Top PDF A novel assay reveals hygrotactic behavior in Drosophila.

A novel assay reveals hygrotactic behavior in Drosophila.

A novel assay reveals hygrotactic behavior in Drosophila.

So far, the molecular and cellular basis for hygrosensation is unclear, however, our findings provide evidence that two sets of molecules and cells function in the moisture-response behav- iors of Drosophila. When allowed to choose between dry air (~3% RH) and moist air (~99% RH), normal wild type flies prefer dry air over moist air [14–15]. However, humidity prefer- ence in wild type flies is adaptive and changes according to the status of body osmolality. Pert- tunen and Erkkila reported that when flies were dehydrated for several hours, they showed a preference for the moist air in their humidity choice assay [8]. Furthermore, the results of our assay demonstrated that thirsty flies are able to detect and locate water sources through sensing the gradient of moisture in their immediate environment. Here we reveal that arista and the third antennal segment play different roles in mediating moisture-induced behaviors, with our data suggesting that there exist two types of hygroreceptors in Drosophila: the hygroreceptors in the arista mediate moisture-aversive behavior in hydrated flies, while the hygroreceptors in the third antennal segment play an essential role in hygrotactic behavior in thirsty flies. Addi- tionally, Liu et al. reported that two channels from TRP family—Nan and WTRW—were nec- essary for normal flies to avoid moist air in their T-maze humidity choice assay [15]. However, we found that nan mutant and wtrw neural knockdown flies still exhibited strong hygrotactic behavior, comparable to that of wild type flies (S5 Fig.), suggesting that two sets of molecules function in the moisture-response behaviors of Drosophila.
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In vivo RNAi screen reveals neddylation genes as novel regulators of Hedgehog signaling.

In vivo RNAi screen reveals neddylation genes as novel regulators of Hedgehog signaling.

Differential neddylation of Cul1 and Cul3 in Hh signaling Our work, together with other studies [22], [59], [66], [67], demonstrates that the activities of both Cul1 and Cul3 are controlled by neddylation in Drosophila. However, it should be noted that there are differences in their respective neddylation patterns in response to reduced neddylation. In hypomorphic dUba3 or dUbc12 RNAi-expressing wing discs, reduced neddyla- tion led to high-level accumulation of un-neddylated Cul1, consistent with the notion that neddylated Cullin proteins are unstable [20]. However, the levels of un-neddylated Cul3 in this sensitized background seemed to be unaffected in wing disc lysates, although the reduction of neddylated Cul3 was evident (Figure 5A and Figure 8A). In contrast, when the neddylation process was compromised in dUba3 or dUbc12 mutant larvae, both Cul proteins were stabilized (Figure 5B and Figure 8B; data not shown). Similarly, much less neddylation of Cul3 is observed than that for Cul1 in our in vitro neddylation assays (Figure 5C and Figure 8C–E) as well as in vertebrate Cullins when tested in an in vitro assay [68]. This differential regulation of Cul1 and Cul3 is also observed in the fly mutants of CSN5 and Int6, two genes that are essential for de-neddylation [22], [66]. Although our results could simply reflect that Cul3 neddylation requires a much higher neddylation activity than Cul1, we believe that intrinsic differences may exist between Cul1 and Cul3 proteins. Neddylated Cul3 might degrade more rapidly than neddylated Cul1, which could explain distinct behaviors of Cul1 and Cul3 in our study. Indeed, the percentage of neddylated Cul3 in the total pool of Cul3 proteins in wildtype wing discs (i.e. 50% lower as determined by Image J densitometry in this study) and in brain lobes and eye discs [67] is significantly lower than that of Cul1, suggesting differential stability of neddylated Cullins. Further analyses are required to test this hypothesis and to elucidate the functional significance of differentially regulated Cullin proteins.
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Proteomics reveals novel Drosophila seminal fluid proteins transferred at mating.

