Mycobacterium tuberculosis (Mtb), is a major world health problem. The production of reactive nitrogen species (RNS) is a potent cytostatic and cytotoxic defense mechanism against intracellular pathogens. Nevertheless, the protective role of RNS during Mtb infection remains controversial. Here we use an anti-nitrotyrosine antibody as a readout to study nitration output by the zebrafish host during early mycobacterial pathogenesis. We found that recognition of Mycobacterium marinum, a close relative of Mtb, was sufficient to induce a nitrosative defense mechanism in a manner dependent on MyD88, the central adaptor protein in Toll like receptor (TLR) mediated pathogen recognition. However, this host response was attenuated by mycobacteria via a virulence mechanism independent of the well-characterized RD1 virulence locus. Our results indicate a mechanism of pathogenic mycobacteria to circumvent host defense in vivo. Shifting the balance of host-pathogen interactions in favor of the host by targeting this virulence mechanism may help to alleviate the problem of infection with Mtb strains that are resistant to multiple drug treatments.Pulmonary tuberculosis (TB), caused by the intracellular bacterial pathogen  Mycobacterium tuberculosis (Mtb), which represents a major global health threat. The current high incidence and associated mortality worldwide is partly explained by the increasing occurrence of multi-drug resistant Mtb strains. This makes TB a key priority for infectious disease research, since understanding of the host-pathogen interactions during Mtb pathogenesis is necessary to develop novel therapeutic strategies Pulmonary tuberculosis (TB) is a common and often lethal infectious disease caused by the pathogenic bacterium 2O2), superoxide anion (O2−), and hydroxyl radical (OH•), is catalyzed by the NADPH-oxidase complex during respiratory burst iNOS transcription plays an important role in the inflammatory and infection response and many cells of the immune system are capable of producing NO The combined efforts of the innate and adaptive arms of the immune system are relatively efficient in containing Mtb infection in immunocompetent individuals. It is estimated that only 5–10 out of a 100 individuals newly infected with Mtb develop active TB during their lifetime •) −), a potent RNS reaction that causes lipid peroxidation, DNA damage, oxidation of thiols, and nitration of tyrosine residues 2O2 and NO2− as substrates Salmonella enterica serovar Typhimurium Escherichia coliCandida albicansAlthough a mutation in the components of the NADPH-complex increased susceptibility to Mtb Mtb that can lead to direct scavenging of reactive species, repair and protection of bacterial proteins as well as DNA An essential role for NO in the killing of Mtb by mononuclear phagocytes was demonstrated in a murine TB model Mycobacterium marinum (Mm) is a close relative of human Mtb and displays similar pathogenesis to the human disease, including the formation of caseating granulomas and development of latency The natural fish pathogen Zebrafish lines were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (zfin.org). The breeding of adult fish was approved by the local animal welfare committee (DEC) of the University of Leiden and adhered to the international guidelines specified by the EU Animal Protection Directive 2010/63/EU. Adult zebrafish were not sacrificed for this study. All experiments in this study were performed on embryos/larvae at 48, 72 and 120 hours post fertilization (hpf) before the free-feeding stage and did not fall under animal experimentation law according to the EU Animal Protection Directive 2010/63/EU. Embryos were anaesthetized with 0.02% Tricaine (MS-222) and subsequently euthanized with 4% paraformaldehyde at 2, 4, and 5 days post-fertilization for immuno-histochemical and microscopy analysis.Tg(mpx:GFP)i114Tg(lyz-DsRED2)nz50myd88−/−hu3568/Tg(mpx:GFP)i114mpx−/−NL144/Tg(mpx:GFP)i114. The mpx−/−NL144 “spotless” mutant line harbors a C to T point mutation at nucleotide position 1126 of the mpx RefSeq mRNA sequence (NM_212779) resulting in a premature stop codon . Adult fish were maintained on a 14 hour light and 10 hour dark cycle at 28°C in accordance with local animal welfare regulations.Zebrafish strains (wildtype strain used was AB/TL) were maintained according to standard protocols. Transgenic and mutant strains used were M. marinum strain M (ATCC #BAA-535) Infection experiments were performed using Injection of bacteria was performed into the blood-forming region above the caudal vein at 28 hpf as previously described At the timepoint of infection 5 µM DAF-FM DA in embryo water was applied to a Petri dish of around 30 embryos as previously described Embryos were imaged at 4 dpi on a Leica MZ16FA Fluorescence Stereo Microscope. Brightfield and fluorescence images were generated with a Leica DC500 (DFC420C) camera. Bacterial loads were analysed using dedicated pixel counting software as previously described Larvae were fixed in 4% paraformaldehyde in PBS overnight at 4°C and nitrotyrosine levels were immune-labelled using a rabbit polyclonal anti-nitrotyrosine antibody  at a 1∶200 dilution of primary antibody, and were detected using an Alexa Fluor  secondary antibody. Mpx activity was labelled with TSA  as previously described For iNOS antibody staining, larvae were fixed in 4% paraformaldehyde in PBS overnight at 4°C and iNOS was immuno-labeled with a rabbit polyclonal iNOS antibody  as previously described Tg(mpx:GFP)i114 line. These measurements assess the cell fluorescence of each individual immune cell, corrected for cell size and background fluorescence of the image.Embryos were imaged at 1 dpi, in the presence or absence of infection, embedded in 1% low melting point agarose  and transferred to a Leica DMIRBE inverted microscope with a Leica SP1 confocal scanhead for imaging with 40 or 63 times lenses. For quantification purposes acquisition settings and area of imaging  were kept the same across the groups. Corrected total cell fluorescence was calculated for each immune-stained cell using Image J measurements as previously described cxcl8-l1 E1/I1 and cxcl8-l2 E1/I1) Il8 signaling was inhibited using a previously described splice blocking morpholino (an injection mixture of hif-1αb (ZFIN: hif1ab) RNA at the one cell stage as previously described Embryos were injected with dominant active All data were analysed  using unpaired, two-tailed t-tests for comparisons between two groups and one-way ANOVA (with Bonferroni post-test adjustment) for multiple group comparisons.Detection of iNOS in the zebrafish model has been previously reported, however detection measures are often limited by either poor specificity for different types of NOS, or by relatively low levels of expression Cyprinus carpio) to study the differential contribution of macrophages and neutrophils to nitrosative stress in response to a parasitic infection Tyrosine nitration is a hallmark of nitrosative stress caused by ROS and RNS produced by macrophages and neutrophils nos2a, one of the zebrafish genes for iNOS Mm are rapidly phagocytosed by macrophages when injected into the circulation of zebrafish embryos After intravenous Mm infection it is primarily the macrophages that phagocytose the bacteria, and yet the upregulation of nitrotyrosine levels is found mainly in neutrophils. This upregulation of neutrophil nitrotyrosine is a systemic neutrophil response, rather than localized around areas of infection, therefore we postulated that chemokine signaling would be a likely candidate responsible for transduction of the signal from infected macrophages to neutrophils. Interleukin 8 (IL8) is a well characterized neutrophil attractant that is released by macrophages and epithelial/endothelial cells iNOS in vitro −/−myd88) was more susceptible to Mm infection −/−myd88 embryos compared to +/+myd88 embryos , can recognize pathogen associated molecular patterns (PAMPs) present on the exterior of mycobacteria and initiate an appropriate immune response, which can be further amplified by signaling through cytokine receptors such as the Interleukin 1 receptor (IL1R)  embryos , indicatcontrols . These fWe evaluated tyrosine nitration at later stages of Mm pathogenesis by performing anti-nitrotyrosine staining on zebrafish larvae with early granuloma structures. At 3 days post infection (dpi) anti-nitrotyrosine staining co-localized with, or was in proximity of, larval granulomas, demonstrating that nitrosative defense mechanisms remain active at this later time point of pathogenesis . Intereshif-1αb. At this stage of infection the infection is systemic and granuloma structures have yet to aggregate. We observed that exposure to heat killed Mm further increased tyrosine nitration levels of neutrophils, while infection with living wild type or ΔRD1 Mm significantly decreased tyrosine nitration levels compared to mock-injected controls . The absence of infection-induced tyrosine nitration in myd88−/− embryos is therefore consistent with the mycobacterial cell wall containing lipoproteins and lipoglycans serving as TLR ligands It has previously been difficult to quantify nitration output in leukocytes, however using an anti-nitrotyrosine antibody staining protocol we can accurately measure the nitration output of neutrophils and compare it to the Mpx-dependent basal levels. Our data demonstrate a robust upregulation of tyrosine nitration levels in neutrophils after Mm infection in a zebrafish host, shown by anti-nitrotyrosine antibody staining, alongside DAF FM-DA and anti-iNOS staining. Although the brightest fluorescence was observed intracellularly, low-level extracellular ‘speckled’ staining was also observed, both with and without infection, which may indicate extracellular tissue nitration as observed in another fish model nos2a gene in zebrafish, nor pan-NOS or iNOS specific inhibitor treatment resulted in significantly increased Mm bacterial burdens hif-1αb we have previously demonstrated an associated decrease in bacterial burden Neutrophils, both bystanders and those infected, have increased nitrotyrosine/iNOS levels at 1 dpi after infection with Mm and Salmonella in the zebrafish embryo Mycobacterium leprae infection, in which the mycobacteria can transcriptionally reprogramme their host cells, Schwann cells, into a stem-cell like state 2 and NO levels and activate the oxidative stress response, which maintains the redox balance and modulates virulence 2O2 or NO initiates a robust transcriptional response, including the expression of cellular repair mechanisms and upregulation of genes that perform ROS and RNS scavenging functions hif-1αb increase in neutrophil NO response as a model to investigate whether this was a bacterial-driven response. Attenuated and heat-killed strains do not replicate and form granulomas at the same rate as live Mm, therefore comparing granulomas from different Mm types at 3 or 4 dpi would be technically difficult. The dominant active hif-1αb model allowed investigation at 1 dpi, a stage at which infection is still systemic before granulomas have begun to aggregate. In the Hif-1α-stabilized model we observed that tyrosine nitration attenuation was independent of the RD1 virulence locus, required for survival and replication inside macrophages, but that this effect was not caused by heat-killed bacteria which increased the nitrotyrosine levels further. These findings are in contrast to the induction of tyrosine nitration, which is induced by ΔRD1 and heatkilled Mm suggesting alternative regulation of induction and subsequent attenuation of tyrosine nitration. Together, these data suggest that Mm infection initially induces increased NO production in neutrophils and that this response is attenuated during the course of infection by a bacterial virulence mechanism. A bacterial protein potentially involved in regulating this oxidative stress response is OxyR Emerging evidence suggests that mycobacteria are able to manipulate cells of the host to allow for intracellular growth. This has been shown in vivo during The search for new anti-TB therapies has intensified with the increasing occurrence of Mtb strains that are multi-drug resistant Figure S1Anti-nitrotyrosine predominantly labels neutrophils in granulomas. Example fluorescent micrographs of anti-nitrotyrosine staining performed on 4 dpi granuloma structures after infection with Mm. The staining colocalized, in the main, with the mpx:GFP fluorescence of neutrophils. Two brightly stained example cells are indicated by the white arrow heads.(TIF)Click here for additional data file.Figure S2M. marinum does not grow on plates. The injection dose of live, heat killed or ΔRD1 Mm were plated for CFU counts. Heat-killed Mm did not grow on appropriate media after incubation for a week.Heat killed (TIF)Click here for additional data file.Figure S3Nitrotyrosine levels in progression granulomas are lower when co-localized with bacteria. (A) Example fluorescence confocal micrographs of anti-nitrotyrosine staining performed on 5 dpi granuloma structures in wild type embryos infected with Mm. A merged image of extended focus is shown in the top left panel while the signal Z-plane used for the measurements is shown in the top right panel. (B) Gray values of the bacterial and anti-nitrotyrosine fluorescence signals measured along a straight line through the center of the granuloma (see white lines in (A)), intensity of the fluorescent signal was measured using ImageJ. Numbered peaks in the graphs correspond to numbered patches of tyrosine nitration in (A).(TIF)Click here for additional data file.


