Carriers of the ApoE ϵ4 allele are at a greater risk for developing Alzheimer’s disease (AD) and those who do develop AD tend to have a much greater neuropathological disease burden. Although several studies have shown significant differences in AD pathology among ϵ4 carriers and non-carriers, few have characterized these differences in terms of brain region and neuropathological score frequency.566 pathologically-confirmed AD cases who were followed prospectively with antemortem dementia diagnoses (312 ApoE ϵ4 carriers and 254 ApoE ϵ4 non-carriers) were compared on the frequencies of neuropathological frequency scores  among several different brain regions  using the CERAD scoring system. Pathology score frequencies were analyzed by carrier status (ϵ4 carrier vs. ϵ4 non-carrier) and by genotype . Both analyses investigated pathology score frequencies among different brain regions .ϵ4 carriers had a significantly lower age at death (p <0.001) and significantly higher Braak scores (p <0.001) than ϵ4 non-carriers. Genotype comparison revealed that plaque and tangle pathologies increased in the following pattern, 2/3<3/3<2/4<3/4<4/4, for several brain regions. When stratified by age and ApoE ϵ4 carrier status, ϵ4 carriers tended to have significantly more frequent scores across most cortical areas. However, non-carriers age 90 and older tended to have greater plaque pathology than carriers. For tangle pathology, ϵ4 carriers tended to have significantly more “frequent” scores than non-carriers, except for the hippocampal and entorhinal areas in individuals age 90 and older.ApoE ϵ4 carriers had a significantly higher percentage of “frequent” scores for plaques and tangles when compared to ApoE ϵ4 non-carriers for several brain regions. However, ϵ4 non-carriers age 90 and older tended to have less plaque and tangle pathology in certain brain regions. These results demonstrate that AD pathology may manifest itself differently based on ApoE genotype and suggest that ApoE carriers and non-carriers may have different patterns of AD neuropathology location and density. It has been shown that the three main polymorphisms , coded by the ϵ2, ϵ3, and ϵ4 alleles on chromosome 19, of the apolipoprotein E (ApoE) gene result in differential risk factors for Alzheimer’s disease (AD) for those who carry them . The ϵ2 In addition to being linked to AD, ApoE ϵ4 has also been strongly linked to increased deposition of the main hallmark proteins in AD brains, amyloid beta (Aβ) and neurofibrillary tangles (NFTs), as well as correlating with certain biomarkers found in plasma and cerebrospinal fluid of AD ,11. WithThe intent of this study is to determine how ApoE genotype affects AD pathology by brain region. By comparing the frequencies of neuropathological scores for several different brain regions, we aim to produce a more refined and specific characterization AD pathology in a large, clinicopathologically confirmed sample that is stratified by age and ApoE genotype.The subjects in this study were recruited from the Banner Sun Health Research Institute Brain and Body Donation Program located in Sun City, Arizona . IndividData from 775 cases autopsied between January of 1997 and December 2011 were available for the analysis. Only individuals who had received both a clinical diagnosis of dementia and a neuropathological diagnosis of AD were included in the study. 209 individuals were excluded due to incomplete neuropathological information, other clinical diagnoses of non-AD dementia or unavailable genotype.The final sample size was 566 and consisted of 312 AD-ApoE4+ and 254 AD-ApoE4- cases who were followed prospectively with antemortem dementia diagnoses. The sample did not differ statistically with respect to numbers of males (n = 295) and females (n = 271). Clinical AD diagnoses were made using DSM-IV criteria .Neuropathological AD diagnoses were made according to National Institute on Aging/Reagan Institute criteria  and inclSenile plaque and neurofibrillary tangle load scores were obtained using the CERAD scoring system with separate semi-quantitative density estimates of none, sparse, moderate, or frequent  using standardized published templates . RegionsDNA for ApoE genotyping from autopsy cases was extracted from pieces of fixed cerebellum tissue. Tissue (100 mg) was digested to completion with proteinase K (1 mg/ml) at 55°C and extracted with phenol/chloroform. DNA was recovered by isopropanol precipitation. For PCR reactions, 500 ng of DNA from each sample was used. PCR primers, amplification conditions employed, and identification of ApoE genotypes by Hha I digestion of amplified material, were carried out according to a published protocol . Digested.For demographic data, chi-square analysis was used to assess the association between ApoE ϵ4 carrier status and gender. The Mann–Whitney U test was used to discern differences on age at death and years of education between ApoE ϵ4 carriers and non-carriers. The effect size of these differences was assessed using Cohen’s For the pathological data, the sample was stratified by ApoE ϵ4 carrier status and age with the frequency of pathological score reported for each stratified group. The sample was divided into the following age groups: ≤69, 70–79, 80–89, ≥90. The data were also stratified by ApoE genotype to examine pathological differences between each ApoE isoform. For the stratified carrier status and age analyses, the Mann–Whitney U test was used to compare plaque and tangle rating frequency differences between ApoE ϵ4 carriers and non-carriers in each age group. For the genotype analyses, the Kruskal-Wallis test was used to compare differences of plaque and tangle pathology rating frequencies. Groupwise comparisons were carried out using the Dwass-Steel-Chritchlow-Fligner test.2 = 1.17, df (1), p = 0.28). Education was not significantly different between carriers and non-carriers, however ϵ4 carriers had a significantly younger age at death and significantly higher Braak score than non-carriers. The effect size for age at death was small, while the effect size for Braak score was moderate. Table For the sample of 566, the average age at death was 82.56 (8.33) years and the average education level was 14.54 (2.67) years. The sample was comprised of 295 males and 271 females. Demographic characteristics broken down by ApoE ϵ4 carrier status are displayed in Table Scores for plaque and tangle frequencies stratified by age group and ApoE ϵ4 carrier status are shown in Tables When analyzed by individual ApoE genotype, cases containing at least 1 ϵ4 allele  had greater frequencies of “frequent” ratings for both plaque and tangle scores when compared to the 2/3 and 3/3 groups. Within the ApoE ϵ4 carriers, the proportion of “frequent” ratings tended to increase (2/4 < 3/4 < 4/4). For all ApoE genotypes, the distribution of plaque pathology scores was skewed toward the “moderate” and “frequent” ratings for the frontal, temporal, and parietal areas Table . For theFor tangle scores, the overall distribution of score frequencies was somewhat more even than plaque scores Table ; howeverIn this study we found within a series of prospectively characterized and clinicopathologically confirmed AD cases, ApoE ϵ4 carriers had a significantly higher percentage of “frequent” scores for plaques and tangles when using the CERAD scale when compared to ApoE ϵ4 non-carriers in several regions of the brain. These results imply that carrying the ϵ4 allele increases the density of pathologies in several brain regions. Since AD is characterized by senile plaques and tangles, our study supports previous evidence demonstrating that the presence of the ApoE ϵ4 allele is a strong contributor to increased AD pathology. However, our study also demonstrates a much more detailed continuum of AD pathology as it relates to ApoE genotype. To our knowledge, the relationship between ApoE genotype and AD pathology has not been reported in this manner before. Previous studies have reported AD pathology and ApoE ϵ4 associations in terms of odds ratios ,19; howeWhen analyzed by individual ApoE genotype, “none”, “sparse” and “moderate” plaque density scores were more prevalent among 3/3 carriers with 3/4 and 4/4 carriers having a higher prevalence of “frequent” scores among the different regions. A similar pattern was also noted for tangle density scores. There was a trend among all ApoE genotype groups for tangle pathology to be less frequent than plaque pathology in the frontal, temporal, and parietal regions. These results suggest that ApoE ϵ4 carries and non-carriers have independent neurodegenerative pathways in which plaque and tangle frequency vary among different cortical regions. Given that ApoE ϵ4 carrier status has been shown to result in differing clinical phenotypes of AD, -21 our rNagy et al.  found thBeffert et al.  found thRichey et al.  found thHowever, a study by Wisniewski et al.  showed tGuo et al.  investigOne positive aspect of this study is the large sample size which allowed for differences to be seen between the different ApoE genotypes. Although the CERAD scoring scheme is commonly used to quantify AD pathology, it uses a semi-quantitative scoring system and does not provide an exact measurement of pathological densities. One weakness of our study was the low number of ApoE 2/4 carriers relative to the other genotypes so the comparisons of pathology score frequencies to the other genotypes may be somewhat biased. However, since the estimated prevalence of the ApoE 2/4 isoform is relatively low (2%) ,31. AlsoAnother point of consideration is whether or not these results are dependent upon the proportion of different clinicopathologic subtypes of AD. Murray et al.  report tAlso, the geographical area from which tissue samples were collected is relatively homogenous with respect to ethnicity and socioeconomic status so it cannot be stated that these results would apply to populations that are more ethnically and intellectually diverse.The results of this study demonstrate that the density and cortical distribution of AD neuropathology differs between ApoE ϵ4 carriers and non-carriers. It is possible that these neuropathologic differences may play a role in determining clinical phenotype of AD. These results also suggest that different disease pathways are associated with ApoEϵ4 carrier status resulting in different distributions of AD neuropathology.The authors have no competing interests to disclose.MNS participated in the study design, performed clinical assessments, made clinical diagnoses, and drafted the manuscript. MM participated in the study design, drafted the manuscript, and carried out the statistical analyses. BND drafted the manuscript and acquired tissue samples. KL participated in the study design and drafted the manuscript. LIS drafted the manuscript and acquired tissue samples. GS drafted the manuscript and acquired tissue samples. DGW performed ApoE genotyping and drafted the manuscript. KD performed clinical assessments and drafted the manuscript. SAJ performed clinical assessments, made clinical diagnoses, drafted the manuscript. TGB drafted the manuscript and acquired tissue samples. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/13/44/prepub


Avpr1a), which exhibits striking differences in neural expression patterns that are responsible for mediating differences in vasopressin-mediated social behaviors. The genomic mechanisms that contribute to these remarkable differences in expression are not well understood. Previous work has suggested that both the proximal 5′ flanking region and a polymorphic microsatellite element within that region of the vole Avpr1a gene are associated with variation in V1a receptor (V1aR) distribution and behavior, but neither has been causally linked. Using homologous recombination in mice, we reveal the modest contribution of proximal 5′ flanking sequences to species differences in V1aR distribution, and confirm that variation in V1aR distribution impacts stress-coping in the forced swim test. We also demonstrate that the vole Avpr1a microsatellite structure contributes to Avpr1a expression in the amygdala, thalamus, and hippocampus, mirroring a subset of the inter- and intra-species differences observed in central V1aR patterns in voles. This is the first direct evidence that polymorphic microsatellite elements near behaviorally relevant genes can contribute to diversity in brain gene expression profiles, providing a mechanism for generating behavioral diversity both at the individual and species level. However, our results suggest that many features of species-specific expression patterns are mediated by elements outside of the immediate 5′ flanking region of the gene.Certain genes exhibit notable diversity in their expression patterns both within and between species. One such gene is the vasopressin receptor 1a gene ( Avpr1a). Specifically, differences in the expression of V1a receptor in the brain have been causally tied to social behavior differences, but the genetic basis of these differences is not understood. Using transgenic mice, we investigate the role of DNA sequences upstream of this gene in generating species-specific and individual variation in Avpr1a expression. We find that, contrary to our expectation, this region has only a modest influence on differences in expression patterns across rodent species. This indicates that DNA elements