Proteomics reveals novel Drosophila seminal fluid proteins transferred at mating.

should be applicable to many taxa. For example, worms, plants, rodents, and microorganisms are all amenable to isotopic labeling [29,41,42]. In any of these systems, differ- ential labeling should readily allow the detection of proteins transferred from one organism (during mating or another behavior, e.g., courtship). Thus, our approach allows trans- ferred proteins in a pre-specified biological process to be identified. Furthermore, our MS- and RACE-based method to identifying novel genes should be applicable to other organisms with sequenced genomes, particularly if their genome sizes are no more than 1–2 orders of magnitude greater than the D. melanogaster genome. Rodents, Arabidopsis, and humans all fall within this range; indeed, recent work in A. thaliana has found new genes using a similar approach [43]. Our results confirm that searching MS data against an entire translated genome, rather than only an annotated or predicted proteome, can identify a considerable number of new genes. Admittedly, this process might have been particularly useful for identifying Drosophila Sfps. As shown here and in previous work, these proteins are short, rapidly evolving, and relatively free of codon bias [19,22], three features making them less likely to be detected by computa- tional gene prediction programs. Nonetheless, our gene Figure 4. Discovery of Unannotated Genes in D. melanogaster Using a Six–Reading Frame Database Search and RACE
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A Follicle Rupture Assay Reveals an Essential Role for Follicular Adrenergic Signaling in Drosophila Ovulation.

A Follicle Rupture Assay Reveals an Essential Role for Follicular Adrenergic Signaling in Drosophila Ovulation.

Ovulation is essential for the propagation of the species and involves a proteolytic degrada- tion of the follicle wall for the release of the fertilizable oocyte. However, the precise mecha- nisms for regulating these proteolytic events are largely unknown. Work from our lab and others have shown that there are several parallels between Drosophila and mammalian ovulation at both the cellular and molecular levels. During ovulation in Drosophila, posterior follicle cells surrounding a mature oocyte are selectively degraded and the residual follicle cells remain in the ovary to form a corpus luteum after follicle rupture. Like in mammals, this rupturing process also depends on matrix metalloproteinase 2 (Mmp2) activity localized at the posterior end of mature follicles, where oocytes exit. In the present study, we show that Mmp2 activity is regulated by the octopaminergic signaling in mature follicle cells. Exoge- nous octopamine (OA; equivalent to norepinephrine, NE) is sufficient to induce follicle rup- ture when isolated mature follicles are cultured ex vivo, in the absence of the oviduct or ovarian muscle sheath. Knocking down the alpha-like adrenergic receptor Oamb (Octoam- pine receptor in mushroom bodies) in mature follicle cells prevents OA-induced follicle rup- ture ex vivo and ovulation in vivo. We also show that follicular OA-Oamb signaling induces Mmp2 enzymatic activation but not Mmp2 protein expression, likely via intracellular Ca 2+ as the second messenger. Our work develops a novel ex vivo follicle rupture assay and dem- onstrates the role for follicular adrenergic signaling in Mmp2 activation and ovulation in Drosophila, which is likely conserved in other species.
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Genome-Wide Analysis Reveals Novel Regulators of Growth in Drosophila melanogaster.

Genome-Wide Analysis Reveals Novel Regulators of Growth in Drosophila melanogaster.

With the exception of the kek1 cluster, all the most significant wing size associations mapped to putative novel growth genes: CG6091, a de-ubiquitinating enzyme whose human ortholog has a role in innate immunity; CG34370, which was recently identified in a GWA analysis of lifespan and lifetime fecundity in Drosophila [72]; and, surprisingly, dsx, a gene well characterized for its involvement in sex determination, fecundity and courtship behavior. dsx showed a significant effect on wing size in both sexes, CG6091 was only validated in males despite reaching a smaller p-value in the female GWAS, and CG34370 did not show a signifi- cant wing size change in either sex. Due to the obvious limitations of RNAi as a validation approach for SNPs we think it important to investigate the roles of CG6091 and CG34370 in growth control by other approaches before discarding them as false associations. As genes affecting growth also impact on general and reproductive fitness of organisms it is not surpris- ing that most of the candidate variants in or close to genes lie in regulatory regions, potentially modulating splicing, RNA turnover or RNA/protein abundance. Our data support the general notion that intergenic SNPs can impact phenotypes, either by affecting transcript abundance of protein coding genes (e.g. through distal enhancer elements) or via noncoding RNAs, which have been shown to regulate many biological functions including cellular processes underlying growth [73–75].
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p120RasGAP is a mediator of rho pathway activation and tumorigenicity in the DLD1 colorectal cancer cell line.

p120RasGAP is a mediator of rho pathway activation and tumorigenicity in the DLD1 colorectal cancer cell line.