Arabidopsis thaliana. Nonetheless, functional evidence for their involvement at telomeres has not been obtained, likely due to functional redundancy. Here we performed genetic analysis on the TRF-like family consisting of six proteins  which have previously shown to bind telomeric DNA in vitro. We used haploid genetics to create multiple knock-out plants deficient for all six proteins of this gene family. These plants did not exhibit changes in telomere length, or phenotypes associated with telomere dysfunction. This data demonstrates that this telobox protein family is not involved in telomere maintenance in Arabidopsis. Phylogenetic analysis in major plant lineages revealed early diversification of telobox proteins families indicating that telomere function may be associated with other telobox proteins.Proteins that bind to telomeric DNA form the key structural and functional constituents of telomeres. While telomere binding proteins have been described in the majority of organisms, their identity in plants remains unknown. Several protein families containing a telomere binding motif known as the telobox have been previously described in  Indeed, functional analysis of TRFL proteins in rice and tobacco has indicated their involvement in telomere length homeostasis  collection and other alleles were from the European Arabidopsis stock centre (NASC). Plants were grown at 22°C in 16 h light/8 h dark cycles.All T-DNA insertions used are shown in Supplementary Table cenh3/GFP-tailswap haploid inducer line was described previously by (cenh3 mutant plants were confirmed by PCR genotyping using derived Cleaved Amplified Polymorphic Sequence (dCAPS) oligos (5′-GGTGCGATTTCTCCAGCA GTAAAAATC-3′ and 5′-CTGAGAAGATGAAGCACCGGCGATAT-3′). Resulting PCR products were digested with EcoRV, cleaved wild type (WT) alleles produced 191 and 24 bp fragments.The ously by . Homozygcenh3/GFP-tailswap lines are mostly male sterile, but can be crossed as female. Heterozygous quadruple or sextuple mutants were crossed to cenh3/GFP-tailswap lines to produce haploid offspring that were homozygous for a combination of insertions derived from the male parent. Only plants that displayed the haploid phenotype as described by Haploid inducer g) and 350 μl was transferred to 350 μl isopropanol. Tubes were inverted to mix and centrifuged for 20 min at 4000 rpm. Supernatant was poured away and the pellet was washed with 70% Ethanol. The remaining pellet was air dried and resuspended in 100 μl dH2O. Telomere length was determined by terminal restriction fragment analysis, and statistical analysis of telomeric smears was performed using the TeloTool software  tubes were centrifuged for 10 min at 4000 rpm .Sequences of telobox containing proteins were obtained from indicated plant genomes by using A. thaliana Group I TRFL proteins result from relatively recent duplication events in Brasicaceae . Therefore, some paralogs may still retain overlapping functions. To begin elucidating the role of TRFL proteins at telomeres, we first examined the published allele of tbp1-1 which has been reported to show telomere elongation . We reasoned that double knockouts could exacerbate tbp1-1 telomere phenotypes. Heterozygous plants containing the published tbp1-1 allele (FLAG_072C05) were crossed to plants heterozygous for the trfl9 (GK-036D11) mutation. Double heterozygous F1 plants were then selfed and First generation WT, double, and single mutants were segregated. DNA from five pooled plants was extracted from second and third generation of double mutants of the same lineage and subject to TRF analysis . To extract data from TRF blots, we used the recently published software TeloTool to measure telomere length and create graphs to better illustrate mean and range of telomeric smears . Double tbp1 trfl9 mutants also did not appear to shown any great change in telomere length over three generations. Previous studies have shown that telomere lengthening occurs gradually in tbp1-1 mutants over four generations . These data argue that absence of TBP1 and TRFL9 does not have any discernible effect on telomere structure.Because of the essential role of telomere binding proteins in telomere protection, we reasoned that their inactivation would lead to resection of blunt ended telomeres. However, no observable difference was seen in the presence of blunt ends in Arabidopsis plants with multiple mutant combinations of the genes in the group I TRFL family. Because generation of sextuple mutants would require extensive screening of a large number of plants in segregating populations, we decided to take advantage of centromere induced genome elimination to produce haploid F2 plants , trfl1 (SALK_052864), trfl2 (SAIL_73_G01), and trfl4 (SAIL_73_F07) mutants were also obtained. In order to combine all alleles into the same plant, we first created three combinations of double heterozygous mutants . Next, we generated two combinations of quadruple mutants, and finally quintuple and sextuple mutants as illustrated in the crossing scheme . Heterozygous quadruple mutants were then crossed as male to cenh3/GFP-tailswap plants; homozygous quadruple haploids were obtained along with the WT combination. Diploid seeds were obtained from mutant and WT haploids by spontaneous diploidization . First, two single doubled haploid plants were tested by TRF analysis for WT, -/- trfl9-/- trp1-/- trfl1-/-tbp1 and -/- trfl1-/- trfl2-/- trfl4-/-trp1 combinations . Seeds were collected from these plants and pooled DNA from 5 plants was used for TRF analysis in the following generation . Terminal restriction fragment analysis of resulting -/- trfl9-/- trp1-/- trfl1-/-tbp1 and -/- trfl1-/- trfl2-/- trfl4-/-trp1 lines showed no effect on telomere length regulation .Centromere induced genome elimination involves generation of haploids by crossing diploid plants as male to the trp1 trfl1 mutations, but segregating for the other four alleles . The haploid induction process was repeated by crossing these plants to the cenh3/GFP-tailswap plants and segregating quintuple and sextuple haploid plants. Individual quintuple and sextuple haploid plants were fully viable and exhibited neither retarded growth in comparison to haploid plants that segregated as WT, nor defects typical for plants with dysfunctional telomeres , although observed variation seen among individual samples suggests that sextuple mutants could display a higher level of telomere length variation compared to WT. The telomere lengths observed here, however, all lie within the natural telomere length limits seen in Col-0 lines and natural variation amongst diverse Arabidopsis accessions was also shown to vary between approximately 1 and 9 kb . This demonstrates ancient origin of the three telobox protein families and their diversification early in evolution of the plant lineage. Hence, telomere function can be associated with either of the remaining two telobox families.Our genetic analysis excluded the possibility that the Group I TRFL protein family harbors functional counterparts of human TRF1/2. Thus, the candidate protein(s) may be encoded by one of the other two telobox families. It is expected that that the Arabidopsis. These proteins form the core part of shelterin and are essential for telomere maintenance and function. Cells expressing dominant negative alleles and conditional knockouts of TRF2 exhibit telomere fusions and telomere length defects demonstrating an essential role of TRF2 in telomere protection . Association with telomeric DNA may, therefore, not be necessarily for telomere specific functions and can similarly colocalize with non-telomeric sequences. Chromatin Immunoprecipitation (ChIP) studies performed within the same paper, however, confirm binding to telomeric sequences in Arabidopsis. With this evident telomere binding capacity and interaction with Pot1b and the N-Terminus of TERT, SMH proteins also show promise as telomere binding components of Arabidopsis telomeres (trb1 mutants although the effect is relatively small after five generations of selfing (The question remains as to what proteins comprise the telomere capping complex in eric DNA . Recentlco cells . One cavelomeres . Telomer selfing . This coNF designed and performed the experiments and wrote the paper. KR designed the experiments, performed phylogenetic analysis and wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mysm1, Vangl1, Trpc4ap, Nom1, Sparc, Farp2, and Prkab1), while others were ascribed new cutaneous functions on the basis of the screening approach . The integration of these skin specific screening protocols into the Sanger-MGP primary phenotyping pipelines marks the largest reported reverse genetic screen undertaken in any organ and defines approaches to maximise the productivity of future projects of this nature, while flagging genes for further characterisation.The skin is a highly regenerative organ which plays critical roles in protecting the body and sensing its environment. Consequently, morbidity and mortality associated with skin defects represent a significant health issue. To identify genes important in skin development and homeostasis, we have applied a high throughput, multi-parameter phenotype screen to the conditional targeted mutant mice generated by the Wellcome Trust Sanger Institute's Mouse Genetics Project (Sanger-MGP). A total of 562 different mouse lines were subjected to a variety of tests assessing cutaneous expression, macroscopic clinical disease, histological change, hair follicle cycling, and aberrant marker expression. Cutaneous lesions were associated with mutations in 23 different genes. Many of these were not previously associated with skin disease in the organ ( Recent developments in high throughput applications to manipulate and inactivate specific genes in mouse embryonic stem cells (ES cells) have allowed for the initiation of large scale reverse genetic screens in the mouse. The immediate connection of a phenotype to a mutated (null) gene represents a paradigm shift in our ability to explore gene function. This study utilized such a screening approach to investigate the genetic contribution to skin development and homeostasis. Not only does this approach provide insight into the genetics of skin biology, it is also instrumental in generating novel models with which to study the genetic underpinnings of skin disease. Initial screening of 562 mutated genes in mice uncovered previously unrecognized genes involved in the biology of this organ and identified novel functions for previously studied genes associated with epidermal phenotypes. Taken together, these results highlight high throughput screening approaches that are valuable in reverse genetic screening and provide a pool of mouse mutants, available to the scientific community, that will serve as the basis for further detailed investigations into skin function and skin disease. To maximise the value of this resource, it is important to develop organ specific secondary screens to complement the broad phenotyping efforts applied in existing production pipelines. To this end, we implemented a multi-parameter, multi-test skin screen to identify mouse models of skin dysfunction and to elucidate genetic pathways important for skin development and function. In this study, we detail the skin assessment pipelines internal to the Sanger-MGP as well as skin analyses performed externally, with the exception of the fluorescent tail whole mount analysis, published elsewhere Recent advances in the high throughput production of targeted mutant mice using gene targeting in ES cells have made it possible to rapidly and effectively produce knockout mouse lines for use in phenotypic screening The skin is the largest of the