Infantile-onset encephalopathy and hypertrophic cardiomyopathy caused by mitochondrial oxidative phosphorylation defects are genetically heterogeneous with defects involving both the mitochondrial and nuclear genomes.To identify the causative genetic defect in two sisters presenting with lethal infantile encephalopathy, hypertrophic cardiomyopathy and optic atrophy.We describe a comprehensive clinical, biochemical and molecular genetic investigation of two affected siblings from a consanguineous family. Molecular genetic analysis was done by a combined approach involving genome-wide autozygosity mapping and next-generation exome sequencing. Biochemical analysis was done by enzymatic analysis and Western blot. Evidence for mitochondrial DNA (mtDNA) instability was investigated using long-range and real-time PCR assays. Mitochondrial cristae morphology was assessed with transmission electron microscopy.OPA1 gene, which resulted in a marked loss of steady-state levels of the native OPA1 protein. We observed severe mtDNA depletion in DNA extracted from the patients’ muscle biopsies. Mitochondrial morphology was consistent with abnormal mitochondrial membrane fusion.Both affected sisters presented with a similar cluster of neurodevelopmental deficits marked by failure to thrive, generalised neuromuscular weakness and optic atrophy. The disease progression was ultimately fatal with severe encephalopathy and hypertrophic cardiomyopathy. Mitochondrial respiratory chain complex activities were globally decreased in skeletal muscle biopsies. They were found to be homozygous for a novel c.1601T>G (p.Leu534Arg) mutation in the OPA1 mutation and human disease. The fatal multisystemic manifestations observed further extend the complex phenotype associated with pathogenic OPA1 mutations, in particular the previously unreported association with hypertrophic cardiomyopathy. Our findings further emphasise the vital role played by OPA1 in mitochondrial biogenesis and mtDNA maintenance.We have established, for the first time, a causal link between a pathogenic homozygous  NDUFV2, COX6B1, and SCO2.1–3Mitochondrial disorders are genetically and phenotypically highly heterogeneous. Due to the limited coding capacity of the mitochondrial genome, the majority of structural and accessory proteins required for the proper assembly and functioning of the mitochondrial respiratory chain are encoded by the nuclear genome. Mutations in an increasing number of these critical nuclear-encoded genes have been identified over the past decade by next-generation sequencing. These advances have allowed the elucidation of the underlying genetic diagnosis in patients with complex phenotypes and also broadened the spectrum of clinical manifestations associated with specific disease-causing genes. Syndromes characterised by infantile-onset hypertrophic cardiomyopathy and encephalopathy are genetically heterogeneous, but mitochondrial dysfunction with impairment of mitochondrial oxidative phosphorylation has emerged as an important pathophysiological mechanism. Accordingly, these specific syndromes may be caused by mutations in various genes involving both the mitochondrial or nuclear genomes. Unsurprisingly, a number of nuclear genes causing isolated or combined mitochondrial respiratory chain defects have been identified in patients with infantile hypertrophic cardiomyopathy and variable forms of encephalopathy, of which recent examples include OPA1 mutations cause autosomal dominant optic atrophy (DOA), which is the most common form of inherited mitochondrial blindness with a minimum prevalence of 1 in 25 000 in the general population.OPA1 gene. Homozygous OPA1 mutations have not been reported previously and the unexpected association with cardiac involvement further broadens the genotypic and phenotypic spectrum associated with syndromic DOA.Heterozygous The subjects of this study are two sisters of Arab Muslim origin and their parents are entirely healthy first-degree cousins with no visual or neurological complaints. Patient I-1 was born prematurely at 35 weeks gestation and failure to thrive was a problem since birth. At 2 months of age, she was also noted to have truncal hypotonia that was replaced by multiple episodes of hypertonia and opisthotonic posturing. Furthermore, she had a weak cry and abnormal eye pursuits. An initial echocardiography and ophthalmological examinations performed at that time were unremarkable. Metabolic investigations revealed elevated serum alanine and lactate levels, but a normal cerebrospinal fluid (CSF) lactate concentration. Disease progression was marked by a persisting failure to thrive with severe neurodevelopmental delay, but brain MRI was normal. The patient subsequently developed a progressive, non-obstructive, generalised hypertrophic cardiomyopathy, in the absence of overt heart failure. The patient died at 10 months of age following an apnoeic episode.Patient I-2 was born at term following a caesarean section due to breech presentation. Two days later, she developed generalised hypertonia and opisthotonic posturing, and feeding was inadequate due to poor suckling. Initial echocardiography showed mild thickening of the left ventricular myocardium. Serum and CSF lactate concentrations were both elevated. Brain MRI performed at 1.5 months of age was normal and no abnormal lactate peaks were detected on functional spectroscopic imaging. Due to gastro-oesophageal reflux and persisting failure to thrive, a gastrostomy tube and Nissen funduplication procedures were performed at 3 months of age. The disease course was further complicated by progressive, hypertrophic, non-obstructive cardiomyopathy, profound neurodevelopmental retardation, hypotonia with significant muscle wasting and sensorineural deafness. There was no nystagmus, ptosis or limitation of eye movements. Ophthalmological examination at 6 months of age revealed mild pallor of both optic nerves. Visual electrophysiology revealed severely attenuated visual evoked potentials, consistent with primary retinal ganglion cell pathology, and the electroretinogram responses were significantly reduced, indicative of more global retinal degeneration. The patient died at 11 months of age following an apnoeic episode while sleeping.c oxidase-deficient muscle fibres. Enzymatic activities of the five respiratory chain complexes and citrate synthase (CS) activity, a mitochondrial matrix enzyme marker, were determined in isolated muscle mitochondria by spectrophotometric assays. This analysis revealed global decrease in all the respiratory chain complexes when normalised to CS activity in both patients, with complexes I and IV being the most affected (A muscle biopsy was performed at 8 months of age (patient I-1) and at 1 month of age (patient I-2). Routine mitochondrial immunohistochemical staining was apparently normal with no cytochrome affected  and compOPA1 gene, predicting the substitution of a highly evolutionary conserved leucine at position 534 with arginine  as the only mitochondrial-targeted gene that could be functionally relevant. This previously unreported OPA1 variant affects the GTPase catalytic domain  and it was predicted to be disease causing by Mutation Taster biotool  and SIFT  prediction softwares. Both parents were heterozygous for this OPA1 variant. They did not report any visual difficulties and a comprehensive neuro-ophthalmological evaluation was normal. Their optic discs did not look pale on dilated fundus examination and optical coherence tomography (OCT) imaging did not show any subclinical thinning of the retinal nerve fibre layer. The parents have two clinically unaffected sons who were 14 and 11 years old when last reviewed in clinic. The older son is heterozygous for the c.1601T>G OPA1 variant, whereas the other son only carries the wild-type OPA1 allele. This novel OPA1 variant was not detected in a cohort of 120 control subjects of similar ethnic origin and it was not present in a database including 63 000 exome sequences .Molecular genetic investigations for both sisters were initiated after obtaining the relevant written informed consents and local ethical review board approval. Because of parental consanguinity, genome-wide homozygosity mapping for both affected sisters was undertaken using Affymetrix Gene-Chip Human Mapping 50 K Xba Array and Gene-Chip Human Mapping 250 K NspI Array. We specifically looked for genes with proven or suspected mitochondrial function in light of the generalised oxidative phosphorylation defects detected in both siblings. We identified four large homozygous chromosomal stretches shared by both siblings (see online supplementary table S1), which harboured >150 genes, of which nine had putative mitochondrial involvement. Whole exome sequencing  and bioinformatic filtering highlighted a homozygous c.1601T>G variant in the OPA1 variant, western blot analysis was performed on homogenate muscle tissue from patient I–1 using standard monoclonal antibodies that bind to the C-terminus region of the OPA1 protein. There was a significant reduction in steady-state OPA1 protein level in patient I–1 compared with age-matched controls  and exon 8 .In order to further assess the pathogenicity of this c.1601T>G controls A,B. ThisLong-range PCR performed on homogenate DNA extracted from the muscle biopsy specimens of the two affected sisters did not reveal any multiple mitochondrial DNA (mtDNA) deletions (see online supplementary figure S3). However, there was evidence of significant mtDNA depletion with a 78% decrease for patient I-1 and a 78% decrease for patient I-2 compared with control values D. TransmOPA1 encodes for a large multimeric dynamin-like GTPase protein, which localises to the inner mitochondrial membrane where it plays a crucial role in mediating mitochondrial fusion.c molecules within these tight junctions and, importantly, mtDNA maintenance.OPA1 mutations account for the majority of cases of non-syndromic DOA, which classically presents in early childhood with progressive visual loss. Although optic atrophy is the defining feature of DOA, non-penetrance has been reported in 10%–20% of OPA1 mutation carriers.OPA1 mutation carriers, up to 20% in a large case series, can develop a more severe syndromic form of the disease (termed DOA plus) where the visual loss is compounded by a cluster of neurological deficits that include chronic progressive external ophthalmoplegia, sensorineural hearing loss, ataxia, myopathy and peripheral neuropathy.OPA1 mutations can sometimes develop in the absence of overt optic atrophy and visual failure.OPA1 mutations result in a more severe phenotype characterised by an earlier age of onset and with a more aggressive neurological course—referred to by some investigators as Behr syndrome.OPA1 mutation was shown to behave as an intralocus modifier that results in DOA plus phenotypes when it occurs in conjunction with an exonic OPA1 missense variant —the latter being insufficient to cause disease on its own.OPA1 variant in our family apparently does not result in clinical or subclinical disease. Taken together, these observation further add to the complexity of genotype–phenotype correlations and it demonstrates the spectrum of OPA1 mutations from the classical dominant mutations (which accounts for the majority of cases), to compound heterozygous mutations causing more severe syndromes (including Behr syndrome), and our current report with a homozygous mutation.OPA1 mutations. First, the two affected siblings harboured a novel homozygous OPA1 variant, which have not been reported previously in association with human disease. These two patients developed a rapidly progressive lethal phenotype beginning in early infancy and characterised by marked multisystemic involvement due to generalised mitochondrial respiratory chain dysfunction. This ultimately fatal clinical presentation is consistent with the rather striking observations made in three Opa1 mouse models. Homozygous mutant mice died in utero during embryogenesis due to severe abrogation of Opa1 protein levels, confirming a critical role for this pro-fusion mediator in early life and development.Our case report provides a number of new insights into the molecular and phenotypic manifestations of pathogenic OPA1 mutations affecting the catalytic GTPase region have a significantly increased risk of developing the more severe DOA plus phenotype,OPA1 mutations. Interestingly, histopathological evidence of cardiomyopathy was apparent in a heterozygous mutant Opa1 mouse model as early as at 5 months of ageCarriers harbouring missense OPA1 mutations lead to human disease is still under intense investigation, a growing body of evidence points towards a synergy between disturbed mitochondrial dynamics, mtDNA instability and impaired mitochondrial oxidative phosphorylation, which eventually precipitates an irreversible molecular cascade towards cell death. Several studies have demonstrated reduced activities of various mitochondrial respiratory chain complexes, in particular complex I and complex IV, in muscle biopsies and fibroblasts obtained from patients with DOA and confirmed OPA1 mutations.Although the precise disease mechanisms by which OPA1 mutation carriers.OPA1 mutations. This observation is mechanistically important and support a number of recent studies linking OPA1 and also its sister protein mitofusin 2 (MFN2), with the complex process of mtDNA replication. OPA1 is thought to physically anchor nucleoids to the inner mitochondrial membrane via the peptide segment encoded by exon 4b, and using an elegant series of mouse models, marked mtDNA depletion was observed in the context OPA1 null cells.OPA1 mutations, MFN2 mutations predominantly cause the accumulation of multiple mtDNA deletions, but a recent case report indicates that mtDNA depletion can occur as well.24In addition to the generalised cellular energy shortage, OPA1 is now well established as a regulatory factor involved in mtDNA maintenance and genome stability. Previous studies have clearly shown that mtDNA deletions can accumulate to high levels in postmitotic tissues of affected OPA1 mutations are not limited to the optic nerve. We have further extended the mutational and phenotypic spectrum of OPA1 disease to include lethal infantile mitochondrial encephalomyopathy and hypertrophic cardiomyopathy secondary to a novel homozygous missense mutation that targets the key functional GTPase domain. Our case report also further reinforces the power of next-generation exome sequencing in elucidating the genetic aetiology of complex disease phenotypes when combined with rigorous phenotyping and a clear a priori list of candidate genes, in this particular family, OPA1 given the clear link with optic atrophy.It is now abundantly clear that the deleterious consequences of pathogenic