Although we were able to transiently overexpress both wild-type and various activated KRAS mutants in DKO4 cells, only the G12V mutation could be stably expressed. It was previously reported that Keller et al. failed to overexpress KRAS G12V in DKS-8, another clone of DLD1 with knockout of the KRAS mutant allele, due in part to proteasomal degradation of the mutant [46]. However, HRAS or NRAS bearing this mutation could be expressed in this cell line, suggesting that the removal of a powerful oncogene may have a myriad of effects of a cell line, causing irreversible changes that cannot be rescued by simple re- introduction [46,47,48]. In our hands, attempts to stably express wild-type or any codon 12 or 13 KRAS mutant (other than Figure 3. RasGAP expression is mediated in part by KRAS. Wild-type (WT) or mutant KRAS was overexpressed in DKO4 cells. mRNA was extracted from cells and quantified using rt-qPCR to measure KRAS (A) or RASA1 (B). C) Western blotting showing levels of these proteins, along with activation status of KRAS. Correlation of mRNA (D) and protein expression using densitometry analysis of Western blotting (E) of KRAS and RASA1 after knockdown of KRAS using 11 different shRNAs. For protein correlation, outliers over 3 standard deviations from the mean were excluded. All quantification is relative to empty vector. Statistical analysis of expression using unpaired t-test, ***p,0.001, *p,0.05.
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Whole genome resequencing reveals natural target site preferences of transposable elements in Drosophila melanogaster.

Whole genome resequencing reveals natural target site preferences of transposable elements in Drosophila melanogaster.

group there was a tendency to have an ANAGT motif on the 5 9 half and an ACTNT motif on the 39 half of the TSM. LTR elements from the Pao group all share a relatively low information content motif characterized by an AWTAWNWTAWT motif. TSMs from the Gypsy superfamily appear to fall into three discrete subgroups, which fall clearly along established phylogenetic lineages represented by the 412, gyspy and Transpac families [8,19,31]. The TSM from the 412 clade (including the 412, blood, mdg1, Stalker2 and Tabor families analyzed here) contains a low information content ATAT motif spanning the TSD flanked by T and A on the 5 9 and 39 ends, respectively. The TSM from the gypsy clade (including Burdock, gtwin, gypsy, HMS-Beagle and opus) contains a central high information content TATA motif spanning the TSD and is flanked by A and T on the 5 9 and 39 ends, respectively. Finally, the TSM from the Idefix clade (including 297, Quasimodo and Transpac) contains a central high information content ATAT motif spanning the TSD and is flanked by C and G on the 5 9 and 3 9 ends, respectively. We note that Transpac is the most divergent member of the Idefix clade in previously published phylogenies of LTR elements in D. melanogaster [8,19] and also presents a divergent TSM relative to other members of the Idefix clade in our data as well. In the context of the wider phylogenetic relationships of the Pao and Gypsy clades, which can be represented in Newick format as (Pao,(412,(gypsy, Idefix))) [8,19,31], our data imply both an increase in target site specificity during the evolution of the more derived gypsy and Idefix clades, and at least one transition from an ATAT to a TATA core TSM. The latter transition may have been facilitated by a simple shift in the preferred target half-site, since the inferred ancestral state ATATAT (core TSM underlined), represented by the 412 clade, is only a 61 base pair edit from the derived state TATATA, represented by the gypsy clade.
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A novel 3D fibril force assay implicates src in tumor cell force generation in collagen networks.

A novel 3D fibril force assay implicates src in tumor cell force generation in collagen networks.