outside of this region play a larger role in species-level differences in expression. We confirm that variation in Avpr1a expression mediated by this upstream region translates to differences in behavior. We also find that variable DNA sequences associated with repetitive motifs within this region subtly influence gene expression. Together these findings highlight the complexity of genetic mechanisms that influence diversity in brain receptor patterns and support the idea that variable repetitive elements can influence both species and individual differences in gene expression patterns.DNA sequence variation underlies many differences both within and between species. In this paper, we investigate a specific DNA sequence that is thought to influence expression of a gene that modulates behavior, the vasopressin V1a receptor gene ( Caenorhabditis elegans, for example, variation in a single nucleotide of npr-1, which alters the neuropeptide receptor protein structure, has been shown to be responsible for strain differences in social feeding behavior Avpr1a) provides an excellent opportunity to explore both of these potential mechanisms of gene expression divergence The genomic mechanisms that give rise to phenotypic diversity across species or among individuals within a species are not well understood. Behavior is a trait that is particularly well suited for exploring genetic mechanisms underlying phenotypic plasticity, as it is an evolutionarily labile trait. Social behaviors, in particular, can be markedly variable among closely related species, and often display significant individual variability within a species Microtus ochrogaster), but not in the closely related, non-monogamous montane voles (M. montanus) M. pennsylvanicus) Avpr1a gene Avpr1a may underlie species differences in AVP-dependent behaviors.Arginine vasopressin (AVP) is an evolutionarily conserved neuropeptide that modulates a wide range of behaviors including stress coping, territorial aggression, mate-guarding, pair bonding and paternal care Avpr1a within the brain contributes to both intra-and inter-specific differences in behavior. For example, increasing V1aR density in the lateral septum using viral vector mediated gene transfer enhances social recognition memory in rats while increasing V1aR density in the ventral pallidum of prairie voles facilitates affiliation and pair-bond formation Avpr1a locus display a pattern of V1aR binding similar to that of prairie voles, and this difference in receptor patterns leads to increased affiliative behavior in response to AVP Avpr1a expression within the brain directly contributes to both inter and intra-species variability in AVP-mediated behaviors. Here we explore the contribution of both the 5′ flanking region and variation in hypermutable microsatellite sequences within this region in the generation of this variability in gene expression.There is substantial direct evidence supporting the hypothesis that diversity in expression of Avpr1a transcription start site have been hypothesized to contain regulatory sequences that contribute to the prairie vole-like patterns of V1aR Avpr1a transgene, comprised of 2.2 kb of 5′ flanking sequence, exons, introns and some downstream sequences from the prairie vole, displayed a receptor pattern more similar to that of a prairie vole than a mouse Avpr1a microsatellite. This microsatellite lies ∼760 bp upstream of the Avpr1a transcription start site and displays polymorphisms in repeat numbers and sequence content across and within vole species, and thus represents a “hot spot” for mutations , prairie vole short (pvKI-short), or meadow vole (mvKI) were identical in sequence except for the composition of the microsatellite element. All three lines were backcrossed to a C57Bl/6J background for at least 5 generations prior to neuroanatomical and behavioral experiments.The targeting strategy is illustrated in Avpr1a gene. This SNP is a G in most lines examined, including C57-related lines, but is a C in 129-related lines Because we performed recombination in hybrid B6/129 ES cells, there was a possibility that recombination could occur at either the C57Bl6/J or the 129SvEv locus. In order to determine the integration site for our three lines, we genotyped rs13480799, which is located outside of the 5′ homology arm upstream of the Avpr1a gene Avpr1a expression) in wildtype (WT) and pvKI-long littermates at post-natal day (PND) 60–70. Because the endogenous mouse 5′ flanking region was replaced with prairie vole sequence, this technique was not subject to random integration effects, as occurs in traditional pronuclear injection transgenics.Previous work had suggested that some of the elements integral to species-specific neural V1aR patterns existed within the 2.2 kb upstream of the transcription start site of the Avpr1a gene expression since this technique is much more quantitative and sensitive than in situ hybridization, provides greater anatomical resolution than qPCR, and accurately reflects Avpr1a mRNA patterns (Young 1997). Furthermore, since the replaced region lies upstream of the transcription start site, variation in that sequence should not affect post-transcriptional processing. The greater signal to noise ratio of this technique allows us to detect relatively subtle differences in V1aR protein binding. Replacement of the 5′ flanking region of the murine Avpr1a locus with the same region from prairie voles yielded qualitative patterns of V1aR with elements of both mouse and prairie vole expression , CeA (p<0.001), PVThal (p<0.001), and DG (p<0.001), but not in the lateral septum  or meadow vole (mvKI) Avpr1a microsatellite, and identified a significant effect of both brain region  = 114.7, p<0.001) and genotype  = 160.9, p<0.001) on V1aR levels. In addition, there was a significant interaction between brain region and genotype  = 74.2, p<0.001), and simple main effects with Sidak-adjusted α showed that pvKI-long animals had significantly higher levels of V1aR than mvKI animals in the CeA (p<0.001), PVThal (p = 0.002), and DG (p<0.001), but not in the lateral septum  but not in the CeA (p = 0.42), PVThal (p = 0.96), LS (p = 0.93) or VP . No spontaneous mutations occurred in the intervening generations. While mutation rates are species specific, this suggests that changes in the microsatellite sequence do not occur every generation, but rather on a longer evolutionary scale, which is in accordance with previously reported mutation rates of 10−2 to 10−5 mutations per locus per generation Microsatellite sequences have been hypothesized to act as evolutionary “tuning knobs,” because they mutate at faster rates than other parts of the genome, potentially due to “slippage” of the DNA polymerase while copying these highly repetitive regions. To investigate the rates of mutation in the Avpr1a expression are not known. Transcriptional differences may ultimately depend on a combination of differences in DNA secondary structure, epigenetic characteristics, and/or differential binding of transcriptional enhancers within the microsatellite-containing region Avpr1a expression would need to be expressed within the brain. While most transcription factors showed at least moderate levels of expression in a few brain regions, one factor that putatively binds uniquely in the long microsatellite, Ras-responsive element binding protein 1 (Rreb1), was particularly notable because it is highly expressed within the dentate gyrus . The homology arms were sequenced and the same homology arms were used in all three targeting constructs. Three versions of the prairie vole Avpr1a 5′ flanking region containing the meadow and prairie long and short microsatellite versions were isolated from previous expression constructs Sbf1.KI mice were generated using a targeting construct illustrated in Acc651 and evidence of recombination was detected using a probe located upstream of the 5′ homology arm  where it was electroporated into hybrid C57Bl/6J/129SV embryonic stem cells. DNA from neomycin resistant/gancyclovir-sensitive clones were screened via southern blot. Specifically, genomic DNA was digested with logy arm . This yi5′ TACAAGTGAGTGGGCCTTTCCTGT and 5′ GAGCCTCGCGGGAAACTCAT for the WT allele (754 bp) and 5′ AGCTCTCTTCCATGCATTCGACCA and 5′ ACAGAAGCAACAGTGACCTTCCCT for the KI allele (334 bp)  .ad libitum access to food and water, and were maintained on a 12∶12 light∶dark cycle. Mouse lines were maintained separately and WT and KI experimental animals were derived from heterozygous breeding pairs in each line .Mice were housed in groups of 3–5 animals with mixed genotypes, had 125I-linear-AVP ligand  in buffer containing 50 mM Tris (pH = 7.4), 10 mM MgCl, and 0.1% BSA for 1 hour. The slides were then washed 4×5 min in 50 mM Tris buffer with 0.2% MgCl at 4°C followed by a final 30 minute rinse in the same buffer at room temperature with agitation. Slides were rinsed briefly in double distilled water and allowed to dry overnight before exposure to BioMax MR film along with an ARC146-F 14C standard. Multiple exposures, ranging from 18 to 72 hours, were performed to ensure all regions of interest could be evaluated within the linear range of the film. All slides were processed simultaneously.N5 and N6 generation mice were euthanized between PND 60–70 via cervical dislocation followed by decapitation. Receptor autoradiography was performed as previously described Receptor densities were quantified by densitometry using MCID software as previously described All statistical calculations are presented as mean ± SEM, and were performed in SPSS version 19. We tested for line differences by comparing WT littermates of all 3 lines  using a 2-way ANOVA with line and brain region  as factors. We found a significant effect of brain region (F (48) = 122.2; p<0.001), but no significant effect of line  (F (48) = 1.095; p = 0.35), and no evidence of interaction between the two (F (48) = 0.823, p = 0.56). Based on these results, WT littermates from all three lines were grouped together in subsequent analyses as the WT comparison group. To compare V1aR density in the brains of mice with different KI genotypes, we again used 2-way ANOVAs with genotype and brain region as factors. When significant main effects of genotype or interactions were observed, we conducted a simple effects analysis for genotype using a Sidak corrected α to account for multiple comparisons.N5 to N6 adult male littermates (4–5 months old) were used to assess novel object recognition. No more than 3 animals of the same genotype were used from a given litter. We used a modified version of the protocol described by Denny et al. Novel objects consisted of (1) a blue, ceramic shoe ; a black plastic box (8×3×9.5 cm); and a clear plastic funnel (diameter 8.5 cm). The mouse could not displace these objects, and the objects were tested previously and elicited the same levels of exploration th session. All animals in the same cage were tested at the same time, and the cage order randomized with respect to the three lines. Testing for all animals was completed within an 8 day period.Novel object consisted of five 5 minute exposures with 3 minute inter-exposure intervals. Mice were place in the center of the arena at the start of each exposure. In between tests, mice were returned to holding cages while the arena was cleaned with a 1% Sparkleen solution and the bedding was replaced. Exposures 1–4 were habituation sessions in which two objects place symmetrically on either side of the arena ∼5 cm away from the wall. In exposure 5, one of the objects was replaced with a novel object. This is referred to as the probe trial. All mice were returned to their home cages following the 5Total locomotion was calculated via automated tracking using AnyMaze Software (Stoelting). Object investigation during the probe trial was scored by an observer blind to genotype using Noldus Observer. Object investigation was defined as orientation of the head toward the object with the nose within 1 cm of the object. Investigation was not scored if the mouse was on top of the object or completely immobile. Novelty preference was determined using a two way ANOVA of Object×Genotype with Object as a repeated measure.N6 to N7 adult male WT and KI littermates (5–6 months old) were used in the Forced Swim test. No more than 3 animals of the same genotype were used from a given litter. Since WT animals between the different lines did not differ in V1aR binding in the CeA or in time struggling  = 0.13, p = 0.88), all WT animals  were combined into one group. Additionally, since pvKI-long (n = 18) and pvKI-short (n = 13) did not differ in the amount of time spent struggling  or in V1aR binding in the CeA, these groups were combined into a single pvKI group. Testing was performed beginning 1 hr after lights on. All animals in the same cage were tested at the same time in three to five separate swim chambers, and the cage order was randomized with respect to the three lines. Testing for all animals was completed in a single day. Behavioral response to forced swimming was measured as described in David et al. Genomic DNA was purified from tail tissue samples using the DNeasy purification kit . The V1a microsatellite region was amplified as previously described. PCR products were gel purified and extracted using the Zymoclean gel DNA recovery kit . Sequencing of PCR products was performed by Macrogen USA .


