 minutes), washed with 10% acetic acid and distilled water, treated in 1% SP (30 minutes), and finally washed with distilled water and dried. Gold grids were examined on a JEOL 1010 transmission electron microscope operating at 80 kV.Visualization of glycogen granules in male gametes was carried out using the Thiéry method51. Tomogram reconstruction and model segmentation were performed with the IMOD software package52. The reconstructed tomogram with an isotropic voxel size of 0.9 nm was filtered for noise reduction by a 3D median filter and stored as an MRC file53. The contrast of virtual tomogram slices was enhanced by using an ImageJ plugin Enhanced Local Contrast (CLAHE) ).Ultrathin sections of 100 nm thickness were placed on 300 mesh copper grids (SPI), and staining was performed using alcoholic uranyl acetate and lead citrate both for 15 minutes. Au nanoparticles 10 nm conjugated with protein-A (BBI) as fiducial markers were placed on both sides and the surfaces were covered by carbon. We performed electron tomography as a dual axis in the range ±70° with a tilt step of 0.8° by means of a JEOL 2100F transmission electron microscope (TEM) equipped with a motorized tilt stage and a Orius SC 1000 Gatan camera automatically controlled by SerialEM software