In utero exposure to diethylstilbestrol (DES) has been associated with increased risk of adverse health outcomes such as fertility problems and vaginal as well as breast cancer. Animal studies have linked prenatal DES exposure to lasting DNA methylation changes. We investigated genome-wide DNA methylation and in utero DES exposure in a sample of non-Hispanic white women aged 40–59 years from the Sister Study, a large United States cohort study of women with a family history of breast cancer. Using questionnaire information from women and their mothers, we selected 100 women whose mothers reported taking DES while pregnant and 100 control women whose mothers had not taken DES. DNA methylation in blood was measured at 485,577 CpG sites using the Illumina HumanMethylation450 BeadChip. Associations between CpG methylation and DES exposure status were analyzed using robust linear regression with adjustment for blood cell composition and multiple comparisons. Although four CpGs had p<105, after accounting for multiple comparisons using the false discovery rate (FDR), none reached genome-wide significance. In conclusion, adult women exposed to DES in utero had no evidence of large persistent changes in blood DNA methylation. More than 2 million American women took the drug during pregnancy, exposing both themselves and their fetuses to high doses of estrogen [exposure .Animal models show that early exposure to estrogen compounds, including DES, can change the expression levels of several genes –8. Numerin utero gives rise to persistent epigenetic changes (detectable in blood) and that these changes are partly responsible for the observed increased risk of infertility and cancer related health outcomes in the offspring. We conducted a study of DNA methylation patterns in whole blood from 100 women reporting that their mothers had used DES during pregnancy, and compared them to 100 women without exposure using the Illumina HumanMethylation450 BeadChip. To our knowledge this is the first study to evaluate possible effects of in utero DES exposure on genome-wide DNA methylation in humans.It is possible that prenatal exposure to DES induces lasting modifications to hematopoietic stem cells if the exposure occurs during early fetal development . We hypohttp://sisterstudy.niehs.nih.gov [All women in the study were participants of the Sister Study—a prospective cohort study of environmental and familial risk factors for breast cancer and other diseases. This cohort includes 50,884 women who were all breast cancer free at enrollment but had at least one sister diagnosed with breast cancer. All participants provided a blood sample at baseline and completed extensive questionnaires including a self-administered family history questionnaire on prenatal exposures. Detailed information about the Sister Study can be found at .nih.gov .Within the Sister Study a sample of the participant’s mothers was also invited to participate in a sub study entitled the Mothers Validation Study (MVS). Sampling for the MVS was restricted to mothers under 60 who were reported to be alive at their daughters’ enrolment into the Sister Study and additionally weighted to include women whose daughters reported rare early life exposures, including DES. A total of 1802 mothers completed the MVS questionnaire which included a question about whether the she had used DES during pregnancy. The concordance between mothers and daughters reports on DES usage and exposure was addressed . In our http://www.cgirb.com/irb-services/).Informed written consent was obtained from all participants prior to participation. The study was approved by the Institutional Review Boards of the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health, and the Copernicus Group (All blood samples were collected using glass blood collection tubes with acid citrate dextrose (ACD). This was done during a home visit at baseline and the blood was stored at-20C prior to DNA extraction. Genomic DNA was extracted using automated equipment  and quantified with Quant-iT PicoGreen dsDNA reagent (Invitrogen). One microgram (μg) of extracted DNA was bisulfite converted using the EZ DNA methylation kit  according to the manufacturer’s protocol.Methylation analysis was conducted at the NIEHS Molecular Genomics Core using the Illumina HumanMethylation450 BeadChip . Samples were randomly assigned to eighteen beadchips with 12 spots on each using a balanced design with respect to exposure and age (<50 vs >+50). We included 6 DNA methylation controls  and 10 duplicate samples, which were placed on different chips. One of the duplicates was eventually excluded due to poor methylation data quality. For the remaining 9 pairs of duplicates we calculated Pearson’s correlation coefficients for Infinium I and Infinium II probes separately . DNA wasWe excluded 48,494 CpG probes based on the following criteria: 1) CpG probes with a common SNP  located at the target CpG site; 2) CpGs with probes mapping to multiple genomic locations; 3) CpGs located on the Y chromosome.U) or methylated (M) CpGs. We pre-processed the M and U intensity values separately for Infinium I and II CpG probes detailed as follows: 1) for Infinium I probes, we separately background corrected (using the Robust Multichip Average (RMA) method [U and M intensity values using the normalizeMethyLumiSet method in the R package “methylumi”, and then performed RMA background correction and quantile normalization separately for U and M intensity values; 3) The methylation level  for each CpG site was calculated as the ratio of normalized fluorescent intensities between methylated and unmethylated alleles β = M/(M+U+100).At each CpG site on the array, methylation status was determined based on intensity measures of probes corresponding to unmethylated  method ) and quawww.sisterstudystars.org, or by contacting one of the authors.The DNA methylation data set used in this publication can be accessed via EMB, WNT11 TGFB1, ERBB2, EGFR, LTF, EGF, FOS, and JUN) that have previously been shown to have differential gene expression in mice uterine or vaginal tissue following pre or perinatal exposure to DES [We also specifically report on nine genes  and DES exposure status. In order to adjust for multiple testing, the false discovery rate (FDR) was calculated using the q-value framework, q<0.05 was used as the cutoff for genome wide significance . In the DES-exposed women were similar to unexposed women in regard to age, ever use of OCs, and smoking status; a greater percent of exposed women had BMI<25, menarche at 14+ years, and were nulliparous than unexposed women .KIFC3, and DCAKD) had nominal p values < 10-5, and an additional 18 CpGs in 18 different genes had nominal p values < 10-4 . Limitations include lack of detailed information on dose, route of exposure, timing, or duration of DES use. Additionally we had limited power to detect small effect sizes . With a sample size of 200 (1:1 case/control ratio) and assuming a p-value threshold of 10in utero DES exposure on blood DNA methylation.In this study we used DNA from blood rather than from a known target tissue such as vagina, cervix or endometrium. Histologic changes in target tissues, including vaginal epithelium, are known to correlate with dose of DES exposure and with increased risk of adverse outcomes such as vaginal carcinoma , 27. AltS1 FigThe 100 exposed women were randomly selected from the 125 eligible exposed women. The 100 unexposed women were selected based on age frequency matching to the exposed women.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Study of human lacrimal cell biology is limited by poor access to tissue samples, heterogeneous cell composition of tissue and a lack of established lacrimal epithelial markers. In order to further our understanding of lacrimal cell biology, we sought to find a better marker for human lacrimal epithelial cells, compared to what has been reported in the literature.We utilized human Muller’s muscle conjunctival resection (MMCR) specimens containing accessory lacrimal gland (ALG) and cadaveric main lacrimal gland (MLG) as sources of lacrimal tissue. Candidate markers were sought using human ALG tissue from MMCR specimens, isolated by laser capture microdissection (LCM). Affymetrix® analysis was performed on total RNA isolated from FFPE samples to profile transcription in ALG. MMCR tissue sections were assessed by immunofluorescence using antibodies for histatin-1, lactoferrin, E-cadherin (E-cad) and alpha-smooth muscle actin (ASMA). Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed to analyze the expression of histatin-1, E-cad and lactoferrin from cadaveric MLG.Histatin-1 is expressed in ALG and MLG, localizes to lacrimal epithelium, and to a greater degree than do other putative lacrimal epithelial markers.Histatin-1 is a good marker for human lacrimal epithelium in ALG and MLG and can be used to identify lacrimal cells in future studies. Dry eye syndrome (DES) is a multifactorial disease of the ocular surface that occurs due to tear film instability, hyperosmolarity, inflammation and loss of main and accessory lacrimal gland function. It is a common disease that affects approximately 11% to 22% of people worldwide. DES is associated with negative effects on quality of life, productivity and has a high cost of treatment [in vitro and in vivo [Understanding of lacrimal gland biology has lagged behind other fields of study and improvements in this area could drive the development of new therapeutic tools. Limitations to investigation into human lacrimal cell biology include inadequate access to tissue, heterogeneous cell populations in samples and physically small samples. In particular, the lack of well-defined cell markers for lacrimal epithelium limits the ability to isolate, identify and investigate the behavior of lacrimal epithelium  in vivo –5.Previous studies have utilized a variety of proteins, such as lactoferrin  and alpha smooth muscle actin (ASMA)  as presumed lacrimal markers –7. HowevMuller Muscle Conjunctival Resection (MMCR) surgery is a commonly used procedure in the treatment of blepharoptosis. Both MMCR surgery and the Fasanella-Servat ptosis procedure involve the resection of the posterior layers of the eyelid in the area of the ALG ,16. ALG In this study, we sought to find a reliable marker for lacrimal epithelium using Affymetrix® gene arrays, coupled with the existing knowledge from the literature on lacrimal gene expression . We valiWritten informed consent was obtained from patients using a consent form specifically approved for this study by the Institutional Review Board (IRB) and processed by The University of Illinois at Chicago (UIC). Completed, signed consent forms were maintained according to the university guidelines following an IRB approved protocol specific for this study. Cadaver donor eye tissues, including MLG cells, included in this study were deidentified and use of these tissues was sanctioned by the UIC IRB.Patient consent and samples were obtained using an IRB approved protocol. Inclusion criteria included age greater than or equal to 18 years old, and plans to undergo blepharoptosis repair via MMCR. Seventeen patients and twenty four eyelid specimens were included.Antibodies to histatin-1, E-cad, lactoferrin and ASMA were used for immunofluorescence staining of MMCR tissue sections. Xylene and rehydration with serial ethanol dilutions were used for deparaffinization. Slides were washed twice for 5 minutes in 0.25% Triton X-100 for permeabilization and blocked for 2 hours at room temperature with 2% BSA and 10% normal donkey serum in PBS. Slides were incubated overnight at 4°C with the primary antibody diluted in blocking solution (1:100) . The next day, the slides were washed twice for 5 minutes in PBS and incubated for 1–2 hours with respective secondary antibodies  diluted in blocking solution (1:200–800). Vecta shield mounting medium with 4′,6-diamidino-2-phenylindole  was placed over the slides and covered with a glass coverslip. Slides were analyzed using the Zeiss LSM 710 Confocal Microscope.Similarly, cultured human MLG derived epithelial cells were cultured on the chamber