Cryptococcus neoformans is a heterothallic fungal pathogen of humans and animals. Although the fungus grows primarily as a yeast, hyphae are produced during the sexual phase and during a process called monokaryotic fruiting, which is also believed to involve sexual reproduction, but between cells of the same mating type. Here we report a novel monokaryotic fruiting mechanism that is dependent on the cell cycle and occurs in haploid cells in the absence of sexual reproduction. Cells grown at 37°C were found to rapidly produce hyphae (∼4 hrs) and at high frequency (∼40% of the population) after inoculation onto hyphae-inducing agar. Microscopic examination of the 37°C seed culture revealed a mixture of normal-sized and enlarged cells. Micromanipulation of single cells demonstrated that only enlarged cells were able to produce hyphae and genetic analysis confirmed that hyphae did not arise from α-α mating or endoduplication. Cell cycle analysis revealed that cells grown at 37°C had an increased population of cells in G2 arrest, with the proportion correlated with the frequency of monokaryotic fruiting. Cell sorting experiments demonstrated that enlarged cells were only found in the G2-arrested population and only this population contained cells able to produce hyphae. Treatment of cells at low temperature with the G2 cell cycle arrest agent, nocodazole, induced hyphal growth, confirming the role of the cell cycle in this process. Taken together, these results reveal a mating-independent mechanism for monokaryotic fruiting, which is dependent on the cell cycle for induction of hyphal competency. Cryptococcus neoformans is a yeast-like fungus that has not been traditionally thought to be dimorphic since hyphae production typically occurs during the mating process between cells of opposite mating types. However, C. neoformans also can generate the hyphal state from haploid cells  in the absence of the opposite mating type. Recent studies have shown that the mechanism behind this process also involves mating, however, the mating reaction occurs between cells of the same mating type. Here we describe a unique mechanism responsible for monokaryotic fruiting that is independent of mating and does not proceed through a diploid intermediate. Instead, the key requirement for hyphal induction appears to be cell cycle arrest. Importantly, arrested cells display an enlarged cell phenotype, which has been observed in vivo in recent reports and has been hypothesized to be a novel protection strategy against host defenses. C. neoformans appears to have an extensive morphological repertoire, which likely contributes to its success as both a pathogen and a saprophyte.Fungi typically grow vegetatively as either yeast or hyphae. Many of the major human fungal pathogens can generate both morphologies and are referred to as the dimorphic fungi.  Cryptococcus neoformans is a basidiomycetous fungal pathogen of humans and animals that typically causes opportunistic infections in patients with cellular immune defects C. neoformans are heterothallic with two mating types, MATa and MATα, with both mating types being pathogenic, although most clinical isolates are MATαC. neoformans variety grubii and variety neoformans respectively, and serotypes B and C being collectively referred to as C. neoformans variety gattii, or more recently, C. gattiiin vitro conditions, however, the fungus can produce two kinds of hyphae; dikaryotic hyphae during MATa×MATα sexual reproduction and monokaryotic hyphae  during monokaryotic fruiting Saccharomyces cerevisiae pheromone response pathway, are required to produce both types of hyphae MATa and one MATα) per hyphal compartment while monokaryotic hyphae have unfused clamp connections and a single nucleus per hyphal compartment. Sexual reproduction in C. neoformans has been characterized in detail and largely follows the pheromone response paradigm that has been developed from decades of S. cerevisiae research Naturally occurring strains of C. neoformans variety neoformans can result from α-α mating C. neoformans variety neoformansα-α cell fusion. However, this assumption proved to be false. Instead, we found that cells arrested in the G2 stage of the cell cycle were competent to undergo monokaryotic fruiting at high frequency, in the absence of α-α cell fusion. Importantly, this mechanism proceeds through enlarged cells, a morphological phenotype that has been increasingly observed in vivo and is hypothesized to serve as a strategy for avoiding host defenses C. neoformans has evolved a number of different mechanisms for modifying cellular morphology to suit its specific environment, with some of these mechanisms contributing to the success of this fungus as a pathogen.Recent studies have concluded that monokaryotic fruiting in Previous studies of monokaryotic fruiting typically were performed by patching cells from a seed culture onto filament agar and then screening for a hyphal fringe around the periphery et al., have demonstrated that same-sex mating is one way in which monokaryotic fruiting can occur Lin α-α mating during monokaryotic fruiting using the α-α cell fusion assay to test different combinations of complementing MATα auxotrophs that would be predicted to yield prototrophic colonies on unsupplemented MIN agar. No fusants were observed from MATα×MATα crosses plated onto MIN agar after growth as a seed culture on YPD at 37°C for 24 h. However, assisted α-α matings showed that each of these strains was capable of undergoing α-α fusion, ruling out an α-α cell fusion defect  with the cpk1Δ mutant (WSA-2126) showed evidence of α-α cell fusion  all revealed only 1n and 2n DNA content peaks, with no evidence of a 4n peak . This reα-α mating or endoduplication as monokaryotic fruiting mechanisms in our system . This mechanism appears to be dependent on the cell cycle and initiates from cells in G2 arrest. It occurs in the absence of α-α mating and/or endoduplication, thereby demonstrating that monokaryotic fruiting can occur asexually. Previous studies have shown that sexual reproduction can occur between cells of the same mating type, resulting in monokaryotic fruiting α-α cell fusion event, nor did we find evidence of endoduplication within the hyphae even though we screened hyphae that had produced basidiospores. During C. neoformans basidiosporogenesis, sexual reproduction results in meiosis in the basidium followed by successive mitotic divisions that yield the nuclei, which ultimately are inserted into spores as they form on the basidial surface et al. observed that when fruiting was derived from an α-α fusant, sporogenesis was robust with spore chains that were long and phenotypically similar to α-a mating during sexual reproduction dmc1 mutants, which are meiotic mutants that still produce spores, but at a much lower frequency than sexually produced spores, and with truncated spore chains that sometimes occur as dyads (two rather than four chains) dmc1 strains was strikingly similar to what we observed in this study and what was previously reported dmc1 mutant and found that it was able to undergo monokaryotic fruiting under our conditions (data not shown).In this study we have identified a novel mechanism in C. neoformans, and their incubation period was for a period of weeks, whereas we screened at 24 hrs and observed hyphae in as little as four hours, although cells also produced hyphae on V8 agar under our conditions. Both filament agar and V8 agar have high agar contents; however, V8 agar is an undefined medium with V8 juice as the basal ingredient. Filament agar, on the other hand, is a low-glucose, defined medium with Yeast Nitrogen Base without amino acids and without ammonium sulfate as the source of vitamins and cofactors. Although both media are starvation media, they are substantially different in composition, which may be one explanation for the differences in hyphal types that we observed. The seed culture conditions, or more precisely, the cell cycle stage may be another explanation.Because our study was done in the same strain background as the study by Lin et al., we reviewed the conditions of both experiments and found some differences that may explain the contrasting differences in ploidy. Our study used a high temperature seed culture condition, which results in rapid hyphae production upon filament agar plating. The seed culture conditions in Lin's study were not clear, however, their plating medium was V8 agar, which is normally used for mating α-α fusion or endoduplication event, which would result in a diploid cell, the actual mechanism that resulted in hyphae production was G2 arrest. The reason for the requirement of G2 arrest to induce hyphae is not clear, and while G2 arrest is required for monokaryotic fruiting, not all arrested cells produced hyphae. We suspect that the subpopulation of non-hyphal, G2-arrested cells consisted of cells that escaped G2, and then proceeded to bud rather than differentiate into a hypha. These two outcomes resemble the decision point that a pheromone-exposed, G2-arrested Ustilago maydis cell faces with regard to which of the two developmental paths it will follow (conjugation tube formation or budding) C. neoformans hyphae produce typical basidiomycete-like clamp connections, the incomplete fusion of these structures in monokaryotic hyphae combined with an aberrant segregation event during the budding of blastospores off of the hyphal compartment may yield the diploid cells that we observed at low frequency.Our initial investigation of the relationship between the cell cycle and monokaryotic fruiting focused on detecting what we presumed would be a transition to diploidy in yeast cells at some point during seed culture growth, which we believed would coincide with the appearance of enlarged cells in the seed culture and the association of this cell type with the hyphal progenitor. The high frequency of fruiting and enlarged cells in the seed culture suggested that detection of the diploidization event would be unambiguous. However, the data showed that instead of an C. neoformans cell cycle have identified a number of stressors that cause G2 arrest, including oxygen depletion C. neoformans, we were able to induce hyphae in all four serotypes. Interestingly, the reports of enlarged cells in vivoC. neoformans cell size in vitro, including cAMP, RAS, and PKA STE12α signal transduction pathway seems to be the major or sole regulator of monokaryotic fruiting as this gene is required for monokaryotic fruiting regardless of inducing conditions. Fruiting still occurred normally in cpkΔ1 (pheromone response pathway), and cacΔ1 (cAMP pathway) mutants, ruling out these pathways as regulators of temperature-induced monokaryotic fruiting. Other pathways, such as the calcineurin signal transduction pathway cannot be ruled out, but are more complicated to test since some mutants in this pathway do not grow at high temperature Previous studies of the in vivo appears to be polyploidy, which has been hypothesized to arise when the M phase of the cell cycle is skipped in vivo via the same mechanisms. It appears that the enlarged cell phenotype can be produced by multiple mechanisms: cell cycle arrest, a-α or α-α cell fusion, and endoduplication, each of which may have a different purpose. The developmental options available after cell cycle arrest may have been selected for in C. neoformans to enhance survival in its specific environmental niche while inadvertently creating an important human fungal pathogen. What remains to be determined is how high temperature generation of the large cell and monokaryotic fruiting phenotypes was incorporated into the evolution of C. neoformans. With the exception of C. neoformans, virtually all members of this genus grow poorly or not at all at mammalian ambient temperature. In contrast, all of the major human fungal pathogens grow at 37°C and virtually all of them have a hyphal phenotype. Perhaps the association of many basidiomycetes with rotting wood or decaying vegetation in general led to high temperature exposure and subsequent genetic selection during self-heating, compost-like conditions caused by microbial metabolism of organic matter. Once nutrients were consumed, hyphal