intermediate sized organs in the human body Mammalian Phenotype (MP) ontology terms http://www.informatics.jax.org/searches/MP_form.shtml), 2 of which are novel  were derived from the EUCOMM/KOMP knockout first conditional-ready targeted ES cell resource on a C57BL/6N background Krt76, Myo5a  followed by general integument phenotypes  (lacZ) reporter gene and found that with the exception of two genes (Myo5a and Prkab1) all were expressed in the epidermis or dermis (Sparc lacZ expression in tail whole mounts was ubiquitous (https://www.mousephenotype.org/data/genes/MGI:98373). Myo6 expression in ear epithelia has been reported using IHC Myo5a and Prkab1 expression were not detected via the lacZ reporter epidermal expression of Myo5a has been previously reported lacZ reporter sensitivity issue.The simplest aspect of the skin screen comprises the visual assay for overt abnormalities of the pelage . This ke6, Myo5a , Mysm1, 6, Lrig1 , Myo6, N6, Nsun2 ) and nai(Prkab1) . A wild The hair follicle is a dynamic mini organ that continually undergoes a controlled program of growth (anagen), regression (catagen), and resting (telogen). Accordingly, hair follicle morphogenesis is maintained by a complex program of proliferation, differentiation and death. Regulators of this process include developmentally important proteins such as members of the bone morphogenetic protein (BMP), hedgehog (HH) and wingless-related MMTV integration site (WNT) signalling pathways, as well as a variety of cytokines, transcription factors, and adhesion molecules  and assessed the colour of a shaved patch of skin daily for 3 weeks from 35 days post-partum. As expected, initially all mice were in the second postnatal anagen phase (and hence were black skinned) and exited anagen by ∼42 days of age, reaching telogen by 7–8 weeks of age. Males in this cohort reached telogen slightly ahead of females and generally had a more pale pink skin colour than females in telogen . This piTrpc4ap/+  with abnormalities in only one or both sexes:   and a n1 Myo5a, . We did Aldh18a1 , hyperkeratosis (Krt76) and aberrant hair follicles or cycling  at which point dorsal skin from 1 to 2 female mice was collected per mutant strain for histopathology assessment. Males were generally not assessed due to a higher incidence of fighting that could result in skin damage. Tissue sections from the mutant mouse lines and control mice were stained with H&E and underwent blinded assessment by an expert pathologist. Abnormalities were then re-classified into MP terms. For an abnormality to be scored as positive, presentation had to be multi-focal or diffuse, thus rare focal events were eliminated and considered part of the normal spectrum. Forty-four wild type control mice and 514 mutant animals  were examined; this encompassed 512 mutant lines with a range of 1–2 mutant mice per line. Three wild type mice and 30 mutant animals (from 30 strains) demonstrated abnormalities and in some cases, multiple abnormalities, giving 35 phenotypes. Strain-related background phenotypes were identified in wild type mice in the MP term categories of abnormal hair shaft morphology , and skin inflammation . Mutant animals also demonstrated these defects, for example, skin inflammation was detected in 17 mutants out of 514 (∼3.3%). Because of their presentation in wild type and test animals these phenotypes were not considered further, although it is possible that a fraction of the flagged mutant phenotypes are directly related to the induced genetic lesion. Further studies of individual lines would be required to establish this. However, the results of this histological analysis flagged other phenotypes not detected in wild type animals including 12 unique abnormalities/MP terms from 30 strains . These iAldh18a1 , Ccdc57 nd Prmt3 ), abnorm , abnormtor pili ), enlargA number of the screens identified new players in cutaneous biology or identified new phenotypes not previously associated with these genes. Some individual examples are discussed.th century and the pioneering work of Cuenot and Castle in the early 20th century utilised coat colour to first demonstrate Mendelian inheritance in mammals Mysm1, which encodes a histone 2A deubiquitinase that regulates stabilisation of linker histone H1 with nucleosomes Mysm1 mutant mice exhibited a white belly patch (lacZ) reporter activity indicates that Mysm1 is expressed in the footpad . However, in the small number of surviving adult knockouts generated, we observed hyperpigmentation of the paw pads specifically in homozygous male animals (3/4 homozygotes) but not in females (0/3 homozygotes)  . Given t the ear .Nsun2, Lrig1, and Farp2 and none of them have previously been associated with this process. Nsun2, ), is a direct downstream target of MYC in the skin and is essential for keratinocyte proliferation Nsun2 are known to affect the entry of hair follicle stem cells into the regenerative program and in regulating epidermal stem cell self-renewal and differentiation Nsun2 expression specifically in the hair follicles of dorsal skin (Nsun2/+ mice can be seen as a detachment of the hair follicle from the bulge in Nsun2/Nsun2 mice (as these mice were in anagen at this time point), their scruffy coat    and the  (pinna) . In thisshedding  which mashedding .Farp2, is a guanine exchange factor known to be involved in osteoclast podosome dynamics lacZ signal for Farp2 expression in the paw pad skin  and a further additional pigmentation phenotype in Myo5a mutants shows that even for the most obvious cutaneous feature, there remains considerable value in ongoing screening for new causative mutations. In particular, clinical and histological presentations vary between alleles based on the type of mutation  or inbred strain background (strain specific modifier genes), and so comparison between these “knockout ready” targeted mouse lines and existing spontaneous mutants present in various public repositories will be particularly informative in respect of gene function.We detail one of the most comprehensive reverse genetic skin screens yet reported in mice. The gene targeting strategy, combined with a robust skin phenotyping protocol, has allowed immediate establishment of a preliminary connection between a genotype and a phenotype. Such approaches have only become feasible through the recent advent of high throughput, targeted mouse mutagenesis screens. Clearly, more detailed follow-up studies are necessary to elaborate on these initial findings and to investigate in greater detail the contribution of a given gene to the biology of the skin. One such follow-up study on Similarly the skin might be considered an “easy” target for phenotypic screens, and that important genes could simply be identified based on a casual observation of the mutant animal in question. Our results argue that this is not the case. Instead we find that there is significant value in including secondary, organ specific screens in large scale projects of this nature. In deciding which phenotypic screens to apply to a given mutant mouse population one has to consider the likelihood that a given test will identify a phenotype not also apparent in other primary phenotyping screens. The object of such an assessment is therefore to reduce the costs and processes associated in examining such large cohorts of mice, whilst maximising the value of the resource (i.e. in robust phenotypes flagged). Interestingly, based on our experience, each of the phenotypic screens utilised added significantly to the base level of hits in the screen . While gAll mouse studies were undertaken by Wellcome Trust Sanger Institute Mouse Genetics Project as part of the International Mouse Phenotyping Consortium and licensed by the UK Home Office in accordance with the Animals (Scientific Procedures) Act 1986. Animal models maintained under the auspices of ethics applications to Monash University were subject to the conditions of the Australian Bureau of Animal Welfare.lacZ reporter cassette was integrated upstream of a floxed essential exon c-BrdTyr background. For mice in the histopathology screen details of the strain background, age of sampling, diet, sex, time of tissue harvest, allele name and heterozygosity/homozygosity are detailed in ad libitum access to food ) and water. Specific details of housing and husbandry for individual lines are available from mouseinterest@sanger.ac.uk. For some lines  cassette was assessed using heterozygous cohorts of mice  generated specifically for expression analysis. The pattern of β-gal reporter gene activity was determined by lacZ labelling of whole mount tissue preparations from heterozygous knockout. Mice were perfused with fresh cold 4% paraformaldehyde (PFA). Tissues were fixed for a further 30 min in 4% PFA, rinsed in phosphate buffered saline (PBS) and labelled with 0.1% X-gal for 48 hours at 4°C. Samples were post-fixed overnight in 4% PFA at 4°C, cleared with 50% glycerol, transferred to 70% glycerol and imaged.All mice generated by the Sanger-MGP underwent a broad primary phenotype screen  and on the Mouse Genome Informatics webpage (http://www.informatics.jax.org/genes.shtml). Samples from 1–2 female animals were reviewed by an experienced, board certified veterinary anatomic pathologist (JPS). Animals intended for histopathology were reserved for this purpose and not subjected to other phenotyping tests. All slides were scanned and images archived using Zeiss Mirax Slide Scanner and associated software. In addition, representative photomicrographs were taken of all H&E stained slides. Images are available immediately through the Dryad online database (doi:10.5061/dryad.mv34v) and progressively through the International Mouse Phenotyping Consortium websiteClick here for additional data file.Figure S2cre and flp mediated recombination.EUCOMM/KOMP knockout first conditional-ready targeting construct. These lines are predicted to produce null alleles and prediction of gene expression patterns using the integrated B-gal reporter (tm1a (EUCOMM)WTSI)). Conditional (tm1c (EUCOMM)WTSI)) and total knockout (tm1d (EUCOMM)WTSI)) alleles can be generated using (TIF)Click here for additional data file.Figure S3Aldh18a1 was seen to have broad but weak expression throughout the skin of E14.5 embryos. B) Krt76 showed expression in suprabasal differentiating keratinocytes of the paw pad epidermis. C) Nf1 showed expression in sub dermal structures in E14.5 skin. D) Pfkl1 exhibited strong epidermal expression in E18.5 skin. E) Rad18 expression was detected weakly in the dermis of E18.5 skin. F) Trpc4ap expression was detected weakly in the dermis of E14.5 skin.LacZ reporting of tm1a allele. A) (TIF)Click here for additional data file.Table S1Genes and phenotypes identified in gross clinical phenotype and hair follicle cycling screens. 7 males and 7 female from 362 different transgenic lines were screened for defects in hair follicle cycling and grossly evident skin abnormalities. Sheet 2 - Genes and phenotypes identified in skin histopathology screen. Paraffin embedded, H&E stained skin from a cohort including 514 mutants and 44 wild types were assessed for histologic lesions. Sheet 3 - Genes and MP terms discovered across all tests. Each screen identified genes that were unique to the specific test, thus increasing the number of genes flagged. Unique MP terms were also described by adding different tests.Sheet 1 - (XLSX)Click here for additional data file.Table S2Screen parameters. The Sanger-MGP pipeline comprises 263 parameters, with 43 directly relating to the integument. Parameters relating to skin, hair, teeth, and nails are indicated with a 1, whereas non skin parameters are indicated with a 0.(XLSX)Click here for additional data file.