slides and fixed with 4% paraformaldehyde for immunostaining with antibodies to histatin-1, lactoferrin, E-cad, and ASMA.LCM (Leica) was performed to isolate ALG from MMCR specimens. Briefly, 2–3 (8 μm) sections from available paraffin embedded blocks of MMCR specimens were placed on PEN membrane slides (Leica). Toluidine blue staining according to previously described protocols  was perfTotal RNA was purified from micro dissected, FFPE samples using RNeasy from QIAGEN using the manufacturer’s protocol. Extracted RNA was analyzed using Qubit quantification and Tape Station 2200 quantification-sizing quality control system.Following RNA extraction from laser capture dissected FFPE tissues, 25ng of total RNA was amplified, converted to cDNA, and labelled with “SensationPlus™ FFPE Amplification and WT Labeling Kit” from Affymetrix®. The labeled double stranded cDNA was hybridized and analyzed on “GeneChip® Human Gene 2.0 ST” Array from Affymetrix® microarray based results.Data obtained from Affymetrix ® microarrays were queried for expression of candidate genes of interest. Genes of interest were determined using a literature search and genes identified in glands of Wolfring . In ordeTotal RNA was isolated from main human lacrimal gland tissue using RNA-easyKit (Qiagen) according to manufacturer's instruction. cDNA was synthesized from 375ng of isolated RNA using iScript cDNA synthesis kit (BioRad). The cDNA was amplified using GoTaq green master mix (Promega) using appropriate primers for histatin-1 (259bp), E-cad (291bp) and lactoferrin (251bp). GAPDH (323bp) was used as a control for intact cDNA. The PCR amplification was performed for 41 cycles at 30 sec denaturation at 95°C annealed for 30 sec at 56°C and extended for 1 min at 72°C. The amplification reaction products were resolved on 1% agarose/TAE gels, electrophoresed at 90mV and visualized by ethidium bromide staining. Primer sequences are noted in http://ndriresource.org/), were inspected and adventitial tissue was dissected away from the lacrimal gland tissue by an oculoplastic surgeon. The MLG was then washed with Hank’s Balanced Salt Solution (HBSS) media (Gibco) to remove any red blood cells. The tissue was cut into 2-3mm pieces (explants) using sterile forceps and scissors. MLG explants were then placed on an uncoated six well plate and 1ml HepatoSTIM ® (Corning) media supplemented with 10ng EGF 10% FBS, 2 mM L-glutamine, 2% penicillin-streptomycin. Cells extending from the explants were then grown on uncoated plates in the same media, after moving explants to other uncoated plates.Human MLG primary culture was performed as follows. Freshly obtained human MLG, provided by the National Disease Research Interchange  and numerous peptides translated from these genes . HTN1 prAlthough histatin-1 localizes well to lacrimal epithelium and is expressed at high levels in both ALG and MLG, it is also present in conjunctival epithelium. All the putative lacrimal epithelial markers used in this study show strong overlap of localization in both conjunctival and lacrimal epithelial cells. We believe histatin-1 has the least amount of co-localization to conjunctival epithelium, as compared with lactoferrin and E-cad. ASMA seems to only identify myoepithelial cells in these samples. Thus, although histatin-1 may be a useful marker for future studies in lacrimal cell biology, there are limits to its ability to differentiate among all cell types. The development of markers to identify sub populations of cells in heterogeneous samples is often limited by the lack of absolutely specific markers and thus must be used in a relative manner based on context. We suggest the use of multiple cell markers and different types of assays in future studies on lacrimal cell biology in order to validate and characterize cell types.It is interesting to note that our Affymetrix® gene microarray analysis of ALG shows the high expression of a number of developmental and stem cell genes which are significant and relevant to future studies. These cells express a number of the baseline markers of pluripotency including Oct4, Sox2, Klf4, c-Myc. This may indicate the suitability of these cells for use in development of regenerative therapies for DES.Limitations of this study include small specimens, and few available samples. Furthermore, previously reported studies demonstrated only low levels of expression of histatin-1 on the ocular surface, and tear proteomic experiments did not demonstrate histatin family members in the tear film. These results may indicate that histatin proteins are not secreted onto the ocular surface, or may reveal the limitations and variability of body fluid proteomic analysis . It may Future studies on lacrimal epithelium to investigate human disease processes, including DES, may utilize histatin-1 as a marker to identify changes in lacrimal cell function or biology. Moreover, the presence of stem cell markers noted in our gene expression array analysis are encouraging and should be investigated in future experiments. Finally, future studies investigating the structure and function of histatin-1 in ocular tissues will need to be considered. Thus, we have demonstrated that histatin-1 is a useful marker of lacrimal epithelium for future studies of lacrimal cell biology and function.


Both mouse and human embryonic stem cells can be differentiated in vitro to produce a variety of somatic cell types. Using a new developmental tracing approach, we show that these cells are subject to massive aberrant CpG island de novo methylation that is exacerbated by differentiation in vitro. Bioinformatics analysis indicates that there are two distinct forms of abnormal de novo methylation, global as opposed to targeted, and in each case the resulting pattern is determined by molecular rules correlated with local pre-existing histone modification profiles. Since much of the abnormal methylation generated in vitro appears to be stably maintained, this modification may inhibit normal differentiation and could predispose to cancer if cells are used for replacement therapy. Excess CpG island methylation is also observed in normal placenta, suggesting that this process may be governed by an inherent program. Both mouse and human embryonic stem cells can be derived from early pre-implantation embryos and grown indefinitely in culture. These cells maintain their pluripotency as indicated by the observation at least in mouse that they can generate a full organism One of the major epigenetic marks of the genome is DNA methylation. In the early pre-implantation embryo, DNA methyl groups derived from the gametes are largely erased and a new bimodal pattern is re-established in every individual at about the time of implantation Although mouse embryonic stem cells are derived from the ICM, genome-wide analysis indicates that, unlike the blastocyst, they are methylated in a manner similar to somatic cells, suggesting that from the epigenetic standpoint, they actually behave like cells at the time of implantation In order to study this problem, we used developmental principles to generate a new approach for assessing what should be considered a normal methylation pattern in ES cells. On the basis of this in vivo perspective, we found that both mouse and human ES cells growing in vitro have aberrant DNA methylation that could have physiological effects on their ability to undergo proper differentiation.+Sox17+ECD+) or mesoderm (Gsc+Sox17−ECD−). Embryoid bodies (EBs) were formed from ES cells (TT2) Mouse endoderm and mesoderm were differentiated from ES-GscgfpSox17huCD25 cells originally derived from line EB5 by sequence targeting Human ES cells (CSES2) Human fetal (20–25 weeks) DNA samples were purchased from Biochain. All mouse DNAs were extracted from C57Bl/6 and mDIP was performed as described previously http://www.genomics.agilent.com) as described previously The Input and Bound DNAs were labeled and hybridized on mouse (105 K) or human (244 K) CpG island microarrays  as determined by Reduced representation bisulfite sequencing (RRBS).Previous published work on undifferentiated and differentiated ES cells and in vitro differentiation was analyzed by downloading data from the appropriate GEO datasets. We used genomic bisulfite data (GSE11034) for mouse DNA https://tcga-data.nci.nih.gov/tcga/) and average methylation values were calculated for every CpG island. Human ChIP-seq data for H3K27 and H3K4 tri-methylation was obtained from the NIH Roadmap Epigenomics Mapping Consortium. We compared 200 CpG islands deemed abnormally methylated in serum-grown ES cells and found them to be about 10% more methylated than the levels seen when cultured in 2i conditions using previously published data (GSE42929) Previous published work on de-novo methylation in cancer was analyzed by downloading data from The Cancer Genome Atlas  of difference between background-island DNA methylation patterns in somatic tissues as opposed to differentiated and undifferentiated ES cells was determined by a two-tailed, non-paired, equal-variance T-test. The significance of H3K27me3 enrichment on target CpG islands as well as comparison between CpG island sets was calculated using the hypergeometric test https://www2.appliedbiosystems.com). Barcodes and adaptors were added to the primers and deep-sequenced using the Ion Torrent (Life Technologies).Single island reads were obtained by Bisulfite conversion of genomic DNA that was carried out using the EZ DNA Methylation-Direct Kit (Zymo Research) according to the manufacturer's instructions. PCR primers were designed using Methyl Primer Express® Software v1.0 .Mouse ES cells were grown in culture and induced to differentiate using a variety of commonly-employed strategies . EmbryoiWe identified about 9,500 CpG islands constitutively unmethylated in every cell type . This pa−132)  undergo de novo methylation (>80%) at over 300 CpG island promoters, even though almost all of these same sequences were completely unmethylated in corresponding neural embryonic cells representing this same approximate stage of development −132) .Since the excess modification observed in differentiated ES cells was originally detected in comparison to normal tissue DNA, we next examined the methylation state of these same CpG islands in undifferentiated mouse ES cells. While the same target sequences appear to be relatively unmethylated in these undifferentiated cells, surprisingly, they were found to be much more methylated than the normal population of CpG islands in the cell , exclusively focusing on CpG islands that are unmethylated in a wide range of different tissue cell types. Here too, differentiated ES cells were found to have a large number of aberrantly methylated sequences similar to those identified by mDIP analysis (P<10−124) and the same was true for induced pluripotent stem (iPS) cells, as well  in the parent line. Furthermore, we have observed that the degree of methylation following differentiation is linearly proportional to the underlying pre-set local concentration of this histone modification in undifferentiated ES cells as determined from several different studies   of them representing polycomb targets   and implantation embryos showing the top 2,000 CpG islands found modified (>25%) in NPCs. It should be noted that 70% of the aberrantly-methylated CpG islands detailed in b. Methylation distribution in undifferentiated ES cells for 9,500 constitutively unmethylated (gray) CpG islands and the 2,000 CpG islands shown in a that were found to be methylated in NPCs (blue). c. Methylation distribution . Group A includes CpG islands that are abnormally methylated (>60%) in at least one undifferentiated or differentiated ES cell type but constitutively unmethylated (<20%) in all normal adult and embryo tissues. Group B includes CpG islands that are methylated in a single tissue type but unmethylated in others. For both groups, the same CpG islands appear to be methylated in iPS cells, as well. Figure S3. Scatter plot of H3K27me3 density in NPCs vs. undifferentiated mouse ES cells for 9,500 CpG islands . The 2,000 aberrantly methylated islands (marked in red) show a dramatic reduction in H3K27me3 density when ES cells are converted to NPCs (a). H3K27me3 density in mouse brain is shown for comparison (b). Figure S4. Markers of de novo methylation. a. Table showing the percentage of abnormally-methylated CpG islands that are marked with polycomb (H3K27me3>2) and their average algorithm score (A2) both in human and mouse ES cells. IMS data are from −63) than the constitutively unmethylated CpG islands. b. Methylation levels of all CpG islands as a function of local H3K4me3 density for human ES cells before and after differentiation to endoderm. The average levels in a collection of fetal tissues is shown for comparison. Figure S5. DNA methylation in placenta. DNA from normal mouse embryos and from placenta were subject to mDIP microarray analysis. Heat map of 1,000 CpG islands methylated in placenta out of 9,500 background CpG islands  and natural β cells purified form fresh pancreatic islets were subject to mDIP microarray analysis. Out of 9,500 background CpG islands . The excess methylation seen in each tumor type is significantly greater than that observed in normal tissues . Table S1. Genes involved in differentiation and development that are associated with aberrantly methylated CpG islands.ands see . An esti islands , IMS HeaES cells  compared(DOC)Click here for additional data file.