extension towards additional nutrients and/or sporulation in the absence of nutrients could have completed the evolution of C. neoformans into a pathogen via development of a mechanism of infectious particle (basidiospores) dispersion combined with the ability to grow at elevated temperatures. Selection for the molecular linkage of pathways controlling cell cycle, nutrient sensing, and ultimately, differentiation, could have been the outcome of this lifestyle and allowed the fungus to coordinately regulate these pathways, thus enabling it to effectively exist as a saprophyte or pathogen.A key characteristic of enlarged cells MATα isolate that was used in the initial characterization of monokaryotic fruiting in C. neoformansMATα lys2), WSA-70 (MATα ade2 lys2), WSA-591 (MATα ade2), WSA-1226 (MATα ura5), WSA-2126 (MATα ura5 cpk1Δ::ura5), WSA-3002 (MATα/MATα fusant from WSA-1226×WSA-70×WSA-68), WSA-3019 (MATα haploid blastospore), WSA-3070 (MATα/MATα diploid blastospore recovered from JEC-21 hypha without basidiospore chains), WSA-3098 (MATα haploid blastospore recovered from JEC-21 hypha with basidiospore chains), WSA-3112 (MATα haploid blastospore recovered from hypha without basidiospore chains), WSA-3145 (rMATα ura5 cpk1Δ::ura5 CPK1::NEO). MATa strains included WSA-65 (MATa ura5 lys1 ade2) and WSA-68 (MATa ura5 ade2 lys2).YPD agar, MIN agar, V8 agar, and filament agar were prepared as described previously The standard assay for monokaryotic fruiting was performed as described 6 cells from a YPD seed culture grown at 37°C in 1 ml sterile water. This suspension was then serially diluted to yield approximately 2000 cells per ml. A 100 µl aliquot was then spread onto filament agar, which was incubated at 25°C for 24 h. The number of hyphal colonies was then compared to the total number of cells to determine the percentage of cells that were capable of undergoing monokaryotic fruiting.Monokaryotic fruiting efficiency was determined by quantitative assay. This assay was performed by suspending ∼10Hyphal ploidy was determined using the blastospore assay 6 cells/ml. Tubes were shaken at 200× RPM at 30°C for 24 hrs. Five µl of overnight culture were then dropped onto filament agar and incubated at 25°C for 5 days. The quantitative assay was performed by growing cells as above, and then plating cells onto filament agar as described in the quantitative monokaryotic fruiting assay. Pictures were taken with an Olympus SZX12 stereo microscope  at 2× magnification.Nocodazole  stock was prepared in DMSO at 1.5 mM and then used to prepare different dilutions in YPD broth . JEC-21 cells were added to 1.5 ml nocodazole-YPD broth in 15 ml snap cap tubes , at a final concentration of 1×10α-α cell fusions, 1×106 cells of complementary, auxotrophic, MATα strains were mixed, cultured on YPD agar at 37°C for 24 hrs, and then transferred onto filament agar plates. The plates were incubated at 25°C for 24 hrs. The mixture was scraped from the plate, suspended in 1.0 ml sterile H2O, and then 200 µl of cells from this suspension were spread onto MIN agar plates, which were then incubated at 30°C for 4 days to screen for fusants. Assisted mating reactions, in which two MATα strains were induced to fuse by including a MATa helper strain, were performed according to previously described methods To perform Cells were harvested, fixed, and stained with propidium iodide , and then sorted as described by Sia et al. et al.7 cells were harvested from 24 h YPD agar plates, which were grown at either 30°C or 37°C, washed twice with phosphate buffer , then resuspended in 400 µl fixative  followed by incubation at room temperature for 90 minutes. The cells were then washed three times in wash buffer and incubated with Lysing Enzymes  for 20 min at 37°C. The cells were then washed once with sterile water, gently resuspended in 400 µl 0.3% Triton X-100 (Sigma-Aldrich), and permeabilized by incubation at room temperature for 15 minutes. The suspension was then pelleted and washed three times with PBS. Cells were stained in DAPI (Sigma-Aldrich)  for 15 minutes, washed twice with PBS, and then resuspended in 200 µl prior to visualization.Cells were stained with DAPI according to the method described by Fuchs Images were captured on a Zeiss AxioImager Z1 microscope  equipped with an AxioCam MR3_2 CCD camera, using the filters for DAPI  and differential interference contrast . Image analysis and adjustments were performed using Axiovision software . Images were adjusted only for frame alignment (to overlay DAPI over DIC), brightness, and contrast (adjustment of +0.01 contrast units over default), and all images received the same treatment.


In spite of extensive freshwater CH4 emissions, most of the CH4 is typically oxidized by methane oxidizing bacteria (MOB) before it can reach the lake surface and be emitted to the atmosphere. In turn, it has been shown that the CH4-derived biomass of MOB can provide the energy and carbon for zooplankton and macroinvertebrates. In this study, we demonstrate the presence of specific fatty acids synthesized by MOB in fish tissues having low carbon stable isotope ratios. Fish species, zooplankton, macroinvertebrates and the water hyacinth Eichhornia crassipes were collected from a shallow lake in Brazil and analyzed for fatty acids (FA) and carbon stable isotope ratios (δ13C). The fatty acids 16∶1ω8c, 16∶1ω8t, 16∶1ω6c, 16∶1ω5t, 18∶1ω8c and 18∶1ω8t were used as signature for MOB. The δ13C ratios varied from −27.7‰ to −42.0‰ and the contribution of MOB FA ranged from 0.05% to 0.84% of total FA. Organisms with higher total content of MOB FAs presented lower δ13C values (i.e. they were more depleted in 13C), while organisms with lower content of MOB signature FAs showed higher δ13C values. An UPGMA cluster analysis was carried out to distinguish grouping of organisms in relation to their MOB FA contents. This combination of stable isotope and fatty acid tracers provides new evidence that assimilation of methane-derived carbon can be an important carbon source for the whole aquatic food web, up to the fish level.Large amounts of the greenhouse gas methane (CH The production of methane is performed by methanogens, which are microorganisms belonging to the domain Archaea.Methane  Aquatic food webs are supported by organic matter derived from phytoplankton, macrophytes or imported from the surrounding land  13C) has been a successful tool in food web studies 4 is much depleted in 13C, low δ13C values in organisms of aquatic food webs have been considered to indicate their consumption of CH4-derived carbon 13C in animal biomass have been reported for invertebrates in various aquatic systems Isotopic distributions in animals are generally closely related to dietary isotopic composition 13C values have been reported for fish, and a few studies have suggested that CH4-derived carbon could be transferred in substantial amounts to higher trophic levels 13C values for zooplankton, aquatic insects and detritivorous fishes in a Brazilian Pantanal lake, being the first study to discuss the importance of methanotrophs for the whole food web in this wetland.Low δ13C values can be an indicator of CH4 carbon, the isotopic signal from other potential sources of 13C-depleted carbon – such as primary production based on CO2 from respiration 13C values are due to assimilation of carbon from biogenic CH4While more negative δThe lipids of the proteobacterial MOB have special fatty acid (FA) composition et al.13C-depleted freshwater invertebrates have been supporting the hypothesis of a link between MOB and animal food webs Similarly, monounsaturated fatty acids, like those diagnostic for MOB, can thus be microbial biomarkers indicating transfer of carbon and energy from methane to higher food web levels. The assumption that animals actually ingest MOB has been supported by the detection of phospholipid fatty acids (PLFAs) diagnostic for MOB in tissues of chironomid larvae 13C and fatty acid composition in fish, benthic macroinvertebrates, zooplankton, and the dominating aquatic macrophyte from a shallow tropical lake in Pantanal, Brazil. We found specific fatty acids of methane-oxidizing bacteria in tissues of aquatic invertebrates and fish, showing that methane can in fact contribute carbon to large parts of aquatic food webs and production of fish biomass.In this study, we tested the hypothesis that methane carbon from MOB can be transferred through food webs all the way to fish, by combining analyses of δ13C values ranged from −42.0 to −27.7‰ and the contribution of MOB FA varied from 0.05 to 0.84% of total FA  organisms with lower content of MOB signature FAs and higher δ13C values. The former group includes Ceratopogonidae and the fish Anadoras grypus with the highest contribution of MOB fatty acids , Chironomidae sp.1 (0.73%), Cyphocharax sp. (0.71%) and the ephemeropteran Campsurus sp. (0.66%). These five organisms were also depleted in 13C . All these organisms can feed at oxic-anoxic interfaces where MOB abundance is likely to be high Chaoborus sp. showed the lowest δ13C values  and a MOB-specific FA contribution of 0.45 and 0.50%, respectively. Calheiros (13C in zooplankton (−42.7 to −31.6‰), benthic chironomids (−62 to −49‰) and ephemeropterans (−41.4 to −34.3‰) in another Pantanal lake, suggesting an assimilation of biogenic CH4. Other taxa in group A include the fishes Potamorhina squamoralevis, Crenicichla sp., Leporinus friderici and Steindachnerina brevipinna which had equally low δ13C values (δ13C<−36‰) and a range of MOB-specific FA content from 0.43 to 0.66%. These fishes can all have benthivorous and omnivorous or detritivorous feeding habits, except Crenicichla sp., which is mainly carnivorous.All δtotal FA . Based o FA  and small variations in isotopic signals among seasons, which could be due to a more specialized diet. Wantzen 13C changes in the diet affected by biogeochemical processes.A higher variability in FA composition than in δ13C-depleted fish species Tetragonopterus argenteus (−27.7‰), Astyanax cf. bimaculatus (−28.0‰) and Parauchenipterus galeatus (−28.4‰), had lower contents of MOB-specific FA . The higher δ13C values in combination with lower MOB FA content of these three fishes indicate a lower incorporation of MOB biomass. This conclusion is in accordance with their feeding habits. These taxa are known to be pelagic or living close to aquatic plants, presenting omnivorous habits with tendency to herbivory-invertivory T. argenteus and A. bimaculatus in Pantanal. Although aquatic C4 plants were not the major carbon source, they suggested that at least part of the A. bimaculatus diet would be derived from aquatic C4 grasses, which have higher δ13C values than C3 plants and occur in small patches among large mats of C3 macrophytes Eichhornia crassipes, a C3 plant that totally dominates the macrophyte community in the studied environment, had a similar δ13C signature (−27.8‰). Therefore, a combination of carbon sources from C3 plants, C4 plants and MOB could yield a 13C signature similar to C3 plants.Group B , formed Sphagnum mosses) E. crassipes is a floating macrophyte and not a moss, we cannot exclude the possibility of this phenomenon.Traces of MOB FA found in plant samples might have two different causes. First, MOB FA in plant samples could be due to the difficulty to completely remove biofilms from the plant surface, especially those attached to the submerged roots. Secondly, there are studies that revealed the presence of MOB inside living tissues of submerged aquatic plants (13C signatures). In contrast to the grouping based on total MOB FA and 13C values illustrated in The dendrogram in 1 grouped organisms with the lowest contributions of MOB FA, i.e. E. crassipes and T. argenteus. This is in accordance with the fact that T. argenteus is an omnivorous fish with tendency to herbivory-insectivory, feeding mainly on vascular plants 2 was formed by all other organisms with higher MOB FA values.At a similarity of 0.4, two distinct clusters can be seen. Cluster 2 was divided into two new clusters: One had a high contribution of the FA 18∶1ω8c, which is highly specific for type II MOB Type II MOB. The other cluster was based on high contribution of specific FA markers for MOB type