A combination of fold change ≥1.4 and false discovery rate (FDR)<5% was used to define differentially expressed genes (DEGs). Overrepresentation of gene ontology terms representing biological processes and signaling pathway impact analysis (SPIA) was performed. Comparison of DEG profiles identified 327 genes unique to females, 585 unique to males and 1882 common genes. The major new findings of this study are the identification of new candidate genes of interest and the sex-specific transcriptomic changes in hyperoxic lung injury. We also identified DEGs involved in signaling pathways like MAP kinase and NF-kappa B which may explain the differences in sex-specific susceptibility to hyperoxic lung injury. These findings highlight changes in the pulmonary transcriptome and sex-specific differences in hyperoxic lung injury, and suggest new pathways, whose components could serve as sex-specific biomarkers and possible therapeutic targets for acute lung injury (ALI)/acute respiratory distress (ARDS) in humans.Exposure to high concentration of oxygen (hyperoxia) leads to lung injury in experimental animal models and plays a role in the pathogenesis of diseases such as Acute Respiratory Distress Syndrome (ARDS) and Bronchopulmonary dysplasia (BPD) in humans. The mechanisms responsible for sex differences in the susceptibility towards hyperoxic lung injury remain largely unknown. The major goal of this study was to characterize the changes in the pulmonary transcriptome following hyperoxia exposure and further elucidate the sex-specific changes. Male and female (8–10 wk) wild type (WT) (C57BL/6J) mice were exposed to hyperoxia (FiO Exposure to high concentration of oxygen (hyperoxia) leads to lung injury in experimental animal models and plays a major role in the pathogenesis of diseases such as Acute Respiratory Distress Syndrome (ARDS) and Bronchopulmonary dysplasia (BPD) in humans Sex-specific differences in lung injury have been established both in animal models of acute lung injury (ALI) and observed in epidemiological studies in human patients. Males with ARDS have a higher mortality rate compared to females Nrf2-ARE (antioxidant response element) signaling pathway has been identified as significant in many acute lung injury models Angpt1) and oxidation resistance-1 (Oxr1) which may determine susceptibility to hyperoxic lung injury Like many other diseases genetic and non-genetic factors play a role in hyperoxic lung injury. Genetic susceptibility is thought to contribute to the development of BPD in human neonates We have recently shown that after hyperoxia exposure, adult male mice show greater pulmonary edema, lung injury, inflammation and cell death compared to female mice ad libitum. Animals were maintained in a 12 h day/night cycle.This study was conducted in strict accordance with the recommendations in the Guide for the Care and use of Laboratory Animals of the National Institutes of Heath. The Institutional Animal Care and Use Committee (IACUC) of Baylor College of Medicine approved the protocol (Protocol number AN-907). All efforts were made to minimize suffering. Breeding pairs of mice were obtained from the Jackson Laboratory . 8–10 week old male (C57BL/6J) or female mice were maintained at Texas Children's Hospital animal facility and used for the study. They were fed standard mice food and water 2 at 5 l/min for 24–72 h in a sealed Plexiglass chamber, as reported previously ad libitum. After hyperoxia exposure, the animals were anesthetized with 200 mg/kg of sodium pentobarbital (i.p.) and euthanized by exsanguination while under deep pentobarbital anesthesia. The lung tissues were harvested for further analysis.Mice were maintained in either room air (21% oxygen) or exposed to hyperoxia (95–100% oxygen) environment using pure OWe used a total of 12 animals per sex per treatment group. Total RNA from lung samples in mice exposed to room air or hyperoxia for 48 h was isolated using the miRNeasy kit as per the manufacturer's standard protocols . Following total RNA isolation, sample concentration was assayed using a Nanodrop-8000 . Sample Quality checks were done using the NanoDrop spectrophotometer and Agilent Bioanalyzer.We used the Ambion  WT Expression Kit protocol and reagents to convert total RNA into sense-strand cDNA. The cDNA was then fragmented and labeled using the Affymetrix GeneChip  WT Terminal Labeling Kit.We used 3 biological replicates in each group. The groups were: 1) Room air-male, 2) Room air-female, 3) Hyperoxia-male and 4) Hyperoxia-female (NCBI GEO accession number: GSE51039). To minimize the impact of individual mouse to mouse variability on the microarray data, each biological replicate was comprised of a randomized pool of lung RNA prepared from n = 4 mice. Gene expression in lung tissue was studied using the Mouse Gene 1.0 ST Array (Affymetrix). Raw gene expression data was preprocessed using ‘Robust Multichip Average’ methodology. A combination of fold-change (FC)≥1.4 and false discovery rate (FDR)<5% was used to define differentially expressed genes (DEGs). Overrepresentation of gene ontology terms representing biological processes among the DEGs was tested using a conditional hypergeometric test . Signaling pathway impact analysis (SPIA), was also performed, as this approach combines two types of evidence: (i) the over- representation of DEGs in a given pathway and (ii) the abnormal perturbation of that pathway, as measured by propagating measured expression changes across the pathway topology Ankrd1, Slc7a5, Slc7a11, Egr1 and Nqo1) from the pool of DEGs in a random-stratified manner. For random- stratified sampling, the entire list of upregulated genes was rank ordered according to fold change; the data were then divided into 5 equal-sized bins and one gene per bin was selected randomly. In addition, we also selected five additional genes from the pool of genes showing sex-specific differential expression in the microarray experiment. RNA (50 ng), isolated as above, was subjected to one step real time quantitative TaqMan RT-PCR using 7900HT Fast Real-Time PCR System . Gene-specific primers purchased from life science technologies  in R programming language To validate microarray results, a subset of genes was validated by quantitative real-time PCR (qRT-PCR). Non-random sampling strategies have limited utility as gene selection procedures because the validation results may not generalize to the entire set of DEGs nologies  in the p2 at 5 l/min for 24 h, 48 h or 72 h in a sealed Plexiglass chamber, as described previously. Tracheotomy was performed on the anesthetized mice (n = 5/group) and the lung tissue was fixed by intratracheal instillation of 10% zinc formalin at constant pressure of 25 cm of H2O. Samples were left in solution for 24 h in formaldehyde, and then transferred to 70% ethanol for long-term storage. Routine histology was performed on lung tissues from individual animals following staining of the paraffin sections with hematoxylin and eosin. Five microns deparaffinized lung sections were immunostained with Anti xCT antibody  followed by staining with biotinylated secondary antibodies . For western blotting, lung whole protein (20 µg of protein) from individual male animals (n = 5/group) exposed to room air or to hyperoxia for 48 h, was prepared in Milliplex MAP lysis buffer (EMD Millipore) and used for apoprotein estimation. The primary antibody was the same as used in immunohistochemistry at a dilution of 1∶3000. For loading controls, the membranes were stripped and incubated with antibodies against β-actin, followed by electrochemical detection of bands. The statistical analysis of densitometric values was done using Students t-test and p value<0.05 was considered significant.The mice were maintained in either room air (21% oxygen) or exposed to hyperoxia (95–100% oxygen) environment using pure OAfter 48 h of hyperoxia exposure, a total of 2209 (upregulated (UR): 1385, downregulated (DR): 824) and 2467  genes were differentially expressed compared to room-air in females and males, respectively. Comparison of DEG profiles identified 327 genes unique to females, 585 unique to males, and 1882 common genes. To identify the biological processes that were enriched in the subsets of DEGs that were common to males and females, males only, and females only, we performed both over-representation and pathway topology analyses in these subsets of genes to highlight the differences in the results obtained by these two methods. There are many drawbacks to the over-representation analysis method (ORA) for pathway analysis. ORA typically only considers the number of genes and ignores any values associated with them such as probe intensities; it typically uses only the most significant genes and discards the others. By treating each gene equally, ORA assumes that each gene is independent of the other genes and that each pathway is independent of other pathways NDE , where NDE stands for the number of differentially expressed genes and PPERT which is calculated based on the amount of perturbation measured in each pathway. The two types of evidence, NDEP and PERTP, are finally combined into one global probability value, GP, which is used to rank the pathways and test the research hypothesis that the pathway is significantly perturbed in the condition under the study. A significance threshold of 5% was used on the FDR corrected p-values (pGFDR) in order to detect pathway significance. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway ID enriched in each group are shown in et al.Ankrd1, Slc7a5, Slc7a11, Egr1 and Nqo1) from the pool of DEGs in a random-stratified manner based on their fold change as described in Microarray results were verified by qPCR. Usually, validation of differential expression of genes is performed on a gene to gene basis. The disadvantage of following this approach is that it is not possible to generalize validation results to the remaining majority of non-validated genes or to evaluate the overall quality of these studies. Miron Slc7a11) in the hyperoxia group, which was common to both males and females. Compared to the room air controls, after 48 h of hyperoxia exposure, intense positive staining is seen in the lung sections in the peribronchiolar epithelial and type II alveolar epithelial cells , was upregulated in males after hyperoxia exposure. Pdk4 is an inhibitory kinase, which phosphorylates and inhibits the activity of pyruvate dehydrogenase. The role of this gene in hyperoxic lung injury has not been tested. Grassian et al (2011) reported that Pdk4 overexpression decreased de novo lipogenesis and cell proliferation. Hyperoxia exposure is known to decrease cell proliferation in pulmonary epithelial cell lines when exposed to hyperoxia Pdk4 could have a potential role in this effect. Another candidate gene, Agr2 was downregulated in female mice after hyperoxia exposure. Agr2 inhibits p53 Agr2 in female could in part explain this phenomenon in female mice. Fhl3  gene was upregulated in males after hyperoxia exposure. The four-and-a-half LIM domain (FHL) proteins have a highly conserved double zinc finger motif and have been implicated in transcriptional regulation Fhl3 has been shown to inhibit the transactivation of hypoxia-inducible factor 1 (HIF-1) HIF-1 in turn has been shown to be downregulated in neonatal mice exposed to hyperoxia Fhl3 could also have effects on cell cycle progression and apoptosis. It has been shown to cause cell-cycle arrest through upregulation of p21 and induce caspase-3 mediated apoptosis in glioma cells BDNF (Brain derived neurotrophic factor) was upregulated in females following hyperoxia exposure. Neurotrophins like BDNF have been implicated in many lung diseases BDNF was upregulated in the lung specifically in the peribronchial smooth muscles in 5- day old rat pups exposed to >95% oxygen for 7 days Ccl17 was upregulated in females after hyperoxia exposure. This gene is one of several Cys-Cys (CC) cytokine genes. The cytokine encoded by this gene displays chemotactic activity for T lymphocytes, but not monocytes or granulocytes. In a study by Bhattacharya et alCCl17 was downregulated.The study reveals potential candidate genes that could explain the sex-specific differences in the pathophysiology