compared with 27 m m for GFP-wt-Src cells. We further show that the cell protrusions contain focal adhesion-like structures and invadopodia core complexes based on GFP-Src colocalization with F-actin and that dynamic membrane ruffling and membrane extensions form associated with these adhesion sites. Traction forces and protrusion lengths and lifetimes were measured from images necessarily obtained with a wide field-of-view, circum- stances under which invadopodia and matrix adhesions containing GFP-Src could not be resolved. We confirmed that GFP-ca-Src localizes to invadopodia and focal adhesions in 2D using higher resolution imaging of the same GFP-ca-Src cell line used in the traction force study. Similarly, cell protrusions in 2D and within 3D collagen contained clustered GFP-ca-Src near membrane protrusions that were highly dynamic. MDA-MB-231 cells and wt- Src transfectants form invadopodia in collagen 3D matrices where fine invadopodia membranes emanate from cortactin/actin/ phosphoproteins-rich core structures [43,45]. Schoumacher et al. found that invadopodia protrusions (stages 1 and 2) in invasive MDA-MB-231 and HCT116 cells were 1–7 m m long and 0.5 to 2 m m in diameter in 3D collagen culture [51]. The relationship of these thin protrusions to invadopodia was determined by the presence of MT1-MMP and cortactin as well as active c-Src [51]. c-Src, MT1-MMP and cortactin are well-documented compo- nents of invadopodia membranes [24,44,47,52,53]. MMP-depen- dent and degradation-associated long membrane extensions have long been observed in other 3D settings [25,47]. We conclude that cell protrusions contain the potential for strong adhesions coupled with dynamic membrane movements that probably possess the matrix-degrading capabilities of invadopodia. The physical presence of Src at the membrane and at sites of adhesion and degradation in 3D collagen cultures together with data derived from 2D studies of cells support the interpretation that Src tyrosine kinase regulates the length and lifetime of protrusions in addition Figure 4. A cartoon depiction of the assay. The bottom layer (blue) is a soft (Young’s modulus , 100 Pa) polyacrylamide (PAA) gel seeded with fluorescent tracer beads (gray). Above that (pink) is an approximately 80 mm thick network of fibrilized collagen I (2 mg/mL). Each imaged data series represents a 3D+time, 3 fluorescence channel movie of a single cell (green), the collagen fibrils, and the motions of the beads. Cell nuclei are typically about 5 mm above the PAA gel.
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Proteomic analysis reveals a novel function of the kinase Sat4p in Saccharomyces cerevisiae mitochondria.

Proteomic analysis reveals a novel function of the kinase Sat4p in Saccharomyces cerevisiae mitochondria.

PCR (data not shown) and overexpression was shown by Western blot analysis (fig. 2A). The Sat4p-signals are significantly stronger in the Tet-Sat4 strain (lane 2) compared to the strain expressing the protein under its native promoter (lane 1), and resemble the pattern described above (fig. 1B). In addition, some faint bands in a lower molecular weight range were detected, probably representing degradation products. To compare the phenotypic consequence of SAT4 overexpression, growth of WT, Dsat4 and Tet-Sat4 strains was compared on different media (fig. 2B). On full media containing glucose (YPD) all strains show a comparable growth. Addition of 1 M sodium chloride (YPD+NaCl) led to a reduced growth of the deletion strain, as was reported previously [13,14]. In contrast, the strain Tet-Sat4 grew better on this medium compared to WT. This observation is in line with a report on a strain overexpressing Sat4p under the control of the GAL1- promoter [14]. Interestingly, growth of the Tet-Sat4 strain on non- fermentable carbon sources like ethanol (YPE) was slightly impaired (fig. 2B, lower panel), indicating a disturbance of the respiratory metabolism or mt function. No apparent growth difference was observed between WT and Dsat4 on this medium. In order to elucidate the impact of deletion or overexpression of SAT4 on the mt proteome, purified mitochondria of the strains Dsat4, Tet-Sat4 and WT were analyzed by 2D-DIGE. Mt proteins were labeled with Cy2 (Dsat4), Cy3 (WT) and Cy5 (Tet-Sat4), respectively, and simultaneously separated in a 3–11 NL isoelectric focussing strip (24 cm) followed by a 12% SDS-PAGE. Fluores- cence images were subsequently acquired and spot intensities were quantified using the Delta2D software. On the merged image 587 spots were detected. Spot intensities did not differ significantly between WT and the deletion strain Dsat4, which is in agreement with their almost identical growth on YPE. In contrast, the intensities of 16 protein spots of the strain Tet-Sat4 differed more than 2.5-fold (fig. 3). These spots were excised from the gel, and proteins were identified by MS as listed in table 1. Peak lists of
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CXCL14 deficiency in mice attenuates obesity and inhibits feeding behavior in a novel environment.