I, and named Type I MOB. Inside cluster Type II MOB, Chaoborus sp. and zooplankton were grouped together since they presented the highest contributions of 18∶1ω8c , followed by A. grypus . Chaoborid larvae are zooplankton predators, whose preferred food source are microcrustaceans, although they eat a wide variety of animals including dipteran larvae, oligochaetes, rotifers, other chaoborids E. crassipes. Anadoras grypus is an invertivore, bottom-feeding fish At a similarity of 0.6, cluster Type I MOB, three clusters were identified at a “cutoff” similarity of 0.8:Within cluster Campsurus sp. which showed the highest content of the FA 16∶1ω5t. Nymphs of Campsurus are bottom collectors/gatherers The ephemeropteran Cyphocharax sp., Chironomidae sp.1, Ceratopogonidae, L. friderici and S. brevipinna). Inside this cluster, two sub-groups emerged: one formed by Cyphocharax sp., Ceratopogonidae and Chironomidae sp.1, and another with L. friderici and S. brevipinna. Species of Cyphocharax pick mainly biofilms from the bottom substrates L. friderici and S. brevipinna were the most 13C depleted fishes and they have similar feeding habits. Both fishes are iliophagous and feed on detritus, benthic filamentous algae and invertebrates associated to bottom substrates A cluster of organisms with the highest contributions of 16∶1ω8c which are mainly benthivorous and iliophagous . The group of Chironomidae sp.2 and P. squamoralevis showed similar proportions of both fatty acids 16∶1ω6c and 16∶1ω5t, while A. cf. bimaculatus, Crenicichla sp. and P. galeatus were similar to each other in terms of primarily 16∶1ω6c. Potamorhina squamoralevis feeds mainly on detritus and benthic algae and invertebrates associated with bottom sediments Astyanax bimaculatus is an omnivorous fish which consumes mainly insects Crenicichla are stalking predators, feeding on insects and fishes P. galeatus consists mainly of terrestrial and aquatic insects, but also aquatic invertebrates, fishes, fungi, algae, higher plants and detritus Two groups with high contributions of 16∶1ω6c and 16∶1ω5t  were not determined and could not be included in this model. The flood pulse also changes the lake dynamics, and may affect the relative importance of MOB for the food chain. We applied the same model for aquatic insects and zooplankton from this lake and obtained a contribution of 16% and 22% as carbon source respectively, a similar value as the results reported by Ravinet et al. The presence of MOB signature FAs in benthic aquatic insects, planktonic organisms as chaoborids and zooplankton, and fishes showed the importance of biogenic methane to different aquatic compartments in this Pantanal ecosystem . We usedectively . However13C seems to be driven more by the MOB type II FA (18C) than by MOB Type I FAs (16C). It is possible that MOB type II dominates over type I in this wetland and the Pantanal floodplain in general. Sundh et al. 4 oxidation in flood-pulsed wetlands In the present study, the relationship between contribution of MOB FAs and δ4 and high oxygen concentrations The two groups of MOB have different growth characteristics and respond differently to variation in temperature, pH and concentrations of oxygen and methane. Low temperatures seem to favor the development MOB type I over type II 4, such as high primary productivity, shallow depth of inundation, and high temperature 13C signatures, we have to be careful to draw the conclusion that MOB type II dominates fish consumption of MOB in Pantanal as the actual levels of MOB type I and type II FAs in the fishes were overall quite similar.The Pantanal floodplain is characterized by conditions that together favor production and emissions of CH13C), that MOB carbon was transferred through the food web up to the fish level. Hence, our data demonstrate that CH4 can be a significant carbon source not only for the microbial food web and invertebrates, but also for higher trophic levels. The Pantanal region is characterized by high CH4 production 4 carbon throughout the food web to various fish species could be a common phenomenon in CH4-rich ecosystems.This study showed, by combining two independent tracers  in the Paraguay River flood plain near Ladário, Mato Grosso do Sul, Brazil. The fishes were provided by local inhabitants using hand-thrown nets. The captured fishes were killed instantaneously to minimize suffering and we were allowed to document the fish taxonomy and take subsamples immediately after fishing, before further use of the fish by the locals. A piece of fish dorsal muscle was cut out and freeze dried. Zooplankton were collected with a zooplankton net (150 µm mesh), rinsed thoroughly with deionized water, handpicked into 2 ml sterile Eppendorf tubes, and freeze dried. Eichhornia crassipes was the totally dominating macrophyte in and around the lake and whole plants including roots were sampled (this plant is floating and roots are detached from the sediment). The plants were rinsed in water and biofilms were removed from their surfaces. Sediment was collected by diving and sieved for sampling of benthic invertebrates. All benthic invertebrates were kept in filtered (Whatman GF/F) lake water for 24 hours for gut clearance, and were then rinsed and freeze dried.Different species of fishes, zooplankton, aquatic invertebrates and the aquatic macrophyte 13C = {(δ13C/δ12C)sample/|(δ13C/δ12C)standard−1|}×1000. Samples with higher isotope values are relatively enriched in the heavy isotope 13C while samples with lower isotope values are relatively depleted in 13C and enriched in the light isotope 12C.Samples for stable isotope analysis were analyzed with a Carlo Erba NC2500 analyzer connected to a Finnigan MAT Delta plus mass spectrometer. Carbon stable isotope ratios were reported relative to the PDB (PeeDee Belemnite) standard and expressed as a per mil (‰) deviation according to the equation δLipids from biological samples were extracted following the method of Hara and Radin A cluster analysis was performed to recognize grouping of organisms according to their contents of the different MOB FA markers 16∶1ω8c, 16∶1ω8t, 16∶1ω6c, 16∶1ω5t, 18∶1ω8c and 18∶1ω8t  and to identify differences among those groups. The hierarchical clustering was carried out on a Bray-Curtis similarity matrix (organisms as objects and MOB FA as descriptors) and represented by a dendrogram produced by UPGMA (Unweighted Pair-Group Method with Arithmetic averages) clustering of this resultant matrix. The cluster analysis was performed using the software package PAST version 2.12 4-derived carbon for fish, macroinvertebrates and zooplankton, a simple two-source mixing model was used (x = {c−b}/{a−b}) as suggested by Trimmer et al. E. crassipes (a) and MOB (b) as dietary end members. The δ13C values of fish, zooplankton and aquatic insects represented the mixture (c). Trophic fractionation was assumed to be negligible. The δ13C values of MOB were derived from methane δ13C values measured for the lake (mean: −80.9‰) by Conrad et al. In order to make an approximation of the relative contribution of CH


The phenotype of these mice is similar to adenylyl cyclase 3 (AC3) knockout mice largely due to the significant down-regulation of AC3 activity in β3GnT2−/− neurons.The defining feature of the main olfactory system in mice is that each olfactory sensory neuron expresses only one of more than a thousand different odorant receptor genes. Axons expressing the same odorant receptor converge onto a small number of targets in the olfactory bulb such that each glomerulus is made up of axon terminals expressing just one odorant receptor. It is thought that this precision in axon targeting is required to maintain highly refined odor discrimination. We previously showed that β3GnT2−/− mice compared to controls. Analysis of OR expression by quantitative PCR and in situ hybridization demonstrates that the number of neurons expressing some odorant receptors, such as mOR256-17, is increased by nearly 60% whereas for others such as mOR28 the number of neurons is decreased by more than 75% in β3GnT2−/− olfactory epithelia. Analysis of axon trajectories confirms that many axons track to inappropriate targets in β3GnT2−/− mice, and some glomeruli are populated by axons expressing more than one odorant receptor. Results show that mutant mice perform nearly as well as control mice in an odor discrimination task. In addition, in situ hybridization studies indicate that the expression of several activity dependent genes is unaffected in β3GnT2−/− olfactory neurons.Microarray analysis reveals that nearly one quarter of all odorant receptor genes are down regulated in β3GnT2−/− mice and further demonstrate that additional axon subsets grow into inappropriate targets or minimally innervate glomeruli in the olfactory bulb. Odor evoked gene expression is unchanged and β3GnT2−/− mice exhibit a relatively small deficit in their ability to discriminate divergent odors. Results suggest that despite the fact that β3GnT2−/− mice have decreased AC3 activity, decreased expression of many ORs, and display many axon growth and guidance errors, odor-evoked activity in cilia of mutant olfactory neurons remains largely intact.Results presented here show that many odorant receptors are under-expressed in β3GnT2 Understanding the organization of connections in the mammalian olfactory system is a challenge because the rules used to generate a map of axon trajectories from the olfactory epithelium to the olfactory bulb (OB) appear to differ considerably from other sensory modalities, such as the visual and somatosensory systems -3. It is−/− axons [−/− mice appear in many respects to phenocopy wiring defects described for AC3 knockout mice where olfactory cAMP signaling is also reduced [We have previously shown that the glycosyltransferase β3GnT2 is expressed in OSNs and is the key enzyme in the synthesis of polylactosamine (PLN) glycans ,8,9. AC3/− axons . The sev reduced -12. In b reduced ,13.−/− mice are anosmic due to the absence of cAMP synthesis and the resulting inability to activate CNG channels in dendritic cilia [−/− mice, AC3 traffics normally to cilia and although reduced amounts of cAMP are produced, adult β3GnT2−/− mice are not anosmic [−/− mice, we compared OR gene expression, examined axon trajectories by immunocytochemistry with specific OR antibodies, and carried out an odorant discrimination task on WT and β3GnT2−/− mice. Results presented here show that many OR genes are significantly down-regulated in β3GnT2−/− mice such that only a small number of those axons make connections in the OB. In addition, other OR-specific axon subsets innervate inappropriate targets in the OB. These findings will be discussed in light of the fact that β3GnT2−/− mice can discriminate odors with considerable fidelity.Despite these many similarities, the major difference between the two mouse models is their respective abilities to detect and discriminate odors. AC3ic cilia . In cont anosmic ,8. In or−/− mice and littermate controls and mRNA was isolated from each sample. Following preparation of cDNA and labeling, the samples were each hybridized to an Affymetrix Mouse Gene 1.0 ST Array . A median value was calculated for each probe set for each OR gene in the 10 samples. Median values of the five null samples were compared pairwise with median values from the five WT samples. A null/WT ratio was generated that represents the mean of the five pairwise comparisons, Of 1,144 probe sets for OR transcripts, results indicated that mRNAs for 867 of the ORs were expressed at similar levels in WT and β3GnT2−/− OEs, including OR37 and M72. Results also indicated that expression of 256 OR genes was decreased by more than 30% in all five samples obtained from β3GnT2−/− OEs compared to the five WT OE samples. In addition, 21 OR transcripts, including mOR 256–17 and mOR 125–1 were increased by greater than 25% in all β3GnT2−/− OEs. The null/WT ratios of several of the 21 ORs that were increased the most, and a few of the many other ORs that decreased or were unchanged are presented in Table −/− OEs compared to littermate controls and performed real time RT-qPCR or in situ hybridization analyses.Five separate samples of OE were dissected from adult β3GnT2−/− OEs