of hyperoxic lung injury. Of particular note are the genes IL-1β was found to be downregulated in males at the 48 h time point and not in females and this could explain the differential regulation of this pathway in males. Similarly, the NFκ-B pathway was inhibited in males by pathway topology analysis. NFκ-B also plays an important role in hyperoxic lung injury et al., 2004). Specifically, IL-1β was downregulated and Iκbα was upregulated (data not shown) both of which could play a role in the inhibition of this pathway. Also, higher levels of oxidative stress can lead to inhibition of NFκ-B rather than its activation through other mechanisms Pathway analysis based on sex-specific gene expression patterns showed sex-specific differences in enrichment of biological processes . The MAPet al in 2003 p21, metallothionein and cysteine-rich protein 61 were the top three upregulated genes in 8–10 week old female mice after hyperoxia exposure for 48 h. We also found these genes to be upregulated both in male and female mice in our study at this time point. We also found similar upregulation in elastin, galectin 3, glutathione reductase, heme oxygenase-1, p53 and connective tissue growth factor. connective tissue growth factor.In addition to the genes, which showed sex-specific changes in their response to hyperoxia exposure, we identified important differentially expressed genes common to both sexes. Gene expression pattern in the lung after exposure to hyperoxia has been reported previously in adult and neonatal mice. In the study by Perkowski Slc7a11 (solute carrier family (anionic amino acid transporter light chain Xc-system), Member 11) was upregulated in both male and female mice in our study and we validated this at the protein level using immunohistochemistry and western blot H:quinone oxidoreductase 1) is a phase II antioxidant enzyme that has been shown to be induced in murine lung in response to hyperoxia by Nrf2 to protect against lung injury Serpine-1 or plasminogen activator inhibitor (PAI)-1 was also upregulated in our study. This gene has been shown to play a major deleterious role in hyperoxic lung injury PAI-1 upregulation impairs fibrinolytic activity in the alveoli and leads to fibrin accumulation.Cyp1a1 shows the most upregulation at the 24 h time point (data not shown), which could explain its absence in the list of DEGs at the 48 h time point.We have reported previously on sex-specific differences in hyperoxic lung injury and the possible protective role of cytochrome P450 (CYP)1A1. We did not find this gene to be significantly differentially regulated at the 48 h time point in this study. In our experiments Although the array data provides a comprehensive overview of gene expression in the lung after hyperoxia, our study has its limitations. First, array data provides information on gene expression levels. Changes in gene expression levels may not necessarily reflect actual protein levels. Also, important functional changes induced by post-translational modifications cannot be identified. In this study, we examined whole lung tissue gene expression that includes endothelial cells, epithelial cells, interstitial cells, as well as circulating or adherent hematopoietic cells. Hence, most of the genes on the array are likely to be expressed by many cell types. In this investigation, we did not time our experiments with the estrous cycle in female mice, which could have led to some of the variability in our findings. The data is from 12 female adult mice whose lung RNA was pooled in this study. However, there is emerging data that mammalian cells differ intrinsically based on the sex and also respond differently to stressors irrespective of the past or current concentrations of sex hormones In conclusion, we present the changes in the mouse pulmonary transcriptome after hyperoxia exposure at the 48 h time point and identified new potential candidate genes of interest. Furthermore, our results support the hypothesis that there is sex-specific modulation of gene expression and biological processes in the lung under hyperoxic conditions, which may contribute to sex-based differences in hyperoxic lung injury. It is increasingly appreciated that gender differences impact disease presentation and clinical trajectory of patients with ALI. While hormonal factors and differences in lung development are described as contributing factors, comprehensive evaluation of the transcriptomic profile as in this study provides a substantial opportunity to advance the understanding of sex-specific differences in the pathophysiology of hyperoxic lung injury, and could lead to insights about sex-related individualized therapy.


Haemophilus influenzae (NTHi) is a common acute otitis media pathogen, with an incidence that is increased by previous antibiotic treatment. NTHi is also an emerging causative agent of other chronic infections in humans, some linked to morbidity, and all of which impose substantial treatment costs. In this study we explore the possibility that antibiotic exposure may stimulate biofilm formation by NTHi bacteria. We discovered that sub-inhibitory concentrations of beta-lactam antibiotic  stimulated the biofilm-forming ability of NTHi strains, an effect that was strain and antibiotic dependent. When exposed to sub-inhibitory concentrations of beta-lactam antibiotics NTHi strains produced tightly packed biofilms with decreased numbers of culturable bacteria but increased biomass. The ratio of protein per unit weight of biofilm decreased as a result of antibiotic exposure. Antibiotic-stimulated biofilms had altered ultrastructure, and genes involved in glycogen production and transporter function were up regulated in response to antibiotic exposure. Down-regulated genes were linked to multiple metabolic processes but not those involved in stress response. Antibiotic-stimulated biofilm bacteria were more resistant to a lethal dose (10 µg/mL) of cefuroxime. Our results suggest that beta-lactam antibiotic exposure may act as a signaling molecule that promotes transformation into the biofilm phenotype. Loss of viable bacteria, increase in biofilm biomass and decreased protein production coupled with a concomitant up-regulation of genes involved with glycogen production might result in a biofilm of sessile, metabolically inactive bacteria sustained by stored glycogen. These biofilms may protect surviving bacteria from subsequent antibiotic challenges, and act as a reservoir of viable bacteria once antibiotic exposure has ended.Non-typeable  Haemophilus influenzae (NTHi), a Gram-negative, rod-shaped bacterium is a common commensal organism in the human upper respiratory tract. However, NTHi is also an emerging causative agent of chronic infections in humans Non-typeable Orally administered beta-lactam antibiotics are the usual treatment for NTHi infections. For otitis media infections beta-lactam antibiotics such as amoxicillin, ampicillin or cefuroxime are used in vitro biofilm formation by Staphylococcus epidermidisPseudomonas aeruginosa biofilm formation is induced by sub-inhibitory concentrations of tobramycin, tetracycline and norfloxacin Escherichia coliE. coli, colanic acid is important for maturation of biofilm architecture, so any increase in its synthesis might exacerbate the formation or persistence of biofilms Recent studies suggest that antibiotics may promote biofilm formation. For example, a broad range of antibiotics can stimulate in vitro formed biofilms.Recognition of the role of biofilm infections in otitis media in vitro to beta-lactam antibiotics. We speculated that the increased risk of NTHi AOM observed after recent antibiotic treatment in vitro by exposing NTHi bacteria to increasing concentrations of beta lactam antibiotic during biofilm formation. We evaluated the formed biofilms using a classical crystal violet biofilm assay and compared the results obtained from biofilms formed in the absence of antibiotic. We also tested the ability of biofilm bacteria previously exposed to beta-lactam antibiotic to resist a subsequent challenge from a lethal dose of cefuroxime. We obtained more accurate measurements of biofilm biomass, protein content and viable bacteria counts for comparisons and have examined the biofilms formed by NTHi bacteria in the presence of beta-lactam antibiotics microscopically. Finally we performed an analysis of mRNA obtained from biofilm bacteria to determine if exposure to beta-lactam antibiotics was able to modulate gene expression in NTHi bacteria.In this study we have used a biofilm assay model to explore the effects of exposing newly forming NTHi biofilms Haemophilus influenzae strains PittAA, PittEE, PittGG, PittII 2 with 95% relative humidity. After 24-hour incubation, multiple colonies were used to inoculate BHI broth  supplemented with hemin  at 10 µg/ml and nicotinamide adenine dinucleotide (NAD)  at 2 µg/ml (sBHI) 9 CFU/ml) at 37°C, 5% CO2, with 95% relative humidity. Biofilms in this study were prepared by diluting 1∶200 bacterial suspensions at stationary phase with fresh sBHI, aliquoting into culture vessels appropriate for subsequent applications, and incubating at 37°C with 5% CO2 and 95% relative humidity for 24 hours. All NTHi strains were isolated from chronic otitis media patients, with the exception of 2019, which was isolated from a patient with chronic bronchitis.50 µl from frozen stock cultures of non-typeable Stock solutions of ampicillin, amoxicillin, and cefuroxime  were prepared as follows: ampicillin trihydrate and amoxicillin were dissolved in pure DMSO, sterilized using a 0.22 µm syringe filter with nylon membrane , and aliquoted into sterile cryovials for storage at −20°C. Cefuroxime sodium salt was dissolved in BHI broth and sterilized as above. All antibiotic stock solutions were frozen and thawed once before use. All were used immediately after thawing and then discarded.H. influenzae ATCC 49247 and ATCC 49766  as control strains for sensitivity to ampicillin and cefuroxime.All NTHi strains in this study were tested for susceptibilities to ampicillin and cefuroxime, using the Bauer-Kirby disk diffusion method Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined on experimental strains by applying BSAC Working Party Report guidelines Biofilms were prepared in 96-well tissue culture-treated polystyrene microplates  with beta-lactam antibiotics at designated concentration ranges. One row of 8 wells was used for each experiment and each well was read individually. Control rows of wells contained sBHI only or bacteria in sBHI with no antibiotics. For ampicillin and amoxicillin assays, additional control wells of bacteria in sBHI containing corresponding dilutions of DMSO were also set up. The bacteria were incubated at 37°C in an atmosphere of 5% CO2 and 95% relative humidity for 24 hours before biofilm quantification using crystal violet assay.In order to investigate the ability of NTHi biofilms to resist lethal doses of beta-lactam antibiotic, biofilms were formed in the wells of 96 well plates (8 wells per experiment) in the presence or absence of specific sub-inhibitory concentrations of selected beta-lactam antibiotics. The formed biofilms were washed with sterile sBHI and incubated overnight in sBHI containing 10 µg/mL cefuroxime. The biofilms were scraped, agitated to disrupt the biofilm and re-suspend the bacteria, and the numbers of viable bacteria estimated using the drop plate counting method 600 of the fully hydrated biofilm-containing wells was measured using a SpectraMax M5 microplate reader . A crystal violet assay was then applied to quantify biofilm formation 600 of crystal violet in DMSO solution (absorption maxima = 600 nm) was measured in a microplate reader.The ODIn order to obtain sufficient material for estimating biofilm biomass (dry weight) and protein content, NTHi biofilms were formed in 20×100 mm tissue culture-treated polystyrene dishes  in the presence and absence of 170 ng/mL ampicillin (3 replicas each). Antibiotics were present during biofilm formation. The culture medium was washed from the formed biofilms with PBS and the biofilm material was freeze-dried under vacuum. Dry weights were measured from batches of pooled biofilms (3 dishes each batch). Protein content was estimated using the