CXCL14 deficiency in mice attenuates obesity and inhibits feeding behavior in a novel environment.

indirectly affected by neuronal abnormalities. In the mouse brain, CXCL14 mRNA is most abundantly expressed in the cortex, hippocampus and cerebellum (http://www.brain-map.org provided by the Allen Institute for Brain Science). Among appetite-related regions, paraventricular hypothalamus, suprachiasmatic nucleus and piriform cortex show a relatively stronger expression of CXCL14 mRNA. Expression levels of CXCL14 in the arcuate nucleus and ventromedial nucleus of the hypothalamus are low. We confirmed the expression of CXCL14 mRNA in the cortex, hippocampus and hypothalamus of adult mice by RT-PCR (YN, SO, YM, TH, unpublished data). As CXCL14 is present not only in the hypothalamus, but also in the cortex and hippocampus, we speculate that CXCL14 may be required for the establishment of neural circuits that are closely linked with feeding behavior. Consistent with this, the feeding behavior of CXCL14 2/2 mice was repressed on the first night after they were transferred to a novel environment. However, the locomotor activity of the mutant mice was not significantly different during this time; thus, it is unlikely that the novelty feeding-suppression phenotype of CXCL14 2/2 mice is a result of fear. This trait may be related to impairment of anti- depressive functions in the brain. It has been previously demonstrated
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Serine proteolytic pathway activation reveals an expanded ensemble of wound response genes in Drosophila.

Serine proteolytic pathway activation reveals an expanded ensemble of wound response genes in Drosophila.

Embryos were collected on apple juice agar plates and aged to 15–17 h at 25uC. Embryos were washed into mesh baskets, dechorionated in bleach for 1 min, then washed copiously with water. Embryos were then transferred to a clean slab of apple juice agar and aligned for 30–60 min at 18uC, transferred to slides with double-sided tape, then covered in a 1:1 ratio of 700:27 weight halocarbon oil. Embryos were then wounded bilaterally with fresh microinjection needles made from an automated puller mounted on a micromanipulator, allowed to recover for 5–6 h at room temperature or overnight at 4 uC, and then visualized under fluorescent light in a compound microscope to determine wound reporter activity. At least 3 independent experiments with at least 30 successfully wounded embryos were performed. Assays involving homozygous deletion or mutant embryos were per- formed in parallel to heterozygous-balancer embryos. A Kr-GFP fluorescent marker on the balancer chromosome, was used to determine the genotype of the embryos [77]. All embryos were impaled using a micromanipulator so that the needle protruded 1 embryo-width from the exit wound. Wound reporter responses were rated on a scale of ‘‘no activity, localized activity, or global activity.’’ Images were obtained by wounding embryos with microinjection needles and imaged on a Leica SP2 confocal microscope, selecting representative embryos to image. Images were resized while constraining proportions to maintain resolu- tion. Adobe Photoshop adjustment functions were used equally on images to enhance clarity, but not to obscure, eliminate, or misrepresent any information. Original images are available on request.
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Genome-wide linkage in a highly consanguineous pedigree reveals two novel loci on chromosome 7 for non-syndromic familial Premature Ovarian Failure.

Genome-wide linkage in a highly consanguineous pedigree reveals two novel loci on chromosome 7 for non-syndromic familial Premature Ovarian Failure.

Among those genes, other possible candidates include SMURF1, the CYP3 gene family and VGF. SMURF1 encodes an E3 ubiquitin ligase specific of regulatory SMAD proteins, that was shown in rat and human granulosa cell lines to ubiquitinylate R-Smad 1 and 5, two of the regulatory Smads activated by oocyte-secreted BMP15, a known POF gene [37]. The CYP3 gene cluster in 7q includes four genes, CYP3A43, CYP3A4, CYP3A7 and CYP3A5, encoding cytochrome P450 enzymes, known to be implicated in drug metabolism and synthesis of cholesterol and steroids. Although some of the CYP3A genes appear to have a restricted expression in the liver and to be mainly responsible for drug detoxification, we cannot exclude a role in steroid synthesis in the ovary [38]. VGF encodes a 68-kDa precursor of multiple bioactive peptides with diverse neuroen- docrine functions, expressed abundantly in the brain, and in peripheral endocrine tissues including the pituitary gland. In addition to a role in the regulation of energy homeostasis, VGF could also regulate reproduction, since homozygous Vgf-null mice are infertile, presenting a delayed sexual maturation, incomplete mammary development and ovaries with only primary and atretic follicles, apparently due to an abnormal pitituary gonadotropin content [39].
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Investigation of Novel Circuits Involved in Virgin Female Receptivity of Drosophila melanogaster

Investigation of Novel Circuits Involved in Virgin Female Receptivity of Drosophila melanogaster