compared to controls and three ORs, mOR13-4, mOR18, and mOR28 were significantly decreased. qPCR analysis showed that mOR256-17 expression was increased by 71% in β3GnT2−/− OEs compared to controls, and mOR28 was decreased by 89%, validating the gene array generated values of a 90% increase for mOR256-17 and a 79% decrease for mOR28.Using the gene array data as a guide, we chose primer sets for six ORs with null/WT ratios that were either greater than 1.9 or were less than 0.6. After isolating RNA from OE samples, we performed RT-qPCR to find the relative differences in the expression of these ORs , mOR256-17 (olfr15) and mOR125-1 (olfr43) were significantly increased in β3GnT2−/− OEs compared to WT OEs  in approximately the correct location of the β3GnT2−/− OB. However, this glomerulus is for the most part I7 negative but synaptophysin positive, indicating that it is principally populated by axons expressing other ORs.Although the level of expression of many ORs is significantly decreased in the OE of β3GnT2 mice, independent analysis using transgenic mice, d Figure , Table 2−/− OEs compared to wild type OEs . Therefore, the data within each day were combined and compared using a two-way repeated measures ANOVA using genotype and day as factors .The significant loss of OR gene expression and the axon guidance errors, coupled with the dramatic decrease in AC3 activity reported earlier , we expe3 Figure . An init−/− mice were able to discriminate all six odor pairings but that they had a significant deficit in their ability to discriminate three of the six odor pairings either on Day 3 or Day 4 compared to littermate controls  but had similar adenylyl cyclase stimulatory capabilities. Furthermore, some of the odors tested were ones that had previously been tested in AC3 transgenic mice. For example, AC3 heterozygous mice display a gene-dose dependent decrease in ability to discriminate citralva [−/− mice was not statistically different from WT mice. Although AC3 is decreased on olfactory axons in β3GnT2−/− mice, the levels of AC3 protein in residual OSN cell bodies and cilia is relatively normal [Although a direct relationship between correct axon targeting and olfactory discrimination has not been established, several studies have shed considerable light on this issue. On the one hand, rats retained most of their olfactory discrimination following surgical removal of significant portions of the target . In contcumented ,8. We, tcitralva , suggesty normal , suggest−/− mice. Using immunocytochemical techniques, we showed that mOR256-17+ axons are mistargeted to multiple glomeruli on the medial and lateral surfaces of the OB. However, some mOR256-17+ axons form a small glomerulus very close to where their normal target should be located. Furthermore, even though only a few I7+ and mOR28+ axons innervate glomeruli in the adult OB, these axons never reach the level required to form their own unique pair of glomeruli. It is possible that just a small number of OR-specific axons connecting to mitral or tufted cells in the correct targeting region of the OB is sufficient to transduce a signal that is correctly interpreted in the olfactory cortex. These results suggest that although a smaller number of neurons expressing a given OR will likely decrease the probability that an odorant-binding event will lead to perception of that odor, only a small number of connections may actually be required for successful transduction [We show here, in addition to the changes in OR expression, further examples of aberrant axon guidance in β3GnT2sduction .−/− OEs, further supporting the possibility that AC3 activity is preferentially preserved in cilia of these null mice. Interestingly, in many CNS neurons and glia where β3GnT2 is not expressed, AC3 expression is restricted to primary cilia [−/− OEs suggest that the remaining AC3 may preferentially localize to cilia [Olfactory axon guidance is orchestrated via a signaling cascade that includes G protein-coupled receptors, AC3 activation, cAMP synthesis and a downstream pathway involving PKA and CREB ,28-30. Try cilia . AC3 is ry cilia . Tectoniry cilia . OSNs apry cilia ,12. In tto cilia . This woto cilia .−/− mice have a modest deficit in their ability to distinguish odors. This is in spite of the fact that hundreds of ORs are underrepresented in the OE and many probably do not innervate normal glomeruli in the OB. Furthermore, axon guidance in β3GnT2−/− mice is aberrant, as shown by the abnormal position and increased number of mOR256-17+ glomeruli in the OB of knockout mice. Moreover, we have previously shown that AC3 activity is decreased by 80 to 90% in β3GnT2−/− mice, such that guidance and adhesion molecules such as neuropilin-1 and kirrel2 are poorly expressed in axons [Olfactory discrimination testing shows that β3GnT2−/− mouse is a unique model of reduced cAMP production in which most of the residual AC3 activity appears to be concentrated in cilia where odor-evoked transduction is largely intact. In contrast, cAMP-dependent transcriptional regulation required for proper axon guidance is virtually absent. An important question that remains unanswered is to what degree glomerular heterogeneity can be tolerated before olfactory discrimination is significantly compromised. Results presented here would suggest that the mouse olfactory system may have a large capacity to withstand down regulation of cAMP signaling, significant deletions in the OR repertoire, alterations in guidance molecule expression and errors in wiring before suffering significant losses of olfactory perception.The β3GnT2−/− mouse littermates were generated from β3GnT2 het/het crosses. PCR was used to genotype the offspring, with a 700-bp fragment amplified from the wildtype β3GnT2 allele and a 1,071–bp fragment amplified from the β3GnT2−/− allele. β3GnT2−/− mice were established from the KST308 embryonic stem cell line, which harbors a secretory trap vector insertion in the β3GnT2 coding sequence as previously described [Wild type and β3GnT2escribed . Mice weDigoxigenin (DIG)-labeled sense and antisense riboprobes were transcribed from cDNAs containing subcloned OR coding sequences using vector-specific RNA polymerases and DIG labeling mix . For in situ analysis of adult mouse OEs, age P28 mice were deeply anesthetized and transcardially perfused with 4% Formaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed and further fixed overnight in 4% Formaldehyde at 4°C followed by immersion in 30% sucrose. Bone thickness in the posterior region of the snout from the orbital area to the OB was reduced using surgical knives. The dissected samples were frozen on dry ice, coronally sectioned at 20-μM thickness with a cryostat, and then mounted on Superfrost plus microscope slides, . After prehybridization and hybridization, the DIG-labeled RNA hybrids were detected with an anti-DIG Fab fragment conjugated to alkaline phosphatase  at a dilution of 1:1,000 overnight at 4°C. The color reaction was produced with nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate with levamisole added to block endogenous phosphatase activity.Tissue was prepared by transcardial perfusion using 2% PLP  fixation in 0.1 M phosphate buffer (PB), pH 7.4. Antibodies to mOR256-17 are sensitive to fixation conditions and we determined empirically that immunoreactivity on frozen tissue sections was enhanced using 2% PLP compared to 4% paraformaldehyde or other stronger fixatives. Heads were subsequently removed and postfixed 16 hrs in the same fixative solution, followed by cryoprotection in 30% sucrose. After embedding, tissue sections were prepared on a Microm HM505E cryostat at 50 μM thickness and were then immediately thawed in transwell boats filled with PBS for staining as free floating sections. Tissues were blocked for one hour in 2% BSA, then incubated overnight at 4°C with the primary antibody for mOR156-17  and dilu−/− and WT control mice were quantified using a Nikon Eclipse E400 microscope  at 100X. The complete OE cavity was serial sectioned at 20 μM and collected on 20 to 22 slides per case. Each in situ hybridization experiment included a pair of β3GnT2−/− and WT slides for each OR probe. Each probe was used on three different pairs of mice. All the sections from one slide of each genotype were counted and used for analysis. These counts were compared using a paired two-way ANOVA to control for any variability, which may occur between each in situ experiment. In addition, pairs of slides within a case, using one probe, were counted and compared and found to be less than 3% different, which was not significant (NS). Thus, we extrapolated the total amount of labeled cells for each OR by adjusting the counts to the total number of slides in each case (Table 2). Statistical analysis was performed using SigmaStat 2.0 .Specific OR-expressing neurons in PD28 OEs of β3GnT2+/+ and β3GnT2−/− mice at six months of age using Trizol Reagent (Invitrogen), and cDNA prepared as described previously . Quantitative PCR (qPCR) was performed with oligonucleotides designed using online Primer3 software (version 0.4.0). Primer sequences used for template amplification are available upon request. Samples were amplified in triplicate using GoTaq Polymerase  in a StepOnePlus Real-Time PCR System . Relative gene expression levels were determined by the Comparative CT (threshold cycle) method after normalization to RNA polymerase 2 as an endogenous reference.RNA was extracted from microdissected olfactory epithelia of 6 β3GnT2OEs from adult mice were collected in PBS on ice. Tissue samples were homogenized using TRIzol  and the RNA was collected in the aqueous phase after chloroform extraction. Subsequently, Isopropyl Alcohol was used to precipitate the RNA, followed by washing with 75% ethanol, air-dried, and redissolved in RNAse-free DEPC H2O. After this RNA isolation, a Qiagen RNeasy mini kit  was used to further purify the RNA, and the yield analyzed with a DU 650 Spectrophotometer . Yields per mouse OE measured at an absorbance of 260 nm were 10 to 20 ug RNA.Affymetrix gene array technology was used to compare the gene profiles between β3GnT2 WT and mutant mouse strains. Individual samples were hybridized to 12 gene chips in a high-density oligonucleotide array /chip) that uses 25-mer probes distributed across the transcribed regions of each gene, with a median of 26 probes per gene. The Ambion WT Expression kit  was used to synthesize first and second strand cDNA, and generate the purified sense strand for the Affymetrix gene chip WT Terminal labeling kit . The single stranded cDNA produced by the Ambion kit was fragmented and labeled using the GeneChip WT Terminal Labeling Kit. The product was then hybridized to the chip for 16 to 17 hours at 45°C, washed, stained and scanned using the Affymetrix GeneChip Array Scanner. The log based signal values were generated using the RMA algorithm in Expression Console. For each pair of samples, results of each probe were compared and an “R” value was calculated. The median of the “R” values of each probe set (R_median) was used for filtering the potentially differential gene expression. No normalization was conducted.+/+ and β3GnT2−/− mice 9 to 13 weeks old were trained in an olfactory discrimination task [β3GnT2ion task . Pairs oion task . The micAC3: Adenylyl cyclase 3; CNG: Cyclic nucleotide gated channel; CREB: Cyclic nucleotide response element binding protein; DIG: Digoxigenin; OB: Olfactory bulb; OE: Olfactory epithelium; OR: Odorant receptor OSN, olfactory sensory neuron; Nrp1: Neuropilin-1; PBS: Phosphate-buffered saline; PKA: Protein kinase A; PLN: Polylactosamine; RT-qPCR: Reverse transcriptase quantitative polymerase chain reaction; Sema3A: Semaphoring 3A; WT: Wild type.The authors declare that they have no competing interests.TKK designed most of the experiments and drafted the initial manuscript. PAM and AAF performed the gene array and RT-qPCR experiments. MX analyzed the gene array data. JS provided the 256–17 antibody and helped to revise the manuscript. TRH supervised the molecular analyses and helped to write and revise the manuscript. GAS supervised the whole study and edited the manuscript. All authors read and approved the final manuscript.