An initial RNAi knockdown screening of MCF-7 human breast cancer cells targeting 6560 proteins identified four signaling molecules required for their fulvestrant-induced apoptosis. Signaling molecules physically or functionally interacting with these four primary node targets were computationally predicted and experimentally validated, resulting in identification of four second-generation nodes. Three rounds of further iterations of the prediction–validation cycle generated third, fourth and fifth generation of nodes, completing a 19-node interaction map that contained three predicted nodes but without experimental validation because of technical limitations. The interaction map involved all three members of the death-associated protein kinases (DAPKs) as well as their upstream and downstream signaling molecules , suggesting that DAPKs play critical roles in the cytocidal action of fulvestrant. The in silico Kaplan–Meier analysis of previously reported human breast cancer cohorts demonstrated significant prognostic predictive power for five of the experimentally validated nodes and for three of the prediction-only nodes. Immunohistochemical studies on the expression of 10 nodal proteins in human breast cancer tissues not only supported their prognostic prediction power but also provided statistically significant evidence of their synchronized expression, implying functional interactions among these nodal proteins. Thus, the Nodes-and-Connections approach to RNAi knockdown screening yields biologically meaningful outcomes by taking advantage of the existing knowledge of the physical and functional interactions between the predicted target genes. The resulting interaction maps provide useful information on signaling pathways cooperatively involved in clinically important features of the malignant cells, such as drug resistance.Although RNA interference (RNAi) knockdown screening of cancer cell cultures is an effective approach to predict drug targets or therapeutic/prognostic biomarkers, interactions among identified targets often remain obscure. Here, we introduce the  However, genome-wide RNAi knockdown screening experiments are technically demanding,24 and integration of the screening results into a biologically informative signaling network is often challenging. To overcome such limitations of genome-wide RNAi knockdown screenings, we propose the use of nodes-and-connections RNAi knockdown screening. This approach starts with a small number of known positive-hit RNAi targets that are designated as the primary node molecules. Then, using bioinformatics tools, molecules that may functionally or physically interact with the primary nodes are predicted, and RNAi knockdown experiments focusing on these predicted node molecules validate them to identify the secondary node molecules. By iterating this prediction–validation cycle, deeper levels of nodes are progressively determined, eventually resulting in the generation of a comprehensive molecular interaction map connecting most, if not all, the primary nodes. A small number of nodes whose involvement in the network is predicted but cannot be experimentally validated because of technical limitations are allowed to be included in the molecular interaction map for effective and flexible prediction of practically useful signaling networks.RNA interference (RNAi) knockdown screening of cell culture models is a powerful approach for identifying molecules involved in drug resistance.nodes-and-connections RNAi knockdown screening approach to generate an interaction map of molecules necessary for the fulvestrant-induced MCF-7 cell apoptosis. The resulting interaction map reveals the critical importance of the death-associated protein kinase (DAPK) family of pro-apoptotic signaling kinases as well as their downstream effectors, including STAT3  and myosin light chains. The in silico Kaplan–Meier survival analysis reveals that not only experimentally validated nodes but also nodes without validation in the interaction map yield promising prognostic biomarkers predicting recurrence of breast cancer. Immunohistochemical evaluation of the nodal protein expression in human breast cancer tissues supports their prognostic predictive power, and statistically significant evidence is presented that these nodal proteins are expressed in a highly synchronized manner, implying organized regulation of their expression. These results demonstrate the usefulness of the nodes-and-connections RNAi knockdown screening for rapid and cost-effective identification of clinically relevant sets of biomarkers and drug targets using cell culture systems.In the present study, we apply the 27 Fulvestrant-induced expression of BIK mRNA requires wild-type TP53 that is strongly expressed in MCF-7 cells.27 To investigate the signaling network required for the fulvestrant-induced MCF-7 cell death in more detail, we performed RNAi knockdown screenings using 6560 arrayed short hairpin RNA (shRNA) expression plasmids targeting the human kinome,28 cell cycle proteins and apoptosis regulators. This preliminary screening yielded four signaling molecules—namely, BIK, ERBB4, DAPK3 and MAP2K2. Signaling by ERBB4 , a member of the ERBB/HER family receptor kinases, promotes differentiation and growth inhibition of breast cancer cells.29 Whereas expression of the other three members of the ERBB/HER family in breast cancer is strongly linked to poor prognosis, increased expression of ERBB4 is more consistently correlated with a favorable prognosis.29 DAPK3 ) is a member of the DAPK family pro-apoptotic signaling kinases.31 The mitogen-activated protein kinase kinase 2 (MAP2K2 or MEK2) regulates the MAPK/ERK protein kinases that directly phosphorylate multiple nuclear proteins involved in cell cycle regulation.32 Although these four signaling molecules did not have any known direct interactions among each other, we presumed that they might be parts of a large network of molecules required for the fulvestrant-induced MCF-7 cell death. To identify this hypothetical network, we extended our RNAi knockdown screenings with the nodes-and-connections approach, as outlined in primary nodes  phosphorylates DAPK3 to activate it,39 and both ROCK1 and DAPK3 phosphorylate myosin light chain subunits to increase the actin-activated myosin ATPase activity.40 DAPK1, DAPK2 and ROCK1, which interact with the DAPK3 primary node, are the second-generation nodes in the molecular interaction map for the fulvestrant-induced MCF-7 cell death. Because RNAi knockdown of MYL2 or MYL9 myosin light chains strongly reduced MCF-7 cell viability even in the absence of fulvestrant,41 their requirement for the fulvestrant-induced apoptosis was not experimentally confirmed. However, RNAi knockdown of the MYLK3 myosin light chain kinase, which phosphorylates MYL2 and MYL9,42 did not damage MCF-7 cell viability and caused their fulvestrant resistance .vestrant . PAG1 inted node . STAT5A,ted node . All theted node . Table 150 we determined whether expression of the mRNA transcripts for the nodal genes is affected by ERα agonists and/or antagonists. Affymetrix microarray analysis  revealed significant increase in mRNA expression for CAMK1D, CSK, ERBB4 and DAPK3 after exposure of MCF-7 cells to 100 nM fulvestrant for 48 h  whose stronger expression correlated with significantly better prognosis in multiple cohorts. Expression of four other nodal genes  and MYLK, encoding a myosin light chain isoform as the nodal gene MYLK3, also correlated with significantly better prognosis in a single cohort (data not shown). These results suggest that the interaction map of molecules required for fulvestrant-induced death of MCF-7 cell culture model may provide insights into clinically useful prognostic biomarkers of breast cancer.To explore the relevance of the interaction map generated in this study to human breast cancer, we examined whether mRNA expression of the nodal genes in tumor tissues of previously published breast cancer cohorts correlates with better prognosis by analysis . PatientP=0.0086). The PME scores were also significantly smaller for recurrent breast cancers when the same analysis was performed only with patients who received endocrine therapy (P=0.046), whereas no statistically significant recurrence-associated difference in the PME scores was observed with patients who did not receive endocrine therapy (P=0.124). These results suggest that expression profiling of the nodal proteins may be useful for predicting prognosis of breast cancer after endocrine therapy.To determine whether nodal protein expression in breast cancer tissues correlates with prognosis, paraffin-embedded pathological slides of surgically excised tumor tissues obtained from 18 age- and stage-matched breast cancer patients , indicating that expression of each nodal protein is not mutually independent. The apparent associations among the nodal proteins for their expression in breast cancer tissues may imply that resistance to endocrine therapy might occur through a coordinated loss of multiple nodes in the interaction map rather than loss or dysfunction of a single nodal molecule. It is unknown as to whether suppression of a single nodal molecule in breast cancer in vivo is insufficient to cause clinically observed drug resistance that is in contrast to the MCF-7 cell culture model showing significant resistance after single molecule knockdown. Alternatively, the apparently coordinated loss of multiple nodal molecule in vivo might reflect an involvement of common mechanisms regulating their expression such as epigenetic suppression.We next performed a permutation test to determine whether expression of the 10 nodal proteins is mutually independent. A bell-shaped distribution of the PME scores was generated by computational simulation without considering mutual effects of the nodal proteins on their expression (red bars). The χ53 Whereas a standard knockdown/knockout study performs screening data generation and bioinformatics data analysis as two separate stages, a nodes-and-connections approach performs multiple cycles of iteration of knockdown/knockout experiments and data analysis. This novel approach has the following merits.High-throughput knockdown/knockout screening of cell culture models is a powerful approach to identify molecules involved in drug resistance.pending and cautiously included in the molecular interaction map if results of later cycles of iteration or other studies support their validity, possibly reducing false negatives caused by technical limitations. Examples of such nodes in our present study are STAT3, STAT5A and myosin light chains  in 10% CO2 at 37 °C. Research-grade fulvestrant was purchased from Tocris . Fulvestrant-induced W2 cell apoptosis was evaluated by crystal violet staining after exposure to 100 nM fulvestrant for 7 days in the presence of 10% nonstripped fetal calf serum. Detailed method of cell viability analysis has been described in our previous paper.46MCF-7 human breast cancer cells (BUS stock) were provided by C Sonnenschein and Ana M Soto ,28 our RNAi knockdown screening experiments defined positive hit as a target with at least two distinct shRNA lentiviral clones inducing a statistically significant resistance to fulvestrant compared with cells infected with the pLKO.1 control virus . Because distinct shRNAs are expected to have different spectra of off-target effects, this criterion is expected to filter out off-target effects. For some positive-hit targets, fulvestrant resistance was observed with only two or three shRNA lentiviral clones among five or larger numbers of viral clones. This incompleteness reflects a commonly known technical limitation of the RNAi knockdown experiments using the TRC shRNA lentiviral library that was thoroughly evaluated in its original literature.28Following the criteria adopted in the original study describing the development and use of the