BCA Protein Assay Kit (Thermo Scientific Pierce) on biofilm material scraped from dishes and tabulated as percent protein content per biofilm. For CFU counts, biofilms were not dried but were scraped and suspended in sBHI, serially diluted and estimates of total viable bacteria calculated after 24 hr incubation Data from experimental and control conditions were analyzed using GraphPad Prism . Groups were compared using an unpaired t test with Welch’s correction. Statistical significance was defined as P<0.05.NTHi biofilms were prepared in 96-well tissue culture-treated imaging microplates . Fluorescent dyes from FilmTracer Live/Dead Biofilm Viability kit  were applied to biofilms at the final concentrations of 5 µM and 30 µM, of Syto9 and propidium iodide, respectively. Biofilms were incubated with the dyes at room temperature for 20–40 minutes before being imaged by LSM710 Meta NLO cLSM . All cLSM images were collected within a window of 40–90 minutes after application of the fluorescent dyes.For better specimen handling for processing and examination in an SEM, plastic coverslips were used as growth substrate for NTHi biofilms; hence, biofilms were prepared in 20×100 mm tissue culture-treated polystyrene dishes  with Thermanox plastic coverslips  placed at the bottom. Then, as previously described Specimen holders for the EMPact2 high-pressure freezer  were autoclaved and immersed in BHI medium containing NTHi bacteria (PittGG strain) in the presence or absence of 170 ng/mL ampicillin and incubated overnight at 37°C. The specimen holders were removed from culture, dried on the base and frozen under high pressure. Biofilm present in the specimen holders were dehydrated while still frozen Glycogen staining was carried out on thin sections before heavy metal contrasting using a previously described protocol Sections were examined with a Tecnai G2 20 transmission electron microscope  and images collected digitally. Images were compiled into figures using PhotoShop. Image modifications consisted of brightness and contrast adjustments applied to the whole image. Image cropping was performed for presentation purposes.www.abcam.com/Glycogen-Assay-Kit-ab65620.html) was used to estimate glycogen content following the protocols supplied with the kit.Replicate preparations (4 replicates) of NTHi biofilms were prepared in Petri dishes using strain Pitt GG in the presence and absence of 170 ng/mL ampicillin. After 24 hr incubation the formed biofilms were washed with PBS, scraped from the plastic substrate, collected in tubes and dried. The dried biofilms were weighed and a commercially available glycogen assay kit  and the experiment was carried out as follows.Briefly, the biofilms were gently washed in PBS to remove culture medium and then removed from the plastic substrate by scraping in 4 mL of PBS. The scraped biofilm suspensions were placed in 50 mL Falcon tubes, evenly dispersed and aliquoted for further processing. The aliquots were centrifuged at 8,000 rpm in a benchtop centrifuge  for 3 min. and the supernatant was discarded. The pellets were re-suspended in RNAwiz and total RNA was extracted using Ambion RiboPure-Bacteria kit following the manufacturer’s instructions .Klenow fragment (3’→5’ exo-).cDNA was synthesized using Invitrogen SuperScript Double-Stranded cDNA Synthesis Kit . Both RNA and cDNA quality was checked using Agilent 2100 Bioanalyzer and Agilent RNA 6000 nano and DNA1000 chips . cDNA (0.5–1 µg) was used to start the amplification and labeling reaction using the Roche NimbleGen One-Color DNA Labeling kit, in which Cy3 was randomly incorporated in the newly synthesized DNA by Two µg of labeled DNA derived from each RNA sample was hybridized to each array for over 16–18 hours. DNA microarray gene expression was carried out using Roche NimbleGen custom whole genome tilling arrays (100910_CW_P_ging_W83_expr_HX12)  according to the standard NimbleGen procedure .http://www.ncbi.nlm.nih.gov/geo).The slides/arrays were washed and spun dry and then scanned using a NimbleGen MS200 Microarray scanner with a resolution of 2 µm. The normalization was done using NimbleScan 2.6.0.0 built-in normalization function. Microarray data analysis was performed using Partek Genomics Suite v6.5 . Differentially expressed genes were determined using fold-change (≥1.5) plus P  with FDR≤0.05. The gene expression data was deposited in NCBI's Gene Expression Omnibus . The two other strains, 2019 and 9274 are considered to be laboratory strains. Crystal violet staining showed that all the NTHi strains used in this study formed biofilms, as all resulted in OD control . The amo control  while Pi control .Antibiotic resistant varied between the strains as tested using Sensi-Disc test discs . Controlp = <0.0001) but not in the presence of ampicillin (p = <0.0001) and ampicillin at 525 ng/mL (p = <0.0001). PittGG and PittII did not appear to react to the presence of amoxicillin or ampicillin in our chosen concentration range, even though there were significant differences between the effects of maximal observed stimulation with unexposed biofilm levels (p = 0.0047) and 170 ng/mL ampicillin (p = 0.0336). PittII reacted to 120 ng/mL amoxicillin (p = <0.0001). However, the stimulated levels of PittGG and PittII in response to amoxicillin were higher than other biofilms formed in the presence of antibiotic but were lower than the untreated biofilms.We examined the ability of these NTHi strains to form biofilms in the presence of increasing concentrations of penicillin-based beta-lactam antibiotics. Some of the NTHi strains reacted to the antibiotics by producing more biofilm . Our exppicillin . Biofilmm levels . PittGG Cefuroxime, a cephalosporin beta-lactam antibiotic produced a biofilm stimulatory effect in the range of 0–950 ng/mL on all of the NTHi strains we studied (p = <0.0001) . Biofilmp = <0.0001) (The results we obtained for PittGG using the biofilm assay showed no apparent reaction in the 150 ng/mL dilution of ampicillin . The hig<0.0001) .In order to determine how beta-lactam antibiotics were affecting the biofilm-forming abilities of NTHi bacteria we assayed the following; a) total biomass of the formed biofilms; b) protein content; and c) total number of viable bacteria in the biofilms. These assays were applied to biofilms formed in the presence and absence of sub-inhibitory concentrations of beta-lactam antibiotics and the results presented in Results from NTHi strain 2019 showed that in the presence of a sub-inhibitory concentration of ampicillin (94 ng/mL) the NTHi bacteria produced an increased amount of biofilm biomass . The dry weight increased from 5.3 mg/dish to 17.3 mg/dish . AmpicilThe presence of ampicillin resulted in a reduction in the relative protein composition in the 2019 and PittGG biofilms . While m8 (no ampicillin) to 2.4×108 (with ampicillin). Viable bacteria in the PittGG biofilms decreased from 8.5×109 (no antibiotic) to 1.8×108 (with antibiotic).The presence of ampicillin reduced the numbers of viable bacteria (in CFU’s per mg biofilm) in forming biofilms . The numPresuming that antibiotic resistance may play a role in the biofilm-stimulatory effect on the NTHi bacteria, we screened all the strains for antibiotic resistance using routine methods . PittAA The Live/Dead stain was applied to biofilms formed in the presence and absence of ampicillin. Untreated biofilms of NTHi strains 2019 and PittGG contained mostly green-staining small dots (live bacteria), with only few red (dead) bacteria . UntreatThe presence of sub-inhibitory concentrations of ampicillin altered the Live/Dead staining patterns in both 2019 and PittGG biofilms . AmpicilReconstructed Z-series stacks through the biofilms showed untreated biofilms composed of mostly intact bacteria, and ampicillin-treated biofilms composed of mostly damaged bacteria . The recIn order to examine the NTHi biofilms at higher resolution, biofilms on Thermanox slides formed under sBHI broth were examined by scanning electron microscopy (SEM). At low magnification, the biofilm formed by strain 2019 appeared as a uniformly thick mat covering the substrate  with thiNTHi strain 2019 biofilms that formed in the presence of sub-inhibitory amounts of ampicillin  were morPittGG biofilms formed on Thermanox slides were approx. 20 µm high, and therefore thinner than the NTHi 2019 biofilms  and had Suspensions of planktonic forms of NTHi strains 2019 and PittGG showed no signs of bacterial growth after an overnight incubation in 10 µg/mL concentration of cefuroxime (data not shown), indicating bacterial killing by the high dose of antibiotic. However, if the two strains of NTHi were first allowed to form a biofilm and the biofilm bacteria then exposed to the 10 µg/mL dose of cefuroxime, the antibiotic was unable to completely eliminate all viable bacteria from the biofilm .p = <0.0001) when compared to bacteria from biofilm alone (p = <0.0059). Exposure to 170 ng/mL of cefuroxime did not change sensitivity to the subsequent lethal dose of cefuroxime . An initial sub-inhibitory dose of cefuroxime did not alter sensitivity to the subsequent higher dose  on microarray whole transcriptome expression profiling showed that the PittGG biofilm samples where bacteria (NTHi) were treated with ampicillin were clearly different from the control samples (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58213).In order to determine the underlying molecular cause for different biofilm phenotype formation in the presence or absence of a sub-inhibitory dose of ampicillin, we performed microarray gene expression profiling using Roche NimbleGen whole genome custom tilling array (12×135 K) for  samples . Signifi samples . Eight g samples , one was samples . However samples . The genA commercial glycogen assay applied to four replicates of ampicillin-treated NTHi biofilms detected up to 0.68 µg of glycogen per mg of biofilm . The lowest calibration point was 0.02 µg on the calibration graph. Glycogen was not detected at any level in four replicate untreated NTHi biofilms (data not shown).Application of a modified Schiff stain to sections through ampicillin-treated NTHi biofilms revealed the presence of an extracellular granular substance that we concluded as being extracellular glycogen . The preAlthough it is possible for processing and staining protocols to produce these contrasting patterns on thin sections we were unable to replicate them on any control experiments. Ampicillin-treated biofilm that was not exposed to the staining reagents did not show contrasted extracellular material . BiofilmAmpicillin-treated NTHi biofilms contained biofilm bacteria with variable sizes . Regularin vitroThe ability of NTHi bacteria to form biofilms Biofilm stimulation was detected using the crystal violet biofilm assay, which measures the amount of biological material sticking to the surface of a container after bacteria have been cultured in it. It is not clear if this assay is an estimator of an increase in biofilm biomass or if it detects an increased ability of the biofilm material to attach to the sides of the plastic wells. For this reason, we examined the NTHi biofilms using other assays to confirm that beta-lactam antibiotic exposure was increasing biofilm biomass. In the presence of ampicillin, two different strains of NTHi showed an increase in dry weight and total protein content. However, antibiotic exposure lowered the ratio of protein to biofilm mass. The antibiotic also resulted in a drop in the number of viable bacteria in the biofilms, an observation supported by light microscopy, which showed more dead bacteria in the treated biofilms. Our direct observation of living biofilms also demonstrated the presence of antibiotic to cause an increase in the thickness of the formed biofilms.Protein content in biofilms has previously been reported to derive from lysed cells or be actively secreted by biofilm-forming bacteria The increase in biomass may offer an explanation for why the ampicillin-stimulated PittGG biofilm was less firmly attached to the plastic surface after drying for SEM examination. The added volume and biofilm bulk could make an already weakly attached biofilm more easily dislodge during specimen preparation. Although it is possible that attachment mechanisms