RT-qPCR is commonly employed in gene expression studies in ectopic pregnancy. Most use RN18S1, β-actin or GAPDH as internal controls without validation of their suitability as reference genes. A systematic study of the suitability of endogenous reference genes for gene expression studies in ectopic pregnancy is lacking. The aims of this study were therefore to evaluate the stability of 12 reference genes and suggest those that are stable for use as internal control genes in fallopian tubes and endometrium from ectopic pregnancy and healthy non-pregnant controls. Analysis of the results showed that the genes consistently ranked in the top six by geNorm and NormFinder algorithms, were UBC, GAPDH, CYC1 and EIF4A2  and UBC and ATP5B (endometrium). mRNA expression of NAPE-PLD as a test gene of interest varied between the groups depending on which of the 12 reference genes was used as internal controls. This study demonstrates that arbitrary selection of reference genes for normalisation in RT-qPCR studies in ectopic pregnancy without validation, risk producing inaccurate data and should therefore be discouraged. Ectopic pregnancy is a common early pregnancy complication in which the embryo implants outside the uterus. It is estimated to complicate 2% of pregnancies and, more importantly, is still associated with significant morbidity and mortality in many parts of the world ,2. The mi.e., that it is susceptible to variation in extraction and experimental processes including differences in RNA extraction, presence of contaminating DNA, efficiency of cDNA synthesis etc.  after written and informed consent. The choice of non-pregnant women  as a comparison group is consistent with current practice as it is unethical and difficult to obtain fallopian tubes from women in the first trimester of pregnancy.Volunteers were recruited from the Women’s Hospital, University Hospitals of Leicester NHS Trust and were either attending as emergencies with a diagnosis of ectopic pregnancy or were non-pregnant volunteers having total abdominal hysterectomy and bilateral salpingoophorectomy (TAH & BSO) for benign gynaecological conditions such as fibroids or dysfunctional uterine bleeding. The non-pregnant were categorised into two groups based on their last menstrual period (LMP) and endometrial histology according to Noyes criteria Tissue biopsies were collected immediately following TAH & BSO or following salpingectomy for ectopic pregnancy, washed (to remove blood) with sterile 1X phosphate buffered saline (PBS) and placed into sterile polypropylene tubes. Samples were immediately transported to the laboratory in liquid nitrogen and stored at −80 °C for later RNA extraction.260/280 absorbance ratios greater than 1.9. RNA integrity was assessed using an Agilent bioanalyser 2100  and samples with RNA integrity number (RIN) of greater than seven were considered suitable for RT-qPCR.Tissues obtained (50 mg) were homogenised in a mortar and total RNA extracted using Qiagen miRNeasy mini kit (50)  according to the manufacturer’s instructions. The RNA obtained was treated with DNAse to eliminate contaminating DNA prior to storage at −20 °C for future use. The yield and purity of isolated RNA was determined using a Nanodrop ND-1000 UV spectrophotometer  and samples selected for RT-qPCR had AFirst strand cDNA was synthesised from 1 μg of DNA free RNA using a high capacity RNA-to-cDNA reverse transcription kit  according to the manufacturer’s instructions. The final 20 μL reaction volume contained 10 μL of 2× RT buffer, 1 μL of 20× RT enzyme and 1 μg of total RNA diluted with molecular grade water to a final volume of 9 μL. A No RT reaction (RT-minus) was included to confirm the absence of contaminating DNA. The thermal conditions for reverse transcription were 37 °C for 1 h followed by 95 °C for 5 min before storage at −20 °C for future use.® 7000 system . Analysis of dissociation curves for all primers showed a single peak. To calculate the PCR efficiency of primers for NAPE-PLD as the test gene of interest, standard curves were constructed which showed a linear relationship between template quantity and target gene expression. The gradient of the standard curve for the target gene was used to calculate the PCR efficiency according to the formula: Efficiency (E)(%) = (A − 1) × 100 where A = 10[−1/slope of standard curve]. The PCR efficiency for NAPE-PLD was 93.1% with a linear correlation coefficient (R2) of 0.972.Experiments were performed using the human geNorm™ reference gene selection kit containing validated lyophilised primer sets for 12 reference genes . All reactions were carried out in a final reaction volume of 20 μL containing 10 μL of 2× SYBR Green PCR Universal Master Mix , 300 nM primer mix of reference genes or 900 nM of NAPE-PLD primers (genbank accession number NM_198990), 1 μL of cDNA and 8 μL of molecular grade water. Amplifications were performed starting with 95 °C for 10 min for enzyme activation, followed by 40 cycles of template denaturation step at 95 °C for 15 s, data collection for 60 s at 60 °C followed by extension at 72 °C. RT-minus and no template controls (NTC) containing molecular grade water instead of template mRNA were included with each run. No product was synthesised in the NTC and RT-minus confirming the absence of contaminating DNA. All reactions for the reference genes were performed in triplicate according to the manufacturer’s protocol using ABI PRISMi.e., geNorm version 3.5 [For stability comparisons of reference genes, two algorithms were used sion 3.5  and Normsion 3.5  accordinsion 3.5 . Detailssion 3.5 –23.® 7000  output data file were converted to relative expression values using the 2−ΔΔCq method [The mRNA expression level for each of the 12 endogenous reference genes was obtained using the mean RT-qPCR threshold cycle (Ct) value. This is defined as the number of cycles needed for the fluorescence to reach a specific threshold level of detection and is inversely correlated with the amount of template nucleic acid present in the reaction. Cq values obtained from the ABI PRISMq method . BrieflyThe highest Cq value was subtracted from all other Cq values for each gene measured. This gave a delta Cq value (ΔCq) with the highest delta Cq value being 0. All other values were less than 0.−ΔCq) was applied. Hence all data were expressed relative to the expression of the least expressed gene. Relative expression values were exported to geNorm and NormFinder for analysis.To each ΔCq value, the equation  or standard deviation (SD) and p values obtained using one-way analysis of variance (ANOVA) followed by appropriate post hoc analysis using GraphPad Prism version 5.00 for Windows . A p valAltogether, our results suggest that certain genes may be more suitable as reference genes in fallopian tubes or endometrium than others. The current widespread use of RN18S1, GAPDH and β-actin in the absence of appropriate validation should be avoided. Despite the limited sample size, at the very least, it does emphasise the importance of a systematic process being needed to choose an appropriate reference gene, and that the arbitrary selection of internal controls genes may lead to misinterpretation of data.