TRC shRNA lentiviral libraries,28 An arrayed shRNA lentivirus library targeting human kinome was prepared by transfecting HEK293T packaging cells with TRC library of 6560 kinome shRNA hairpin plasmids  together with the pMD2G expression plasmid for vesicular stomatitis virus glycoprotein and the pCMV-dR8.91 plasmid for the core lentiviral genome. MCF-7 cells grown at 5–10 × 104 cells/well density were infected with 4–8 multiplicity of infection titers of lentiviruses in the presence of 8 μg/ml polybrene under 1200 g gravity for 60 min. Medium was changed 48 h after infection, and successfully infected cells were selected by exposure to 2.5 μg/ml puromycin for 48 h.Lentiviruses expressing shRNA species targeting human mRNA transcripts were produced using the pLKO.1 vector harboring a puromycin resistance marker gene as previously described.54 Microarray data of MCF-7 cells exposed to fulvestrant and 17β-estradiol are available from the Gene Expression Omnibus (GEO) database of the National Center for Biotechnology Information (NCBI) accession numbers GSE14986 and GSE50705, respectively. The 17β-estradiol dose-dependent mRNA expression data are also retrievable from our website (http://mplwebserver.partners.org).Transcriptomal profiles of MCF-7 cells determined by Affymetrix microarray has been described in our previous studies.Human breast cancer tissues were surgically excised from patients admitted in the Rinku General Medical Center  between 2005 and 2008 and processed for the standard formalin fixation and paraffin embedding. Informed consent was obtained from the 18 patients involved in this study, whose clinical profiles are shown in in silico prediction of interactions between nodes was performed using the Ingenuity Pathway Analysis tool . Kaplan–Meier analysis of recurrence-free survival was performed using the ‘survival' package of the R programming language  and publicly available mRNA expression and survival data , a distribution of 50 000 theoretical PME scores was computationally generated, and its 1% and 5% two-tail cutoff values were determined. If expression of the 10 antigens is mutually independent, the majority of the observed PME scores are expected to follow this theoretical distribution. The χ2 tests were performed to determine whether the observed PME scores follow such a theoretical distribution.To generate a theoretical distribution of the PME scores, an intensity score was randomly sampled from the 18 scores of each antigen, and the sum of these scores for all the 10 antigens were calculated to obtain a theoretical PME score. To test a null hypothesis that expression of each antigen is mutually independent (that is,


The Atlantic or Arctic origin of water masses had a strong impact on the AAP bacterial community structure whose populations mostly belonged to the Alpha- and Gammaproteobacteria. A majority (>60%) of pufM sequences were affiliated to the Gammaproteobacteria reasserting that this class often represents the major component of the AAP bacterial community in oceanic regions. Two alphaproteobacterial groups dominate locally suggesting that they can constitute key players in this marine system transiently. We found that temperature is a major determinant of alpha diversity of AAP bacteria in this marine biome with specific clades emerging locally according to the partitioning of water masses. Whereas we expected specific AAP bacterial populations in this peculiar and newly explored ecosystem, most pufM sequences were highly related to sequences retrieved elsewhere. This observation highlights that the studied area does not favor AAP bacteria endemism but also opens new questions about the truthfulness of biogeographical patterns and on the extent of AAP bacterial diversity.Despite an increasing number of studies over the last 15 years, aerobic anoxygenic photoheterotrophic (AAP) bacteria remain a puzzling functional group in terms of physiology, metabolism, and ecology. To contribute to a better knowledge of their environmental distribution, the present study aims at analyzing their diversity and structure at the boundary between the Norwegian, Greenland, and Barents Seas. The polymorphism of a marker gene encoding a sub-unit of the photosynthetic apparatus ( This finding inspired an increasing number of studies on this functional group and on its expected key role in the carbon cycle. However, the metabolic potential of these microorganisms is still enigmatic and their contribution to the marine carbon cycle remains poorly understood.More than 10 years ago, Kolber et al.  reported2) as carbon source. Although it has been suggested that AAP bacteria display a light enhanced CO2 incorporation  identify AAP bacterial populations in this newly explored area and (ii) determine the influence of the origin of water masses and of the local environment on the AAP bacterial community structure.Johan Hjort (Norwegian Institute of Marine Research)  concentrations were determined on board. An overview of position, bottom depth and hydrology for each sampling station was previously reported  and proteinase K (0.21 mg mL−1)], lysates were recovered and nucleic acids were extracted twice , and were concentrated using Centricon-100 concentrators (Millipore).DNA extraction was performed as described by Lovejoy et al. . BrieflypufM gene (245-bp fragments). The following PCR conditions were used: 95°C for 5 min followed by 40 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s, using a final extension step at 72°C for 10 min. PCR reactions (50 μL) contained 5 X buffer (10 μL), 2 mM MgCl2, 10 pmol of each dNTP (Eurogenetec), 10 pmol of each primer, 2.5 U of GoTaq Flexi DNA polymerase (Promega) and 50–100 ng of template DNA. Amplified products were checked by electrophoresis in 1.5% agarose in 0.5 X Tris-acetate-EDTA buffer and further quantified with a DNA quantitation fluorescence assay kit (Sigma-Aldrich). For each library, amplicons were sequenced on an ABI 3130 POP7 sequencer (Applied Biosystems) at the Biogenouest Sequencing-Genotyping Platform (Roscoff site) after being ligated to the TOPO-TA vector (Invitrogen) and cloned into Escherichia coli following the manufacturer's recommended protocol . Sequences were manually curated, subjected to BLAST search against publicly available sequences, grouped into Operational Taxonomic Units (OTUs) according to a cut-off value of 94% using BOSQUE  are deposited in the GenBank database under accession numbers pufM genes was performed by Temporal Temperature gel Gradient Electrophoresis (TTGE) after PCR amplification using a Cy5-labeled pufM_forward primer and a primer pufM_WAW with a 40-bp GC-clamp (CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCG) added at the 5′-end. The electrophoresis conditions were similar as described by Lehours et al. , Canonical Correspondence Analyses (CCA) and correlations were performed using PAST 1.81  available at a as variables, explained a huge part of the variance between stations. It mainly discriminated waters of the N/S transect from those of the E/W transect and grouped separately Arctic waters  were locally highly variable with depth, they did not discriminate waters of Arctic or Atlantic origin    were characterized by low temperatures (range 1.78°C–4.78°C), low salinities (ca. 34.5 psu) Figures  and show) Figure . The Atl) Figure . Chl a c) Figure . Differe) Figure . Accordi) Figure . Stationt Figure .Considering the hydrological context of the sampled region, 3 hypotheses could be drawn about the distribution of AAP bacterial populations: (H1) different patterns in relation to the S/N, E/W transects and Arctic waters might be observed in AAP bacterial community composition, (H2) the sampled area being a frontier zone where Atlantic and Arctic waters meet, its AAP bacterial community composition should reflect that of the water masses, and (H3) the distribution of AAP bacterial populations should be dependent on the environmental local context  which is highly variable with depth Table .pufM gene was analyzed by TTGE profiling. Among the 4 groups identified by PCA  to that of Roseobacter sp. LE17  were also found between Chla concentrations and the relative abundance of SPIT1 (r = 0.62), SPIT28 (r = 0.71), SPIT29 (r = −0.6), but also between salinity and the relative abundance of SPIT19 (r = −0.91), SPIT21 (r = −0.65) and SPIT31 (r = −0.65). However, understanding the factors that govern the environmental distribution AAP bacteria is not so simple, as illustrated by the relationship between the relative abundances of OTUs SPIT3, SPIT7, and SPIT10 and temperature  negative correlation observed suggests that the corresponding AAP populations are favored by colder waters  was performed using relative proportions of OTUs to determine if specific AAP bacterial populations are associated with specific environmental parameters. Consistent with the clustering deduced from TTGE analyses the sampled stations grouped in the CCA according to Atlantic or Arctic water masses Figure  reinforce Figure . The sigs Figure . Howevers Figure  and theis Figure  illustras Figure .pufM sequences were similar to sequences from other oceanic regions, we must acknowledge that AAP bacteria in the Norwegian and Barents Seas are not endemic. In a recent exploration of the AAP diversity in the western Arctic Ocean, pufM sequences of the dominant OTUs were also similar to sequences already detected elsewhere  retrieved from the Mediterranean Sea (Figure We acknowledge that the Arctic Ocean, as an enclosed ocean connected to the Pacific and Atlantic oceans Jones, , is not a Figure  whose hya Figure  and raisa Figure .pufM OTUs in newly explored ecosystems. It could be considered that a hidden part of the AAP diversity is not accessible through classical cloning-sequencing approaches and that high-throughput sequencing methods could reveal a “rare biosphere of AAP bacteria” as most of the prokaryotic diversity is in the less abundant taxa (Sogin et al., pufM genes from seasonal water samples at a coastal oligotrophic environment of the northwestern Mediterranean Sea. Whereas pyrosequencing approaches are expected to increase the number of OTUs by at least one order of magnitude (Crespo et al., Investigations on the diversity of AAP bacteria are relatively recent, and although the number of sequences in public databases has greatly increased over the past 10 years, one might expect to find populations exhibiting new Overall, results gathered from recent sampling expeditions and sequencing efforts suggest that a large part of the AAP bacterial diversity has already been sampled in marine environments.pufM sequences were similar to sequences retrieved elsewhere with, for example, a large number of gammaproteobacterial AAP sequences closely related to sequences from the Mediterranean Sea. These observations highlighted several issues to be addressed, such as the existence of biogeographical patterns for AAP bacteria and the yet known extent of their diversity in marine systems. These questions, which cannot be resolved on the basis of this study, will need to rely on comprehensive analyses integrating comparable datasets of pufM sequences from different oceanic regions.The present analysis of the AAP bacterial diversity demonstrated conclusively that variation of AAP bacterial populations across transects was much greater than along depth profiles, contrasting with previous observations in other oceanic provinces. Temperature was found to have a major role in structuring AAP bacterial populations in the present biome but as this factor is linked to water mass origin, other physical and/or chemical variables may also be contributing and synergetic factors. Understanding the factors that govern AAP bacteria diversity patterns is therefore not simple as these factors seem dependent of the oceanographic context. Surprisingly, most 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.