of the biofilm or biofilm bacteria have been modified by exposure to antibiotic, any such modifications were not evident in the mRNA data we collected.The loss of bacterial cells after antibiotic exposure may indicate the expected massive cell death, and subsequent lysis, that theoretically may influence biofilm formation by chemical and physical aggregation lacking a biological component The composition of the partition walls we observed in the biofilms at the ultrastructural level is still a mystery. Increased production of glycogen might suggest that as the source of the partition wall material, a speculation that is currently being tested. The TEM examination of ampicillin-treated biofilms to detect glycogen did not reveal cross sections of partition walls, rather the glycogen in the ECM appeared globular and in close proximity to bacterial cells. The loss of bacterial cells in biofilms exposed to antibiotic might be due to massive cell death, which would liberate cytoplasmic proteins into the ECM and which could aggregate around nucleation sites such as provided by extracellular DNA We selected beta-lactam antibiotics to study because these are the class of antibiotics most commonly used to treat patients with otitis media in vitro biofilm stimulatory effect are in the range of 230–450 ng/mL. These in vitro amounts are much lower than recorded peak concentrations of amoxicillin in the middle ear fluids taken from otitis media patients, where the mean concentration was 9.5 µg/mL Amoxicillin concentrations that have an in vitro system, we performed a preliminary experiment to mimic the treatment protocol where bacteria are first exposed to a sub-inhibitory dose of one antibody and then challenged with a higher dose of cefuroxime, a common antibiotic used to treat recalcitrant NTHi infections. Both the 2019 and PittGG strains showed that biofilm formation is important for protecting bacteria from a subsequent high dose of cefuroxime. Amoxicillin exposure appeared to increase the resistance of 2019 biofilm bacteria to a subsequent lethal dose of cefuroxime. Ampicillin exposure protected PittGG biofilm bacteria from the subsequent cefuroxime exposure. A sub-inhibitory dose of beta-lactam antibiotic influencing a subsequent antibiotic challenge is an effect that might occur under conditions in patients undergoing treatment for otitis media. It is thus worth further investigation. The phenomenon of increased resistance to a second course of antibiotics may help explain the increased occurrence of acute otitis media caused by NTHi Recommended treatment protocols for otitis media consist of oral administration of amoxicillin, or amoxicillin combined with clavulanic acid, a beta-lactamase inhibitor in vitro observations, it is possible that an immediate and less desirable side effect of beta-lactam treatment of otitis media would be to stimulate bacterial attachment to surfaces within the body. Such a stimulus for attachment may signal a change in the NTHi causing it to become a pathogenic form. As such, these biofilm bacteria could then become a source of future chronic infections. Beta-lactam-treated patients would be expected to have an increased risk of recurrent infection. In fact, observations of otitis media patients do indicate an increased risk of recurrent otitis media after antibiotic treatment Outpatient beta-lactam antibiotics are mostly orally administered. Thus, the pharmacological bioavailability of the antibiotics may not be homogeneous throughout the body, with local concentrations being influenced by body weight, percent fat, gender, and drug accessibility. Bacterial biofilms are present throughout the human body Staphylococcus epidermidisPseudomonas aeruginosaEscherichia coliWhile it has been reported that azithromycin can decrease established NTHi biofilms and inhibit NTHi biofilm formation We have shown that sub-inhibitory concentrations of amoxicillin, ampicillin and cefuroxime (beta-lactam antibiotics) will stimulate NTHi bacteria to produce more biofilm biomass even as the numbers of culturable bacteria decline and as the biofilm protein content decreases. Biofilm bacteria were protected from the toxic effects of a high dose of cefuroxime. The biofilms formed in the presence of sub-inhibitory concentrations of antibiotic were thicker, had altered ultrastructure and contained increased numbers of dead bacteria. Sub-inhibitory concentrations of antibiotic up-regulated genes involved with glycogen biosynthesis, and glycogen was detected in antibiotic-treated biofilms. Genes encoding transporters and one gene involved with pyrimidine metabolism were also up-regulated. Surprisingly, genes involved with stress responses were not up-regulated. A total of 51 genes were down-regulated and were involved in a variety of metabolic processes, including cell-wall biosynthesis. Our results suggest that exposure to sub-inhibitory concentrations of beta-lactam antibiotic cause NTHi bacteria to increase glycogen synthesis while turning down a broad range of other metabolic processes. Beta-lactam-stimulated NTHi biofilms may protect embedded bacteria from subsequent high concentrations of cefuroxime.Table S1A) Statistical analysis for crystal violet assays in (DOCX)Click here for additional data file.Table S2A) NTHi sensitivity to Sensi-Disc Antimicrobial Susceptibility Test Discs. Reaction to antibiotic classified using manufacturers recommendation: R = Resistant, S = Susceptible, I – Intermediate. NTHi strains 49249 and 49766 are resistant and susceptible controls respectively. B) Minimum Inhibitory Concentrations (MIC) and Minimum Bactericidal Concentrations (MBC) in µg/mL antibiotic.(DOCX)Click here for additional data file.Table S3Statistical analysis for (DOCX)Click here for additional data file.Table S4Significant gene list with all probe IDs identified by microarray analysis in the PittGG biofilm bacteria (NTHi) exposed to 170 ng/mL ampicillin.(DOCX)Click here for additional data file.


The manipulation of plant seed oil composition so as to deliver enhanced fatty acid compositions suitable for feed or fuel has long been a goal of metabolic engineers. Recent advances in our understanding of the flux of acyl-changes through different key metabolic pools such as phosphatidylcholine and diacylglycerol have allowed for more targeted interventions. When combined in iterative fashion with further lipidomic analyses, significant breakthroughs in our capacity to generate plants with novel oils have been achieved. Collectively these studies, working at the interface between metabolic engineering and synthetic biology, demonstrate the positive fundamental and applied outcomes derived from such research. Current Opinion in Plant Biology 2014, 19:68–75Physiology and metabolismThis review comes from a themed issue on Sarah E O’Connor and Thomas P BrutnellEdited by Issue and the EditorialFor a complete overview see the Available online 15th April 2014http://dx.doi.org/10.1016/j.pbi.2014.04.001http://creativecommons.org/licenses/by/3.0/).1369-5266/© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license  that the developing seed accumulates as energy reserves for catabolism during germination. The ability to harness and use these storage compounds has historically underpinned the transition from hunter-gather to agricultural-based society, and now continues to feed the ever-increasing global population. The predominant storage oil in seeds are neutral lipids such as triacylglycerol and given their significance for nutrition and industry, considerable effort has focussed on the desire to improve both the composition and yield of vegetable oils. However, the apparent simplicity of a seed (as the inert container of useful storage reserves) is misleading, since there is still much to learn about how these compounds are co-ordinately synthesized and compartmentalised. On the other hand, our ability to manipulate and tailor the composition of these reserves is steadily increasing, driven forward by advanced plant metabolic engineering/synthetic biology and informed by detailed metabolite analyses. The adoption of such multidisciplinary approaches has extended our understanding of seed lipid metabolism and, as will be discussed below, generated new specialized platforms for oil production. Indeed, progress in vegetable oil production is now extending beyond the seed and exciting insights are emerging about the possibility of oil production in vegetative tissue. Given the pressures on the carbon economy, exemplified by an increased demand and a declining supply of conventional fossil oil, progress in meeting the requirements for vegetable oil is timely. In this article, we will focus on the synthesis and manipulation of one specific type of storage reserve — triacylglycerol – since this represents one of the best examples of complex metabolic engineering in transgenic plants.Camelina sativa) has emerged as a particularly attractive metabolic engineering host because it can be readily transformed using an Agrobacterium-based floral infiltration method [FAD2 (Fatty Acid Desaturase 2) that controls Δ12 desaturation of oleic acid and FAE1 (fatty acid elongase 1) that mediates oleic acid elongation to C20 and C22 chain lengths [Crambe abyssinica) as a dedicated industrial oil crop, and the recent development of an Agrobacterium-mediated transformation system has enabled its use for metabolic engineering of seed oil traits [Over the last two decades, the challenge for researchers has been the accumulation of novel fatty acids, which have beneficial functional groups or properties, into oilseeds with good agronomical traits. Although, metabolic engineering of oil-related traits has largely relied principally upon Arabidopsis as a host to test individual genes and gene combinations for modifying seed oils and more recently for engineering of oil production in vegetative tissues as described below. As proof of principal has been established in Arabidopsis, interest has grown in translation of these oil traits in established oilseed crops. For specialty oils, including those enriched in fish oil-type long chain polyunsaturated fatty acids (LC-PUFAs) and industrially valuable unusual fatty acid structures, attention has centred on non-food oilseed crops to mitigate the unintended mixing of food and specialized oil traits. Camelina . As the terminal point in seed oil biosynthesis, TAG is comprised of a glycerol backbone onto which three fatty acids are sequentially esterified. Plant oils are rich in C18 fatty acids, including the essential fatty acids linoleic acid 18:2Δ9,12,n−6; LA) and α-linolenic acid , but are devoid of LC-PUFAs, such as arachidonic acid , eicosapentaenoic acid  and docosahexaenoic acid , which typically only enter the human diet as oily fish. The health benefits of the omega-3 LC-PUFAs EPA and DHA are now well-established , reviewe,n−6; LA Ricinus communis), which has limited commercial cultivation because of the high content of ricin toxins in it seeds, accumulates ricinoleic acid to 90% of the fatty acids of its seed oil through this pathway. To date, the transfer of the castor FAD2-related hydroxylase together with specialized castor acyltransferases, including the castor diacylglycerol acyltransferase 2 (DGAT2) and phospholipid-diacylglycerol acyltransferase 1 (PDAT1), have yielded only 20–30% ricinoleic acid and other derivative hydroxy fatty acids in transgenic Arabidopsis seeds [rod1 mutant defective in phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT)-mediated flux of DAG through PC, displayed reduced hydroxy fatty acid synthesis in seeds engineered for castor bean hydroxylase expression [A long-term goal of oilseed metabolic engineering has been the generation of fatty acid traits targeted for industrial applications. A particular focus has been the transfer of biosynthetic and metabolic pathways for unusual fatty acids, such as hydroxy and epoxy fatty acids  from non-agronomic plant species to existing oilseed crops. After more than a decade of gene discovery efforts and numerous basic and translational breakthroughs , many chis seeds . Resultsis seeds •. Consispression . The subpression .Escherichia coli cyclopropane synthase illustrated how the transgenic expression of this enzyme