The influence of serous retinal detachment (SRD) on visual acuity, macular sensitivity, and macular thickness is unclear after intravitreal injection of triamcinolone acetonide (IVTA) for macular edema with branch retinal vein occlusion (BRVO).Twenty-one eyes of 21 BRVO patients with macular edema received IVTA. Patients were divided into two groups by optical coherence tomography findings: 11 patients who had cystoid macular edema (CME) with SRD (SRD (+) group) and 10 patients who had CME without SRD (SRD (−) group). Microperimetry was performed with a Micro Perimeter 1 before and at 3 and 6 months after IVTA. Macular thickness was measured by optical coherence tomography. We exchanged the superior and inferior regions to separate the regions into those with and without occlusion. As a result, the superior region was always the occluded region and the inferior region was non-occluded.In both the SRD (−) group and the SRD (+) group, the mean macular thickness within the central 4° field and the 10° and 20° fields of the occluded region decreased significantly from baseline to 3 and 6 months after IVTA . Visual acuity also improved significantly in both groups from baseline to 3 and 6 months after IVTA (both P <0.05). In both groups, the mean macular sensitivity (measured with by microperimetry) within the central 4° field and the 10° and 20° fields of the occluded region showed a significant increase from baseline to 3 and 6 months after IVTA . The trend profiles of macular thickness within the 10° and 20° fields of the occluded region showed significant differences, but there were no significant differences with respect to the trend profiles of visual acuity and macular sensitivity within the central 4° field and the 10° and 20° fields of the occluded region.These results suggest that IVTA may achieve more marked improvement of macular morphology in BRVO patients with SRD than in those without SRD, while this therapy may have a similar effect on macular function in BRVO patients with or without SRD. Branch retinal vein occlusion (BRVO) is a common retinal vascular disease that often leads to macular edema, which is the chief reason for visual impairment in BRVO patients ,2. An inOptical coherence tomography (OCT) has revealed that macular edema secondary to BRVO is frequently associated with serous retinal detachment (SRD), as well as with cystoid macular edema (CME) and inner retinal thickening -15. SomeThis study was approved by the Institutional Ethics Committee of Tokyo Women’s Medical University and adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from each patient. IVTA was performed as part of standard care because it has been reported that macular edema and visual acuity can be improved in BRVO patients by this procedure . At our The inclusion criteria were eyes with a foveal thickness greater than 300 μm and a visual acuity equal to or less than 20/30. The exclusion criteria were (1) previous ocular surgery, (2) diabetes mellitus with diabetic retinopathy, (3) previous macular laser photocoagulation, (4) previous intravitreal injection of anti-VEGF agents or triamcinolone acetonide, (5) a history of ocular inflammation, (6) marked retinal hemorrhage , (7) coexisting ocular disease , and (8) retreatment during the 6-month follow-up period. Twelve patients had superior vein occlusion and 9 patients had inferior occlusion.All patients had undergone a comprehensive ophthalmologic examination, including best-corrected visual acuity measurement, intraocular pressure determination, indirect ophthalmoscopy, and slit-lamp biomicroscopy with a contact lens before and at 3, and 6 months after treatment. In addition, retinal sensitivity was investigated by microperimetry, and retinal thickness was measured by OCT.In the current study, 21 patients received IVTA under local anaesthesia. For intravitreal therapy, the most common dosage of triamcinolone acetonide was 4.0 mg in a volume of 0.1 ml. Injection of triamcinolone into the vitreous fluid was done via the pars plana at 3–4 mm posterior to the limbus. All injections were performed with a sterile technique, and prophylactic topical antibiotics were applied for 1 week after injection. All patients were followed up for at least 6 months postoperatively. Recurrence of macular edema was defined as an increase  of foveal thickness when the thickness had once decreased to <300 μm at 3 months after initial IVTA . RecurreAs baseline screening, patients underwent ophthalmoscopy and biomicroscopic examination using a slit-lamp with a fundus contact lens. They also underwent standard fundus color photography and fluorescein angiography, which was performed with a Topcon TRC-50EX fundus camera, an image-net system , and a preset lens with a slit-lamp.A masked grader independently assessed ischemic retinal vascular occlusion on the fluorescein angiograms by measuring the ischemic area of the retina with the public domain Scion Image program, as reported previously -8. On di2, and chart contrast was more than 74%. The results were converted to the logarithm of the minimum angle of resolution scale (log MAR).Each patient underwent measurement of best-corrected visual acuity (BCVA) with an SC-2000 System chart . BCVA was measured in decimal units on a Landolt chart by the orthoptists. The chart brightness was set at 80–320 cd/mOCT was performed with an instrument from Zeiss-Humphrey Ophthalmic Systems  to measure the foveal thickness. At each visit, all patients underwent Stratus OCT examination in the vertical cross-section with the instrument centered on the fovea and in the fast macular thickness mode. On these views, retinal thickness was defined as the distance between the inner surface of the neurosensory retina and the retinal pigment epithelium. Foveal thickness was calculated as the average retinal thickness within a circle of 500-μm radius centered on the fovea. A retinal thickness map and retinal volume map were obtained by scanning 6×6 mm (20°×20°) areas of the macular region, which was divided into the following nine subfields: 1) fovea, 2) superior inner macula, 3) nasal inner macula, 4) inferior inner macula, 5) temporal inner macula, 6) superior outer macula, 7) nasal outer macula, 8) inferior outer macula, and 9) temporal outer macula . The diaIn this study, we exchanged the superior and inferior regions to separate the regions into those with occlusion and those without it. As a result, the superior region was always the occluded region and the inferior region was always non-occluded. We then analyzed the occluded and non-occluded regions separately. For the occluded region, the mean macular thickness was determined across four subfields  covering the central 3×3 mm (10°×10°), as well as across seven subfields  covering the central 6×6 mm (20°×20°). For the non-occluded region, the mean macular thickness was determined in one subfield (inferior inner) covering the central 3×3 mm (10°×10°), as well as across two subfields (inferior inner and inferior outer) covering the central 6×6 mm (20°×20°). The mean macular thickness was also determined in one subfield (fovea) covering the central 1×1 mm (4°×4°).SRD was defined as being present if OCT revealed typical accumulation of subretinal fluid resulting in neurosensory retinal detachment with low or absent reflectivity anterior to a clearly distinguishable outer band, irrespective of the coexistence of CME . CME was2) in 1-dB steps, and the duration of each stimulus was 200 ms. The fixation target was varied in size according to the patient's visual acuity. Retinal sensitivity maps were obtained by using the macula 20 degrees program of the MP-1. During the examination, background illumination was set at 1.27 cd/m2. Mean retinal sensitivity was calculated from the sensitivity for each of nine subfields on the retinal map generated by OCT. We analyzed data for the occluded and non-occluded regions separately. For the occluded region, we determined the mean macular sensitivity at 23 sites in the central 10° field  and at 44 sites in the central 20° field . For the non-occluded region, we determined the mean macular sensitivity at 6 sites in the central 10° field (inferior inner subfield) and at 13 sites in the central 20° field (inferior inner and inferior outer subfields). The mean macular sensitivity was also determined at five sites in the central 4° field.Microperimetry with the MP-1  is performed using an infrared fundus camera with a liquid crystal display controlled by special software. The MP-1 software contains an automatic tracking system for fundus movements; this evaluates every acquired frame for shifts in the x and y directions of the fundus with respect to a reference frame obtained by an infrared camera at the beginning of the examination. Each patient underwent fundus-monitored microperimetry with the MP-1 system . Its software performs automatic tracking of fundus movements and evaluates every frame acquired for fundus shift in the x and y directions relative to a reference frame obtained with an infrared camera at the beginning of the examination. Microperimetry settings were identical for all examinations: Goldmann III stimuli were presented in random order according to a 4-2-1 double staircase strategy. The stimulus intensity ranged from 0 to 20 decibels (dB) . Results are presented as the mean ± SD or as the frequency. One-way or two-way repeated measures ANOVA was used to evaluate changes in visual acuity, macular sensitivity, and macular thickness. To examine differences in the changes of parameters over time, trend profiles were determined by repeated measures analysis. Two-tailed P values of less than 0.05 were considered to indicate statistical significance.P = 0.448, P = 0.537, P = 0.890 P = 0.801, P = 0.287, P = 0.890, and P = 0.932, respectively).The characteristics of the SRD (−) group and the SRD (+) group are summarized in Table In both groups, IVTA achieved a reduction of clinical macular edema that was confirmed by OCT. In both groups, the mean macular thickness within the central 4° field decreased significantly from baseline to 3 and 6 months after IVTA Figure a. In botIn both groups, postoperative improvement of visual acuity was noted after IVTA and persisted for at least 6 months. The visual acuity of the SRD (−) group showed significant improvement from baseline to 3 and 6 months after IVTA Figure . LikewisIn both the SRD (−) group and the SRD (+) group, improvement of macular function after IVTA was detected with the MP-1. In both groups, the mean macular sensitivity within the central 4° field increased significantly from baseline to 3 and 6 months after IVTA  group and the SRD (+) group with regard to the trend profiles of macular thickness within the central 4° field or within the 10° and 20° fields of the non-occluded region Figures a-e.Up to 6 months after IVTA, two of the 21 patients (9.5%) showed an increase of intraocular pressure, but this could be controlled by a change of medication. Neovascular glaucoma was not detected in any of the patients, so trabeculectomy or shunt surgery was not performed. There were also no new cases of cataract or progression of existing cataracts and no infectious endophthalmitis after IVTA.In the present study, we found that the mean macular thickness within the central 4° field and within the 10° and 20° fields of the occluded region showed a significant decrease from baseline to 3 and 6 months after IVTA in both the SRD (−) group and the SRD (+) group. These results suggest that macular thickness is improved by IVTA in BRVO patients with SRD, as has already been demonstrated in BRVO patients without SRD. SRD is thought to be caused by transudation of extracellular fluid into the subretinal space -15,22,23We also found that visual acuity and macular sensitivity within the central 4° field and within the 10°, and 20° fields of the occluded region were improved significantly from baseline to 3 and 6 months after IVTA in both the SRD (−) group and the SRD (+) group. These results suggest that visual acuity and macular sensitivity can also be improved by IVTA in BRVO patients with SRD as has been shown in BRVO patients without SRD. In eyes with BRVO, fluid leaking from affected retinal capillaries accumulates around the fovea and produces retinal thickening, which may cause the internal limiting membrane at the clivus of the fovea to protrude with formation of foveal cystoid spaces . The celInterestingly, this study demonstrated a significant difference in the trend profiles of macular thickness within the 10° and 20° fields of the occluded region between the SRD (−) group and the SRD (+) group, while there were no significant differences between the two groups with regard to the trend profiles of visual acuity and macular sensitivity within the central 4° field and within the 10° and 20° fields of the occluded and non-occluded regions. In other words, macular thickness within the 10° and 20° fields showed a better response to IVTA in BRVO patients with SRD than in those without SRD. The greater improvement of morphology in the SRD (+) group compared with the SRD (−) group may have been due to the fact that SRD causes more extensive morphologic change compared with CME, thus allowing a stronger response to IVTA. On the other hand, visual acuity and macular sensitivity showed similar trend profiles after IVTA regardless of the presence or absence of SRD. This discrepancy between the trend profiles of macular thickness versus visual acuity and macular sensitivity may have arisen because SRD did not have much influence on macular function since it is caused by transudation of extracellular fluid into the subretinal space -15 and tThe limitations of this study were a short follow-up period, a small sample size, and lack of a control group. Although the results of long-term follow up could be interesting, additional treatment with further IVTA or anti-VEGF agents for recurrence may bias the outcome. Despite this risk, further investigations should be done to evaluate the long-term outcome in the future. In addition, we could not evaluate the relation between macular function and the inner segment/outer segment ratio of the photoreceptor layer because we did not have access to spectral domain OCT.There was a significant difference between the SRD (−) group and the SRD (+) group with respect to the trend profiles of macular thickness within the 10° and 20° fields of the occluded region, but there were no significant between-group differences with regard to the trend profiles of visual acuity and macular sensitivity within the central 4° field and within the 10°, and 20° fields of the occluded and non-occluded regions. These results suggest that IVTA may achieve more marked improvement of macular morphology in BRVO patients with SRD than in those without SRD, while this therapy may have a similar effect on macular function irrespective of the presence of SRD.No conflicting relationship exists for any author.HN and HF were involved in the design and conduct of the study. Collection and management of the data were done by HN and KS while analysis and interpretation of the data were performed by HN HF TM and KS Preparation of the first draft of the manuscript was done by HN and review and approval of the manuscript was performed by HF and TM All authors read and approved the final manuscript.None.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2415/12/39/prepub