High-throughput sequencing (HTS) of antibody repertoire libraries has become a powerful tool in the field of systems immunology. However, numerous sources of bias in HTS workflows may affect the obtained antibody repertoire data. A crucial step in antibody library preparation is the addition of short platform-specific nucleotide adapter sequences. As of yet, the impact of the method of adapter addition on experimental library preparation and the resulting antibody repertoire HTS datasets has not been thoroughly investigated. Therefore, we compared three standard library preparation methods by performing Illumina HTS on antibody variable heavy genes from murine antibody-secreting cells. Clonal overlap and rank statistics demonstrated that the investigated methods produced equivalent HTS datasets. PCR-based methods were experimentally superior to ligation with respect to speed, efficiency, and practicality. Finally, using a two-step PCR based method we established a protocol for antibody repertoire library generation, beginning from inputs as low as 1 ng of total RNA. In summary, this study represents a major advance towards a standardized experimental framework for antibody HTS, thus opening up the potential for systems-based, cross-experiment meta-analyses of antibody repertoires. High-throughput sequencing (HTS) of antibody repertoires offers the potential to study the humoral immune system in a quantitative and systems-based approach One essential component of all amplicon library preparation methods for HTS is the addition of sequencing adapters. To date, the impact of adapter addition methods on antibody HTS has not been thoroughly determined. Adapters are dual-purpose, platform-specific oligonucleotide sequences required for nearly all HTS technologies . On the Illumina platform, they are essential to the sequencing biochemistry, enabling flow cell binding, cluster generation, and reaction priming. They also permit indexing of samples to perform efficient multiplexed sequencing runs. Adapters are attached to the 5′ and 3′ ends of the genetic fragments of interest to yield the sequencing-ready library. Commonly used methods are based on ligation or PCR-addition of the sequencing adapters. In the ligation method, the antibody libraries are first amplified by PCR using a primer set specific for the targeted variable heavy or light chain regions. Subsequently, double-stranded oligonucleotides partly containing the adapter sequences are attached by ligation and then followed by a low-cycle PCR amplification step , which completes the addition of full-length adapter sequences H) libraries were prepared for Illumina-based HTS using ligation-, DA-, or PE-based adapter addition. Additionally, using the PE method, a titration of total RNA was conducted to determine a potential lower limit of total RNA input. HTS datasets were compared based on reliably detected clones This study investigated the influence of methods of adapter addition on both the PCR and sequencing level in terms of experimental efficiency, practicality, and impact on resulting HTS datasets. Using the same starting total RNA source isolated from pooled antibody-secreting cells (ASCs) from immunized mice, antibody heavy chain variable region . Nine female BALB/c mice (Charles Rivers Laboratories) 8–10 weeks old were housed under specific pathogen-free conditions and were maintained on a normal chow diet. Mice were immunized with 50 µg alum-precipitated chicken gamma globulin (CGG) conjugated to 4-hydroxy-3-nitrophenylacetyl  and were sacrificed 14 days post-immunization. Spleens and bone marrow (from femurs and tibia) were harvested and antibody-secreting CD138-positive cells were enriched as previously described Total RNA extraction from the pooled ASC population was performed using the TRIzol Plus RNA Purification Kit (Life Technologies) according to the manufacturer’s protocol. RNA concentration was determined on a Nanodrop 2000c Spectrophotometer and RNA integrity and concentration were evaluated on a Bioanalyzer 2100 (Agilent). Isolated total RNA was homogeneously mixed, aliquoted, and stored at −80°C. Next, first-strand cDNA was synthesized with Maxima Reverse Transcriptase (Fermentas) using 500 ng total RNA and Oligo(dT) primers (Thermo Scientific) following the manufacturer’s protocol. cDNA from multiple reactions was pooled, mixed, and stored in aliquots at −80°C to serve as PCR template for all tested methods. PCR amplification of the variable heavy IgG genes was performed using a set of 19 forward primers with the gene-specific regions annealing to framework 1 of the VDJ-region Ligation method: Libraries were generated using the aforementioned gene-specific primer set and PCR conditions, with the distinction that primers contained 5′ restriction sites  as previDirect addition (DA) method: The Illumina TruSeq universal adapter constituted the 5′ portion of the forward primers, while the IgG-reverse primer contained the Illumina index adapter, which resulted in direct addition of Illumina adapter sequences to the PCR products . Ten PCRPrimer extension method: Pre-amplification was achieved in a first round PCR using the gene-specific primer regions containing a 5′ GC-rich overhang . DiffereFor the RNA titration analysis, PE was performed using 100 ng and 50 ng total input RNA following the same PCR conditions as sample PE3 and the final product was gel-purified using the QIAquick Gel Extraction kit (Qiagen). PCR conditions of low RNA samples  were extended by two additional cycles in the last annealing temperature step of PCR2  and the final product (≈600 bp) was gel-purified . Only libraries giving a single clear peak of appropriate fragment length were submitted for Illumina HTS. Calculated amounts of sample DNA libraries were pooled to a 4 nM concentration for read equilibration of parallel multiplexed sequencing.All samples were sequenced using the Illumina MiSeq platform with 2×250 bp paired-end reads. Raw data can be accessed from the European Nucleotide Archive (ENA Study Accession ERP004622). Forward and reverse reads were paired using PANDAseq with default parameters Starting from IMGT/HighV-QUEST output, data analysis was performed using the R statistical programming environment Filtered and IMGT detected CDR3s were ranked in decreasing order of frequency and tested for simultaneous presence in the other dataset(s). We regarded all clones as “reliably detected” that belonged to the highest frequency set of clones with 95% of the contained clones present in the other dataset(s). Specifically, a list of reliably detected clones was generated from each dataset by sequentially adding the highest-frequency clones to the list, until the percentage of present clones fell below the set threshold of 95% .In order to generate sufficient amounts of antibody RNA for comparisons of Illumina sequencing adapter addition methods, we collected and pooled splenic and bone marrow-derived ASCs of 9 mice 14 days after immunization with the common antigen NP-CGG. Total RNA was extracted, followed by first-strand cDNA synthesis. For all library preparation methods, we used an antibody variable heavy chain gene-specific primer set that binds to the first 20–22 nucleotides of framework region 1  in terms of yield, amplification efficiency, and overall practicality. For the ligation method, we followed the manufacturer’s recommendation of a minimum DNA input of 1 µg in order to produce a library of sufficient size for HTS. However, with almost 4 µg of total RNA required to yield 1 µg of amplified DNA library, a much higher number of pooled parallel PCR reactions was necessary than for the other methods  and libraries were prepared using the PCR conditions of PE3. The lowest three RNA samples  were amplified with two additional cycles in the last amplification step of PCR2 . We were able to show that even with as little as 1 ng of starting total RNA the PE method could produce a visible and gel-extractable band  and the ligation method for adapter addition and found experimental advantages and drawbacks for each choice, which are summarized in 6 250 bp paired-end reads for each sample with high mean quality Phred scores ranging from 35 to 37  by means of a previously established method for the reliable determination of antibody clones, which is based on the simultaneous existence of clones (CDR3s) in each of the investigated datasets que CDR3 . These rque CDR3  demonstrque CDR3 . While tque CDR3 , ranges que CDR3 . Pairwisque CDR3 . Of inteque CDR3 ; samplesque CDR3 . To summFurthermore, in order to assess the influence of degenerate primers on VDJ sequence diversity, the full-length amino acid sequences of both complete antibody variable regions (VDJ) as well as primer-trimmed VDJs were used as clonal identifiers and reliable detection analyses were performed. We found differences between complete and primer-trimmed VDJs in average numbers of reliably detected VDJs  and CDR3 ranking  within which CDR3 ranking was highly conserved - and one ligation-based method for the addition of Illumina sequencing adapters to antibody variable gene libraries examining the effects with respect to yield, practicality, and effect on resulting HTS datasets. We compared HTS datasets of antibody repertoire libraries generated with the three methods of adapter addition and determined the extent of CDR3 repertoire similarity by leveraging the concept of reliable detection  r≥0.79, . This re r≥0.79,  showing  r≥0.79,  by emplo r≥0.79, .The finding that all three methods of adapter addition resulted in comparable and statistically equivalent HTS datasets allowed for their evaluation based on experimental performances: yield , efficiency (yield related to RNA input and PCR cycle number), and overall practicality. Of the methods tested, ligation required the highest amount of starting RNA material due to the fact that a minimum input of 1 µg of preamplified DNA library was necessary as input for the ligation reaction (manufacturer’s recommendation). Despite the high amount of input RNA and the high number of PCR cycles, the final extracted product yield was low ≈20 ng,  and did The DA method readily allowed for library preparations with a more standard input of 500 ng total RNA  [16], [3The PE method was designed similarly to Wang and colleagues −3%) with a diverging CDR3 repertoire composition below a CDR3 frequency of ≈2.5×10−3% and abundance of 32 reads per unique CDR3 , where consequentially RNA amounts were rarely limited Furthermore, this report is highly relevant for amplicon HTS regarding experimental library preparation and statistical interpretation of HTS datasets. We described two PCR-based methods of adapter addition, both of which exhibited strong experimental advantages compared to the ligation method, thereby predestining them as future standard of adapter addition. On the level of HTS, employing the framework of reliable detection, we concluded that all three examined methods yielded comparable and nearly equivalent datasets, thus underlining the advantages of the PCR-based methods of adapter addition. The framework of reliable clonal detection did not only enable the quantitation of absolute numbers of reliably shared CDR3s across HTS datasets but also gave rise to ranges of reliable detection. Specifically, these ranges implicated the determination of minimal clonal frequencies, below which HTS datasets of tested methods and conditions showed a divergence in clonal composition. We believe that these strict, objective, and unbiased reliability thresholds will become standard and essential components of future comparative HTS analyses.File S1File S1 includes the following: Figure S1. Amplicon libraries on 1% agarose gel. Figure S2. DA duplicates  yield highly comparable HTS datasets. Figure S3. Pairwise comparison of HTS datasets from antibody repertoire libraries prepared using the DA and PE method. Figure S4. Pairwise comparison of HTS datasets from antibody repertoire libraries prepared using the ligation and DA method. Figure S5. Pairwise comparison of HTS datasets from antibody repertoire libraries using the ligation and PE method. Figure S6. Variations in the PE protocol have minimal effects on HTS datasets. Figure S7. Comparison of HTS datasets from antibody repertoire libraries prepared using the PE method and lowering the amounts of total RNA input (500–1 ng). Figure S8. VDJ primer trimming reduces VDJ diversity. Table S1. RNA titration (500–5 ng) using the PE method. Table S2. The 30 highest ranked CDR3 amino acid sequences of all RNA titration datasets are shown in a color-coded manner with respective frequencies. Table S3. List of all primers used for ligation, DA, and PE.(DOCX)Click here for additional data file.