The only known volatile pheromone to date is cis-vaccenyl acetate (cVA). It was initially described as an aggregation pheromone (Bartelt, Schaner, & Jackson, 1985), but is now shown to decrease courtship and promote aggression in males, and increase receptivity in females (Kurtovic, Widmer, & Dickson, 2007; Wang & Anderson, 2010). Its cognate receptor is OR67d (Ha & Smith, 2006). The respective, fruitless-positive, ORNs project to the DA1 glomerulus. This is one of the three sexually dimorphic glomeruli, DA1, VA1 and VL2a that are larger in males (Vosshall & Stocker, 2007). From here fruitless-positive projection neurons innervate the LH in a stereotypic, sex-specific manner and contact local fru+ third-order neurons of five different clusters, two of which have differing connectivity in male and female (Cachero et al., 2010; Datta et al., 2008; Kohl, Ostrovsky, Frechter, & Jefferis, 2013; Ruta et al., 2010).
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RYR2 sequencing reveals novel missense mutations in a Kazakh idiopathic ventricular tachycardia study cohort.

RYR2 sequencing reveals novel missense mutations in a Kazakh idiopathic ventricular tachycardia study cohort.

RYR2 mutations have been attributed to a spectrum of variable and converging clinical phenotypes such as patients with reproducible bidirectional VT and polymorphic VT at exercise stress testing; patients presenting only with polymorphic VT; and patients with idiopathic VT. Even more, the occurrence of CPVT1 and ARVD2 even within the same family raised the suggestion that the two entities might correspond to different degrees of phenotypic expression of the same disease [13]. Considering the various phenotypes and the variable penetrance of the numerous RYR2 mutations, we thought to investigate 35 patients, 33 of them diagnosed with idiopathic VT for mutations in the RYR2 gene in a pilot study. We observed a heterozygous missense mutation at c13892A.T (p.D4631V; Case #239) with high pathogenic potential (high in-silico prediction scores, de- novo, reference databases negative) in a patient with classical clinical characteristics of CPVT. The penetrance of this de-novo variant has to be considered high due to the early age of onset and the distinctive and severe clinical course. Due to ongoing symptoms and risk of SCD index patient #239 is considered to undergo the selective sympathectomy. The ARVD2 variant p.T2504M has been observed in a 20-year old patient with idiopathic ventricular tachycardia. Index patient #444 had a successful RFA and anti-arrhythmic drugs need not be prescribed. However, based on the identification of the ARVD2 associated RYR2 gene mutation, the patient management strategy was modified. MRI of the heart is planned to perform to detect any arrhythmogenic dysplasia.
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Predicting the progress of colon cancer by DNA methylation markers of the p16 gene in feces - Evidence from an animal model

Predicting the progress of colon cancer by DNA methylation markers of the p16 gene in feces - Evidence from an animal model

In a normal intestinal tract, the epithelium is con- stantly and rapidly renewed by the turnover of 5 x 10 10 epi- thelial cells per day (Mehl, 1991). The shedding rate of carcinoma colonocytes is faster than that of normal cells, and we can use this characteristic to identify tumor cells and assay the status of genetic or epigenetic abnormalities. Promoter hypermethylation analysis of stool DNA is a promising noninvasive test for early diagnosis of CRC, and this area has received much research interest. An increasing number of genes is found to undergo promoter region hypermethylation in the tissue and stool of CRC patients, and Glockner et al. (2009) found a higher sensitivity to TFPI2 methylation in patients with CRC (73-89%), in pa- tients with adenomas (21-43%), and a high specificity to- ward TFPI2 methylation by patients with either CRC or adenomas (93-100%). Other reports observed high sensi- tivity (> 68%) and specificity (> 84%) towards SFRP2 and GATA4 methylation (Oberwalder et al., 2008; Wang and Tang, 2008; Hellebrekers et al., 2009)
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Goodness of fit to a mathematical model for Drosophila sleep behavior is reduced in hyposomnolent mutants

Goodness of fit to a mathematical model for Drosophila sleep behavior is reduced in hyposomnolent mutants

Insomniac demonstrates a robust phenotype in terms of total time slept. Insomniac animals tested in this experiment sleep significantly less than controls in the 24-hour period. Mean total sleep in insomniac is 770.1, with SD = 210.0, compared to 1069.0 with SD = 81.7 in wild type (Fig. 1A). According to a two-tailed, two-sample heteroscedastic (allowing for unequal variance) Student’s T -test, probability that measures of total sleep per 24 h in insomniac and controls came from the same distribution is given by p < 0.0001. Separate consideration of daytime and nighttime sleep reveals that nighttime total sleep in insomniac, at 394.7 (SD = 148.7) is significantly less than nighttime sleep in wildtype, at 672.6 (SD = 29.9) (Fig. 1A). Daytime total sleep in insomniac is unchanged as compared to control (Fig. 1A).
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The Drosophila melanogaster methuselah gene: a novel gene with ancient functions.