in Arabidopsis seeds effectively converted the Δ9 double bond of oleic acid into a cyclopropane ring, a result which confers vegetable oils with a wide range of industrial functionalities [sn-1 position of PC as a substrate [E. coli cyclopropane synthase with a lysophosphatidic acid acyltransferase (LPAT) from Sterculia foetida seeds, which naturally accumulate high levels of cyclopropane fatty acids, resulted in small increases in cyclopropane fatty acid accumulation. LPAT catalyzes the acylation of the sn-2 position of the glycerol backbone in the TAG biosynthetic pathway. Consistent with this activity, increased amounts of cyclopropane fatty acids in TAG in the engineered Arabidopsis seeds was due primarily to enhanced accumulation at the TAG sn-2 position [Defective flux of acyl chains from PC to TAG in engineered seeds appears to be a common bottleneck for the accumulation of unusual fatty acids. This was previously noted for conjugated fatty acid accumulation in Arabidopsis and soybean seeds engineered to express FAD2-related fatty acid conjugases that convert either the Δ9 or Δ12 double bond of linoleic acid linked to PC into two conjugated double bonds, which enhances the drying properties of vegetable oils . More renalities . This reubstrate . The seeposition . Despiteposition . These ret al. demonstrated the feasibility of co-expressing an endoplasmic reticulum (ER)-localized mouse WS with a mouse peroxisomal FAR retargeted for ER localization to generate wax esters in Arabidopsis seeds with principally C18 and C20 fatty acid and fatty alcohol components [fad2/fae1 mutant that has high levels of oleic acid in its seeds [Recent efforts to produce oils with industrial functionality have also targeted pathways that use only acyl-CoA substrates to produce novel oils, bypassing the intricacies of unusual fatty acid biosynthetic pathways involving PC-linked substrates. One such pathway is that for wax ester biosynthesis. Wax esters lack diacylglycerol backbones and consist of a fatty acid linked through an ester bond to a fatty alcohol. These molecules are desirable for use as high temperature lubricants and are synthesized in a two-step biosynthetic pathway involving conversion of a fatty acyl-CoA to a fatty alcohol via a fatty alcohol reductase (FAR) and condensation of the fatty alcohol with an acyl-CoA via a wax synthase (WS). Recently, Heilmann mponents . By linkmponents . In addits seeds .Brassica napus FAE1 to enhance elongation of oleic acid to erucic acid; and a specialized Limnanthes douglasii LPAT to increase erucic acid incorporation into the sn-2 position of TAG [Carthamus tictorius) seeds, Crambe seeds are particularly effective at producing high levels of erucic acid through acyl-CoA reactions, due to a low PDCT activity that effectively precludes exchange of fatty acids between DAG and PC [Metabolic engineering of very long-chain fatty acid production also offers an opportunity for generating industrial oils through acyl-CoA reactions that bypass PC-linked biosynthetic pathways. Crambe seed oil is naturally enriched in erucic acid , a C22 monounsaturated fatty acid •. This fn of TAG •. The ren of TAG •. AdditiG and PC •. LabeliG and PC •.The pressing need to produce more energy from plant biomass has encouraged attempts to produce oil in vegetative tissues. Although seeds and some fruit pericarps  are by far the largest source of plant produced oils, many other tissues are capable of synthesizing triacylglycerols and a number of studies have reported the presence of cytosolic lipid droplets in leaf mesophyll cells . TAGs noLEC1), LEC2 or WRINKLED1 (WRI1) [CTS2), SUGAR DEPENDENT1 (SPD1) or COMPARATIVE GENE IDENTIFICATION-58 (CGI58) [LEC2 in the cts2 β-oxidation mutant [WRI1 or LEC2 [The possibility of producing TAGs for biodiesel in leaves and other vegetative tissues has recently attracted considerable interest . A numbe1 (WRI1)  that con (CGI58) ••,39,40.n mutant ••. Recen or LEC2  or an en or LEC2  with DGA or LEC2 . However or LEC2 ••,41••.  or LEC2  has idenIt will be interesting to investigate whether oil accumulation in green biomass can be further improved without severely impacting photosynthesis and plant development. One possibility for achieving this could be the use of senescence induced promoters to engineer plants in which TAG accumulation is initiated only after leaves have reached their maximum size . Anotheret al. [The engineering of economically viable levels of LC-PUFAs in camelina seeds and ‘ultra-high’ levels of erucic acid in crambe seeds represent recent successes in the translation of specialty fatty acid traits to oilseed crops. The ability to achieve high amounts of unusual fatty acid production by transfer of PC-linked biosynthetic and metabolic pathways from seeds of non-agronomic species to seeds of either the Arabidopsis model or existing oilseed crops remains elusive to metabolic engineers. Solving bottlenecks that limit the synthesis and accumulation of these fatty acids will require more in-depth understanding of fatty acid metabolic pathways in seeds that naturally accumulate high levels of unusual fatty acids. It will also be necessary to determine the relative contributions of different enzymes specialized for these pathways in the native species and to possibly down-regulate non-productive, competing pathways in seeds of host oilseeds- this is summarised in et al. ••. Similet al. • and tobet al.  are prov• of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as:


Uterine Fibroids  are the most important benign neoplastic threat to women’s health. They are the most common cause of hysterectomy imposing untold personal consequences and 100s of billions of healthcare dollars, worldwide. Currently, there is no long term effective FDA-approved medical treatment available, and surgery is the mainstay. The etiology of UFs is not fully understood. In this regard, we and others have recently reported that somatic mutations in the gene encoding the transcriptional mediator subunit Med12 are found to occur at a high frequency (∼85%) in UFs. UFs likely originate when a Med12 mutation occurs in a myometrial stem cell converting it into a tumor-forming stem cell leading to a clonal fibroid lesion. Although the molecular attributes underlying the mechanistic formation of UFs is largely unknown, a growing body of literature implicates unfavorable early life environmental exposures as potentially important contributors. Early life exposure to EDCs during sensitive windows of development can reprogram normal physiological responses and alter disease susceptibility later in life. Neonatal exposure to the EDCs such as diethylstilbestrol (DES) and genistein during reproductive tract development has been shown to increase the incidence, multiplicity and overall size of UFs in the Eker rat model, concomitantly reprogramming estrogen-responsive gene expression. Importantly, EDC exposure represses enhancer of zeste 2 (EZH2) and reduces levels of histone 3 lysine 27 trimethylation (H3K27me3) repressive mark through Estrogen receptor/Phosphatidylinositide 3-kinases/Protein kinase B non-genomic signaling in the developing uterus. Considering the fact that distinct Mediator Complex Subunit 12 (Med12) mutations are detected in different fibroid lesions in the same uterus, the emergence of each Med12 mutation is likely an independent event in an altered myometrial stem cell. It is therefore possible that a chronic reduction in DNA repair capacity eventually causes the emergence of mutations such as Med12 in myometrial stem cells converting them into fibroid tumor-forming stem cells, and thereby leads to the development of UFs. Advancing our understanding of the mechanistic role epigenetic regulation of stem cells plays in mediating risk and tumorigenesis will help in pointing the way toward the development of novel therapeutic options. Exposure to environmental toxicants and toxins causes epigenetic changes that play a role in the development of disease . IdentifA striking feature of UFs is their dependency on the ovarian steroids estrogen and progesterone . A numbeThe biological effects of 17β-estradiol are mediated by two isoforms of the ERs (ERα and ERβ). Hormone-activated ERs form dimers. Since the two forms are coexpressed in many cell types, the receptors may form ERα (αα) or ERβ (ββ) homodimers or ERαβ (αβ) heterodimers. Although ERs are widely expressed in different tissues types, some notable differences in their expression patterns occur. For instance, the ERα is found in endometrium, ovarian stromal cells, and breast cancer cells. ERs mediate the effects of 17β-estradiol under physiologic and pathologic conditions. ERs trigger 17β-estradiol-sensitive gene transcription by binding to specific estrogen response elements  . In the Some ERs can be rapidly activated to downstream kinase cascades by exposure of the cells to estrogen . Epigenetic writers catalyze the addition of chemical groups onto either histone tails or onto the DNA itself . PRC2 methylates H3K27 via its EZH2 subunit. This modification, in turn, provides a binding site for the chromodomain-containing Pc subunit of PRC1, which subsequently leads to ubiquitination of H2AK119 via its Ring1a/1b subunit . In recent years, these proteins have raised considerable interest, due to their regulatory mechanisms and for the variety of key roles they play in normal cellular and disease processes (Table 1) through canonical and non-canonical activity .Polycomb group proteins form multimeric complexes that exert their functions by modifying chromatin structure and by regulating the deposition and recognition of multiple post-translational histone modifications . Two majrocesses . For insFigure 2), confer heritable memory by sustaining active gene expression states, but they antagonize the function of the PcG  promoter CpG Island (CGI) in the uteri which leads to persistent overexpression throughout life. Since the Nsbp1 encodes a nuclear protein similar to the HMG 14, this protein may alter the gene expression pattern in utero, in response to early life EDC exposure leading to an increased risk of uterine cancer in adulthood  encodes a component of prostatein, a major androgen-binding protein secreted by rat prostate, and hence suggests potential implications for cancer risk and response to chemotherapeutics associated with prostatein binding  tumor suppressor gene . In a mouse model that conditionally expresses a Med12 missense variant (c. 131G > A), it has been demonstrated that this alteration alone promotes fibroid formation and drives genomic instability  gene amplification in BTICs occurred, which activates p-ERK-β-catenin signaling to promote BTIC expansion  were found to be detected in the serum of patients with pancreatic cancer, distinguishing healthy subjects from those with benign pancreatic disease . MelanomUterine Fibroids are thought to be monoclonal tumors arising from the myometrium, and tumor stem cells are considered to play pivotal roles in the tumorigenesis of UFs. It is possible that cell-to-cell interaction between myometrial stem cells and differentiation cells is involved in the development of UFs. Although the role of myometrial stem cell-derived exosomes is unknown, increasing, studies have suggested that stem cell-derived exosomes containing important effectors of Wnt , Hedgehoin utero polyunsaturated fatty acid supply, along with marked alterations in miRNA expression (The importance of stem cell-derived miRNAs in response to environmental factors has recently been identified . Shah etpression . AlthougCurrently, there is a remarkable lack of knowledge regarding the involvement of chromatin assembly in the process by which adverse environmental exposures increase the overall risk of UF development. The precise mechanism underlying EDC-dependent effects on myometrial cell physiology are not adequately understood. Accordingly, in response to EDC administration, no single PcG or TrxG-target genes have been discovered in myometrium tissues as well as in myometrial stem cells . In addiQY and AA, conception of the manuscript; QY, drafted the manuscript; QY, MD, and AA, revised the manuscript. All authors approved the revised version of 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.