FgfFgfFgfFgfFgfKl−/− mice, suggesting that loss of FGF23 activity impacts the auditory system via mechanisms at least partially independent of KL. Given the extensive middle ear malformations and the overlap of initiation of FGF23 activity and Eustachian tube development, this work suggests a possible role for FGF23 in otitis media.Fibroblast growth factor 23 (FGF23) is a circulating hormone important in phosphate homeostasis. Abnormal serum levels of FGF23 result in systemic pathologies in humans and mice, including renal phosphate wasting diseases and hyperphosphatemia. We sought to uncover the role FGF23 plays in the auditory system due to shared molecular mechanisms and genetic pathways between ear and kidney development, the critical roles multiple FGFs play in auditory development and the known hearing phenotype in mice deficient in klotho (KL), a critical co-factor for FGF23 signaling. Using functional assessments of hearing, we demonstrate that  FgfFibroblast growth factor 23 (FGF23) is a circulating hormone, typically secreted by osteoblasts and osteocytes, which regulates renal phosphate handling Kl−/− mice appear normal FgfKl−/− mice and double knockouts. Bioactive FGF23 injection results in phenotypic rescue of FgfKl−/− phenotypes This study sought to characterize the murine auditory phenotype due to FGF23 deficiency because other members of the FGF superfamily are critical for normal development of the auditory system in humans and animal models Kl−/− auditory phenotype is due to altered FGF23 signaling because KL and FGF23 can exhibit functions independent of each other FgfFgfFgfIt is currently unclear if the FgfFgfFgflacZ gene. Male, 6-week old mice were generated via the breeding of FgfFor this study, we utilized a previously developed FgfFgfFgfPhysiologic performance of the auditory systems of DPOAEs were measured in anesthetized mice via simultaneous presentation of two tones  and xylazine (0.02 mg/g) before intra-cardiac perfusion. 4% paraformaldehyde (PFA) was used for paraffin embedding and 1.5% PFA/2.5% gluteraldehyde for araldite work. A ventral approach was performed to expose the bulla and tympanic membrane for photography and extraction. The round and oval windows were perforated and intra-cochlear perfusion was performed to improve tissue preservation. Cochleae were extracted and submerged in fixative for 2 hours at room temperature. Samples for paraffin embedding were decalcified in EDTA for 2 days and stained with hematoxylin and eosin. Cochleae for araldite embedding were incubated in 1% osmium tetroxide for 1 hour and decalcified in EDTA with 1% gluteraldehyde for 3–4 days at room temperature.Slides for FGF23 immunostaining were deparaffinized in xylene and rehydrated with an alcohol ladder . Sections were immersed in 3% FgfFgfμCT). Mice were anesthetized and intra-cardially fixed as above. Soft tissues were removed from the head to expose the skull. The cranium and hard palate were carefully dissected, leaving only the portion of the skull base between the bullae and extending forward to contain the bony ear-canals. The tympanic membrane of each ear was perforated and samples were fixed overnight in 4% PFA.Gross morphology of the bulla, ossicles and cochlea of μCT scans were performed on a Scanco Medical μCT35 System . Scans were conducted in 70% ethanol with parameters: voxel size - 7 μm, X-ray tube potential - 55 kVp, X-ray intensity - 0.145 mA and integration time - 600 ms . For the 2D reformats, the raw data for each ear were separated and then brought up on a clinical imaging work station viewing console, where the raw data were displayed in three orthogonal planes. The data were first reformatted into a dataset of 7 μm thick images rendered in a plane parallel to the posterior semicircular canal (PSCC) and sagittal to the head. An orthogonal coronal data set was then made in a plane perpendicular to the PSCC and coronal to the head. For the 3D reconstructions, the raw data for each ear were displayed using 3D volume rendering with preset window level settings that were optimized for middle ear analysis (settings provided by the software developer). Regions of interest were manually segmented to exclude the walls of the middle ear and to isolate the ossicles.Raw FgfFgfThe sagittal and coronal images, and segmented 3D reconstructions from the FgfFgfFgfFgfFgfFgfFgfFgfFgfFgfMorphological assessments of middle and inner ear anatomy revealed that bullae from gf mice . The audgf mice . In contd whiter . The bulFgfFgfThe membranous labyrinth of the inner ear appears anatomically normal, healthy and properly organized in gf mice . Howeverrization .FgfFgfFgfFgfImmunostaining of cochlear slides revealed the widespread presence of FGF23 throughout most cell types of the inner ear . ImmunorFgf  and Fgfgf mice , or in Fantibody , indicatµCT sections from FgfFgfFgfFgfFgfFgfFgfComparison of 2D pecimens , as doespecimens . The incgf mice . The masgf mice  and petrgf mice  are subsgf mice . The incgf mice . Similargf mice .FgfFgfFgfFgfµCT contrast with surroundings due to lower ossicular density.Three-dimensional reconstruction of the ossicles highlights the lack of structural refinement in gf mice . RegionsFgfFgfFgfFgfFgfABR measurements  demonstrr trends : Fgf mithreshold measurements are known to be insensitive to certain types of neural damage amplitude and latency were also analyzed. Changes in the amplitude of ABR wave I can be more sensitive than threshold measurements to certain forms of auditory pathology and signal neuronal malfunction months before histological observation of neuronal loss FgfFgfFgfBecause ABR urements  is affecThreshold-adjusted ABR wave I latency values are presented in FgfFgfFgfFgfFgfFgfFgfFgfThese results demonstrate profound hearing loss in FgfKl−/− mice have been shown to exhibit hearing loss; 14–18 dB threshold shifts have been detected in ABRs elicited by clicks as well as tones at 4, 8, 16 and 32 kHz in 3 week old 129/SvJ mice non-threshold adjusted ABR wave I latency in response to 100 dB SPL tones and found a significant delay of Kl−/− mice.Our findings of significant conductive impairments in FgfKl−/− phenotypes. Conversely, the observed ABR wave I latency delays were more severe in Kl−/− mice than the FgfKl−/− mice were non-threshold adjusted, these metrics are not directly comparable. ABR latency decreases as tone intensity (in dB SL) increases; therefore the latency difference observed in Kl−/− mice may have a significant contribution owing to the The Klotho mutations presented hearing results from +/−Kl mice; thus comparison with the unexpected FgfFgfFgfKl phenotypes is made more interesting by recent research which has suggested a calcineurin-mediated FGF23 signaling pathway Unfortunately, neither of the above studies of FGF23. However, diseases with known elevations in serum concentrations of FGF23 co-present with hearing loss Our results raise questions concerning the prevalence of hearing loss among individuals with FGF23 deficiencies. No auditory phenotype has been reported in FGF23-mediated familial tumoral calcinosis, which results from a missense mutation in FgfOur findings also suggest a potential role for FGF23 in otitis media, a clinically important disease that afflicts 75–90% of Americans at least once before 3 years of age


In SynCAM 1 knockout mice the survival rate of existing spines was reduced and fewer filopodia-like structures were converted into stable spines. SynCAM 1flag overexpression resulted in more stable spines and fewer filopodia-like structures. When SynCAM 1flag overexpression is turned on the spine density rapidly increases within a few days. Interestingly, the spine density stayed at an elevated level when SynCAM 1flag overexpression was turned off. Our data indicate that the SynCAM 1 induced altered spine density is not caused by the formation of newly emerging protrusions, instead SynCAM 1 stabilizes nascent synaptic contacts which promotes their maturation. Concomitant with the synaptic stabilization, SynCAM 1 generally prolongs the lifetime of spines. In summary, we demonstrate that SynCAM 1 is a key regulator of spine stability.Formation and stability of synapses are required for proper brain function. While it is well established that synaptic adhesion molecules are important regulators of synapse formation, their specific role during different phases of synapse development remains unclear. To investigate the function of the synaptic cell adhesion molecule SynCAM 1 in the formation, stability, and maintenance of spines we used 2-photon  Dynamic changes of synaptic spines are associated with cognitive functions such as learning and memory. The formation and maintenance of synaptic spines is critically dependent on synaptic adhesion molecules24Members of the neuroligin, SynCAM and EphB receptor families are involved in the morphologic and functional differentiation of synapses45671011121314flag leads to an increase in synapse density and the loss of SynCAM 1 causes a decrease in synapse density in hippocampal CA1 pyramidal cellsHere we focus on the function of SynCAM 1 in spine formation and maintenance. SynCAM 1 belongs to the immunoglobulin superfamily (IgSF). The SynCAM family comprises four members in mammals (SynCAM 1–4)17in vivo microscopy of layer V pyramidal neurons in the visual cortex. We used Thy1-GFP mice that express the green fluorescent protein (GFP) in a sparse subset of cortical neuronsIdentifying the physiological role of a synaptic adhesion molecule is still a challenging question. The time domains of synapse formation and maturation are quite diverse; while forming a spine might take some minutes to hours, spines can be stable for several months. To cover the extended time domain we tracked individual spines in living animals by two-photon flag off. However, our data do not support the idea, that SynCAM 1 directly induces the formation of nascent protrusions. In summary, we show that SynCAM 1 is a key regulator of the spine stability.Here, we demonstrate that SynCAM 1 directly stabilizes newly formed contacts, thereby impacting on the maturation state of spines. Furthermore, SynCAM 1 improves the stability of mature spines. Inducing the overexpression of SynCAM 1 increases the spine density within a few days. Interestingly, the spine density does not rapidly return to control levels after turning the overexpression of SynCAM 1in-vivo two photon microscopy to follow the fate of individual spines over a long period of time in adult SynCAM 1 knockout (KO) mice completely lacking SynCAM 1flag overexpressing mice (OE)flag expression is driven by a