The Drosophila melanogaster methuselah gene: a novel gene with ancient functions.

In D. melanogaster, variability at the mth gene has been reported to influence lifespan (see introduction). In order to gather further evidence that gene GJ12490 could be playing the same role as the mth gene, we looked for an association between the N/S polymorphism (see above) and lifespan. A significant association between genotype and lifespan was found (Non-parametric Kruskal-Wallis Test; P,0.005). The average and standard deviation for genotypic classes N/N, S/S and N/S is 49.4620.0 days (N = 54), 59.2619.8 days; (N = 83), and 60.6621.1 days (N = 155). There are no significant differences regarding lifespan when genotypes S/S and N/S are compared (Non-parametric Mann-Whitney Test; P .0.05) but significant changes are observed when comparing genotypes N/N and N/S (Non- parametric Mann-Whitney Test; P,0.001), and when comparing genotypes N/N and S/S (Non-parametric Mann-Whitney Test; P ,0.01). These results are significant after applying the sequential Bonferroni correction for multiple testing (P,0.05). Therefore, N/ N genotype seems to be associated with short lifespan. Individuals with genotypes S/S and N/S show a 21.7% average increase in lifespan in comparison with individuals with genotype N/N. Under the assumption that the N allele is recessive over the S allele, the N/S polymorphism explains 3.8% (Pearson correlation coefficient = 0.200; P ,0.005; the 95% lower and upper confi- Figure 7. Amino acid variation at the D. americana mth -like GJ12490 protein. The amino acid replacement that is segregating in the F2 association experiment is highlighted in grey. C1– the first typical cysteine; N1– putative N-glycosylation site. Amino acid positions are given relative to the first typical cysteine.
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Confocal Analysis of Nuclear Lamina Behavior during Male Meiosis and Spermatogenesis in Drosophila melanogaster.

Confocal Analysis of Nuclear Lamina Behavior during Male Meiosis and Spermatogenesis in Drosophila melanogaster.

This suggested connection between chromatin organization and NL is strongly supported by our observations of the post-meiotic stages of spermatogenesis. Indeed, at the end of meiotic divisions, spermatids enter into a differentiation program characterized by a series of chroma- tin condensation and decondensation events [20], which are accompanied by modifications of NL pattern. At the early stage of spermatid differentiation, where mitochondria aggregated and chromatin started to condense, the NL lost the compact appearance of the previous stage and exhibited a punctuated pattern. At onion stage, the highly condensed nucleus was encircled by a well-structured, thick, lamina signal that presented small gaps. Finally, a diffused intranuclear distribution of the lamina accompanied the chromatin decondensation that, together with Nebenkern elongation, characterizes the late stages of spermatid differentiation. This apparent ability of the NL to reorganize in parallel with chromatin reorganization emphasizes again the strong dynamics of this structure. The observed pattern is in accordance with functions of nuclear lamins in determining and maintaining the nuclear shape (for a review see [1]). In fact, the chromatin condensation/decondensation events imply nuclear conformational changes that may likely require NL reorganization. In mammals, the differences in the composition of lamins of spermatogenetic cells with respect to its somatic counterpart may reflect the nuclear organization changes during spermatogenesis. Mammalian germ line-specific lamins B3 and C2 are shorter and in minor amounts, with respect to their somatic counterparts, thus allowing the formation of a more flexible NL structure, suited to address the nuclear changes character- izing gametogenesis (For a review see [36]).
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ENU mutagenesis reveals a novel phenotype of reduced limb strength in mice lacking fibrillin 2.

ENU mutagenesis reveals a novel phenotype of reduced limb strength in mice lacking fibrillin 2.