tet-off system under the control of the CaMKII promotor allowing to suppress ectopic overexpressionTo study the effect of SynCAM 1 on spine dynamics, we employed First, we analyzed the spine density of layer V pyramidal neurons, which was significantly reduced in adult SynCAM 1 KO mice compared to control (CTR) littermates  are persistent .flag overexpressing miceflag overexpressing is regulated by a tet-responsive element; the necessary tTA is expressed under the control of the CaMKII promotorflag mice received doxycycline-containing water (1 g/l). Littermates lacking the SynCAM 1flag transgene served as controls. Accordingly, SynCAM 1 Knockout micead libitum. Mice were separately housed after the craniotomy.Previously described SynCAM 1Cranial surgery was performed on 12 week old mice as described elsewhereFor imaging the mice were lightly anesthetized with an i.p. injection of Ketamine  and Xylazine  and head-fixed under the microscope. The body temperature was kept constant and the eyes protected from dehydration with eye ointment . We used a custom built two-photon microscope driven with a Ti:sapphire Laser  running at 910 nm for GFP excitation. The setup was controlled by ScanImageImages were analyzed with a custom written software . In total we analyzed 14723 individual spines present for up to eight different time points over a total dendritic length of 14.347 mm. Only dendrites of cells of which the soma was localized deeper than 350 μm measured from the cortex surface were analyzed. Scoring criteria were similar as describedFor Western blots cortex samples were sonicated and lysed in RIPA buffer. Samples were then separated using 8–10% SDS-PAGE and blotted on a polyvinylidene difluoride membrane . The following antibodies were used: Mouse anti-FLAG 1:1000 , mouse anti-actin 1:1000  and HRP-linked horse anti-mouse 1:1000 . Chemiluminescence signals were detected using a PeQlab Solo Imaging System .All imaging data were quantified blind to experimental conditions. Data were tested for normal distribution and statistical significance using Prism 5 . We used paired student’s t-tests to compare the means of different groups and unpaired t-tests for comparison of individual time points. Significance levels are denoted as follows: *p < 0.05; **p < 0.01; ***p < 0.001.How to cite this article: Körber, N. and Stein, V. In vivo imaging demonstrates dendritic spine stabilization by SynCAM 1. Sci. Rep.6, 24241; doi: 10.1038/srep24241 (2016).


Here we review current knowledge specifically on layer 5 (L5) inhibitory microcircuits, which play a critical role in controlling cortical output. We focus on recent work from the well-studied rodent barrel cortex, but also draw on evidence from studies in primary visual cortex and other cortical areas. The diversity of both deep inhibitory neurons and their pyramidal cell targets make this a challenging but essential area of study in cortical computation and sensory processing.Inhibitory neurons play a fundamental role in cortical computation and behavior. Recent technological advances, such as two photon imaging, targeted  Before surveying the existent literature on layer 5 (L5) inhibitory neurons, we briefly review current knowledge on the connectivity and physiological properties of L5 pyramidal cells (PCs), as it provides important context for understanding L5 inhibitory circuits. One feature that distinguishes L5 from other cortical layers is the diversity of its PCs, which send a myriad of long-range projections to other cortical and sub-cortical structures  the pyramidal tract (PT) neurons, which project to subcortical regions, are located mainly in layer 5B, and display impressive “thick-tufted” apical dendritic morphologies; and (2) intratelencephalic (IT) neurons, which project mainly to other cortical regions and striatum, are found mainly in layer 5A, and have smaller and less complex dendrites  cells in L5, and especially L5B, encode a high degree of information about the temporal features of a sensory stimulus and may play an important role in initiating precisely timed sequences of spikes during sensory responses  between L5 PCs. FDDI is generated when an L5 PC fires a high-frequency burst of action potentials and excites a postsynaptic L5 Martinotti cell  suggest that inhibition onto L5 PCs most strongly originates from interneurons within L5 , is a nearly ubiquitous motif in cortical microcircuits where afferent excitatory input simultaneously recruits local inhibitory neurons that provide inhibition to the same cells receiving excitation. Circuits for FFI have been well studied at the projections from thalamus to L4  can recruit both L5 PV and SOM cells driving various forms of FFI. Interestingly, as described for FFI circuits in hippocampal CA1  can also lead to unintended problems. First, these lines are not completely non-overlapping. Up to ~10% of Cre expressing cells in the widely used SOM-IRES-Cre line may actually be PV expressing, fast-spiking interneurons (Hu et al., However, there are potential solutions to these problems. The first would be the development and adoption of new transgenic lines that target highly specific inhibitory cell types (Taniguchi et al., in vivo has mostly focused on superficial interneurons due to the technical difficulty of imaging deeper cells in the cortex due to light scattering, and the practical challenges of maneuvering recording pipettes into deeper layers under visual guidance. A number of recent technical advantages should overcome these challenges. These include adaptive optics for deeper in vivo imaging (Wang et al., As we alluded to above, the recent explosion of functional information on inhibitory subtypes New optical methods will also open new avenues for manipulation of interneuron activity. Novel techniques for spatial light modulation are poised to provide unprecedented optogenetic precision, enabling experimenters to arbitrarily target manipulations to single cells or even specific ensembles of neurons (Bovetti and Fellin, AN and HA researched, wrote, and edited the manuscript.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.


Genetic variants of pulmonary surface glycoproteins like Muc5a and Muc5b are known to affect mucociliary clearance, control of infections and maybe associated with pulmonary fibrosis –3. We tePreterm infants with a birth weight below 1500 grams born in the GNN were genotyped for rs35705950 (n=8029). Furthermore, 1272 infants were chip-genotyped , and candidate polymorphisms were replicated in two additional groups of preterm infants (n=3839 and n=2830).-5). In GWA, we identified 10 candidate polymorphisms however, only one minor allele proved to be associated with a reduced need for surfactant treatment in preterm infants .Frequencies for rs35705950 were in the expected range . The GT/TT-genotype was a strong genetic risk factor for the development of BPD in ventilated infants with sepsis .


The design is simple, making fabrication straightforward and relatively inexpensive. Reactor operation is characterized using two infrared absorption measurements to determine exhaust times for tetrakis(dimethylamino)titanium and water, proto-typical ALD precursors, in a pressure and flow regime commonly used for ALD.The design and operation of a simple, optically-accessible modular reactor for probing thermal thin film deposition processes, such as atomic layer deposition processes (ALD) and chemical vapor deposition (CVD), is described. This reactor has a nominal footprint of 225 cm Atomic layer deposition (ALD) is a thin film deposition technique  increasiIn situ optical diagnostics have been demonstrated as valuable tools for investigating numerous ALD chemistries [in situ diagnostics is reactor design, which must permit optical access without significant modification of the desired deposition chemistry under investigation. A variety of reactor chamber designs have been reported for ALD growth [mistries . An inteD growth ,5,6, eacIn past works we presented the design and characterization of a diagnostics-accessible ALD reactor , which pThe expansion cone (see (b) The reactor body (see (c) The exhaust manifold (see (e) Gasses were introduced into the expansion cone using four symmetric ≈4.8 mm ID, ≈6.3 mm OD (quarter inch OD) tubes (see (a) 2 windows for TDMAT and water measurements, respectively.The reactor was instrumented using a double window configuration (see (d) −1 using a broadband infrared source in conjunction with a nominal 10.5 µm center wavelength bandpass filter, as previously described [The TDMAT concentration as a function of time was determined by measuring the absorbance of the TDMAT feature at ≈948 cm−1 using a distributive feed-back diode laser and a wavelength modulation detection scheme, as previously described [The water concentration as a function of time was determined by monitoring the rovibrational transition of water at 7181.156 cmescribed . Data pr0 = 1) with an exponential decay constant equal to the ratio between volumetric flow rate of the purge gas  and the calculated reactor volume upstream of the face plate (V = 1.16 L):6 after a 3.5 s purge. This purge time accounts for both reactor exhaust times and time required for each precursor to traverse the injection system.Agreement between the model and the measurement results for water is much better than that for TDMAT. This difference is likely the result of TDMAT being more efficiently entrained in the carrier gas flow due the smaller diffusion coefficient of TDMAT relative to water, resulting from the larger collision diameter and mass of TDMAT. In both cases agreement between the model and precursor concentration converge as the precursor concentrations approaches zero, suggesting the model is a useful tool for approximating purge times. For reactor pressures of 133 Pa and purge gas flow rates of 300 mL/min (at 273.15 K and 101.3 kPa) the model predicts a reduction of precursor concentrations by greater than a factor of 10A diagnostic-accessible ALD reactor capable of depositing device quality oxides using thermal ALD while being small enough to be easily integrated into an optical train was described. The operation of the reactor was demonstrated by measuring the time-dependent concentration profiles of TDMAT and water, two prototypical ALD precursors with significantly different mass diffusivity values. For both precursors, in a typical pressure and flow regime for ALD, purge times could be estimated using a simple exponential decay model with an exponential decay constant equal to the ratio between volumetric flow rate of the purge gas and the cell volume.