Physiological tests on the muscle indicate that the myofibre structure is not altered in Fbn2-null mice (as their was no difference in peak isometric stress). The muscle itself generated less force, probably due its relatively smaller size in Fbn2-null mice. However, the loss of muscular strength in Fbn2-null mice is more extreme than the reduction in the maximum force produced by the muscle itself, suggesting an extra-muscular involvement. Our finding that the muscle itself shows signs of degeneration/ regeneration suggests that some of the myofibres of Fbn2-null mice are in a regenerative phase. However, there was no gross inflammation and the level of regeneration can probably not account for the drastic reduction in the strength of these mice. A reduced level of collagen cross-linking has been identified in the tendons of Fbn2-null mice [17]. As this provides structural integrity to the tendons, the mechanical properties of the tendon, such as Figure 6. Skeletal muscle histology in Mariusz mice. (A) Morphometric analysis of muscle fibre cross-sectional area in the soleus and quadriceps muscles in Mariusz (n = 3) and wild type controls (n = 3). Two-way ANOVA was not significant. (B) Typical H&E sections of Mariusz quadriceps muscle from mice at 4 and 9 weeks of age. Scale bar = 100 um. (C) Quantification of central nucleation. The number of muscle fibres with central nuclei is represented as percentage of the total number of muscle fibres analyzed in .100 fibres. Arrows indicate centrally displaced nuclei. Mice were aged 4 weeks and 9 weeks (n = 3 Mz/Mz; n = 3+/+). P-values (Student’s t test): * P,0.05, ** P,0.01.
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Targeted deletion of fibrinogen like protein 1 reveals a novel role in energy substrate utilization.

Targeted deletion of fibrinogen like protein 1 reveals a novel role in energy substrate utilization.

Fgl1 Null Mice are Heavier than Wild Type Controls To initiate studies on the physiological functions of Fgl1, we generated a mouse null for Fgl1. The Fgl1 gene comprises 8 exons spanning more than 24 kb of genomic DNA. The initiator ATG resides in the second exon. To disrupt Fgl1, we deleted exons 2 and 3 which removed the initiator ATG and abolished the secretory signal recognition peptide (Figure S1A in File S1). Genotyping confirmed the insertion of the neomycin cassette in the Fgl1 2/2 mouse (Figure S1B in File S1). To ascertain whether Fgl1 was absent in the knockout mouse, we performed qPCR assays and found that Fgl1 mRNA and protein were absent in the livers of Fgl1 2/2 mice at baseline and after PH (Figure 2A and B). We also observed that Fgl1 mRNA was absent from both brown and white adipose tissues of the Fgl1 2/2 mice (Figure S2 in File S1). Despite the loss of Fgl1 expression, Fgl1 2/2 mice appeared grossly normal when compared to wild type mates except that they were persistently heavier. This difference in size was obvious at 3 weeks, the earliest we could genotype our mice, at which point the Fgl1 null mouse were 37% heavier than wild type mates (Figure 2C, P ,0.0001). We monitored a group of mice beginning at 7 weeks and find that the size difference remains as the mice age (Figure 2D). Indeed in a small cohort of mice we kept for 11 months this difference persisted (not shown). Considering Fgl1’s presumptive role in liver proliferation, we carefully examined liver tissue, but observed no differences between the livers of Fgl1 +/+ and Fgl1 2/2 mice by: i) liver weight (1.15+/ 20.28 grams versus 1.03+/20.05 grams respectively, Figure 2E), ii) histologic analysis by hematoxylin and eosin staining (not shown), iii) immunohisto- chemistry for PCNA and BRDU incorporation (not shown). However we found that the liver weight to body weight ratio, a quotient used to determine appropriate liver size for body weight [24] is smaller in Fgl1 2/2 when compared to wild type mates (Figure 2F, P = 0.0081). These findings confirm that the Fgl1 mice were not heavier as a result of increased liver size. At 48 h after PH, Fgl1 2/2 livers appeared grossly steatotic (Figure 2G, top panels). Histologic analysis demonstrated the presence of marked macrovesicular steatosis following PH at 48 h (Figure 2E, compare middle panel images) with resolution by 96 h (Figure 2G, compare right middle panel to right lower panel). In addition, lipid analysis showed that the triglyceride (TG) content of liver extract was markedly elevated at 48 h post PH (Figure 2H, P = 0.014). This suggests that Fgl1 has lipid regulatory roles that became manifest during recovery from liver injury. We next determined the expression levels of hepatic lipid regulatory genes; lipolysis: hepatic lipase (HL), oxidation: peroxisome proliferator-activated receptor alpha (PPARa, PPARc1, PPARc2, PPARd and carnitine palmitoyltransferase (CPT1)), fatty acid transport genes: cluster
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