WFS1) mutation in a family with autosomal dominant optic neuropathy and deafness. The study is designed as a retrospective observational case series.To describe the phenotype of a novel Wolframin  were excluded. In the affected individuals, a novel missense mutation c.2508G>C (p.Lys836Asn) in exon 8 of WFS1 was identified.All affected individuals showed loss of neuroretinal rim of the optic nerve at fundoscopy with enlarged blind spots at perimetry. They showed a red-green color vision defect at color vision tests and deviations at visually evoked response tests. The audiograms of the affected individuals showed hearing loss and were relatively flat. The unaffected individuals showed no visual deviations or hearing impairment. The affected family members had no glucose intolerance. Leber hereditary optic neuropathy (LHON) mitochondrial mutations and mutations in the Optic atrophy-1 gene (WFS1.This study describes the phenotype of a family with autosomal dominant optic neuropathy and hearing impairment associated with a novel missense mutation in  In later stages, visual loss becomes severe, usually worse than 20/200 . HON is  [(WFS1) gene. WFS1, on chromosome 4p16.3, contains eight exons. Mutations in this gene are reported to be responsible for Wolfram Syndrome, Deafness Autosomal dominant type 6/14 , psychiatric disorders, and diabetes mellitus (OMIM 606201). After examination of the family with autosomal dominant optic neuropathy and deafness, Eiberg et al. [WFS1.The combination of autosomal dominant optic neuropathy and deafness has been reported in families and in isolated cases with a heterozygous missense mutation in Optic atrophy-1 gene  605290) –6. Eiber 605290)  describeg et al.  concludeg et al. . There wThe proband, a 57-year-old man, was referred to our tertiary referral hospital for progressive hearing loss, which coexisted with optic neuropathy. The question was raised whether he was a good candidate for cochlear implantation. Medical history showed that both his mother and brother also had optic neuropathy and hearing loss. All family members were examined in our outpatient clinic and medical history was taken. For all family members except patient II:2, the ophthalmological examinations included best corrected visual acuity measurements, slit-lamp microscopy and ophthalmoscopy. Goldmann perimetry was performed to evaluate visual field size. A morphometric analysis of the optic disc was performed using the Heidelberg Retina Tomograph II  . Color v1C were analyzed in the proband and plasma glucose was analyzed in all affected individuals).The affected family members underwent fasting serum testing to exclude diabetes mellitus . The coding exons and the flanking intronic sequences were PCR amplified and subsequently sequenced on a 3730 automated sequencer using Dye terminator chemistry . For primer information and PCR conditions see OPA1 and three known LHON mutations  were screened by a combination of dHPLC  and direct sequencing analysis.Blood samples of all living individuals were collected in EDTA tubes and kept at room temperature. DNA was isolated within five days after withdrawal on a Chemagen MSM1 platform using the Chemagic DNA blood 10k kit . Mutation analysis of From the pedigree in The results of the ophthalmological examinations are shown in The results of otological and audiological examinations are shown in The proband had normal vestibular function. CT scan of the temporal bone showed no anatomic abnormalities. Central causes of sensorineural hearing loss were excluded by ASSR testing, which revealed normal function of the auditory nerve. The proband underwent successful right ear cochlear implantation with a 22-electrode implant . Seven months after implantation, he was found to have 83% speech recognition at 70 dB sound pressure level (SPL). Prior to implantation, aided thresholds with a conventional aid on the right were tested: no speech recognition was found at 70 dB SPL and the maximum speech recognition of about 20% was found at 80 dB SPL.WFS1 and OPA1 revealed no mutations in all coding regions of OPA1. In exon 8 of WFS1, a heterozygous mutation was identified in all three affected patients. This variant was not detected in the unaffected family members. At position 2508, a substitution of a cytosine for a guanine (c.2508G>C) leads to the amino acid substitution p.Lys836Asn. Based on the medical history and clinical information available, we assume that this is a de novo mutation. Screening of mtDNA positions m.11778, m.3460 and m.14484 demonstrated that the three most frequent LHON mutations were not present. Screening of 1C . In the proband, insulin  and C-peptide  were assessed as well, and found to be in the normal range.Diabetes mellitus was excluded in the affected individuals by assessing fasting serum glucose  and HbAWFS1 that is associated with autosomal dominant optic neuropathy and deafness. The disease shows 100% segregation with the mutation. The three affected individuals did not have diabetes mellitus or any other symptoms. Since there was no autosomal recessive inheritance, they did not present with Wolfram syndrome spectrum. They also did not have DFNA6/14, the nonsyndromic autosomal dominant low frequency sensorineural hearing loss that is caused by WFS1 mutations. The affected participants did show more severe sensorineural hearing loss with involvement of all frequencies, and they did have optic atrophy.In this article we report a novel missense mutation (p.Lys836Asn) in exon 8 of OPA1, OPA3, OPA4, and OPA5. OPA1 (OMIM 165500) is the most frequently encountered type of isolated autosomal dominant optic neuropathy. Symptoms usually manifest in the first decade of life, and, in most cases, visual impairment develops gradually over many years. The reduction in visual acuity tends to be mild or moderate [HON is a clinically and genetically heterogeneous condition. Isolated autosomal dominant optic neuropathy is caused by four genes: moderate ,11. Visumoderate .Optic neuropathy is also a sequela of LHON. LHON is the result of maternal mitochondrial DNA mutations. Visual loss usually begins painlessly and centrally in one eye, and the second eye is affected weeks to months later with an acute or subacute course. However, the duration of progression of visual loss in each eye varies and may be difficult to document accurately. Eventually, optic atrophy bundle supervenes .WFS1 on chromosome 4p16.3, Wolfram syndrome is an autosomal recessive neurodegenerative syndrome [Optic neuropathy is also one of the main symptoms in Wolfram syndrome. Caused by mutations in syndrome . The minThe affected individuals of the described family have severe optic nerve damage as well as sensorineural hearing loss that is inherited in an autosomal dominant way. In 1977, Deutman presented a family with autosomal dominant optic neuropathy and deafness similar to our family: the affected individuals had sensorineural hearing impairment and optic atrophy but no other symptoms. All affected individuals had hearing loss with a relatively flat audiogram, an enlarged blind spot at Goldmann perimetry, deutanomaly, and a deviating VER . There wOPA1 [OPA1. However, OPA1 mutations were excluded by sequencing analysis. In addition, the OPA1 mutations seem to cause a broader phenotype with ptosis, ophthalmoplegia, ataxia, axonal sensory-motor polyneuropathy, and mitochondrial myopathy [OPA1 mutation (p.Tyr582Cys) is responsible for progressive hearing loss that necessitated cochlear implantation, macrocytic anemia, and hypogonadism [Autosomal dominant optic neuropathy and deafness has also been reported in several families and in isolated cases with a heterozygous missense mutation  in OPA1 . In our myopathy ,15. Anotgonadism . InteresThe combination of hearing loss and LHON has not been ascribed to a single mutation so far . HoweverWFS1 encodes for wolframin, a protein known to contain nine predicted transmembrane domains. So far, about 110 mutations in WFS1 are believed to cause the Wolfram syndrome [WFS1 missense mutation (p.Gln864Lys) in the same conserved region as the mutation that we found. This mutation caused an autosomal dominant clinical triad: congenital hearing impairment, diabetes mellitus, and optic atrophy. In the study by Valéro, however, the proband (thus far) had no optic atrophy [WFS1 are also responsible for other conditions such as psychiatric disorders and diabetes mellitus (Lesperance laboratory database). The pleiotropy of this disorder can possibly be explained by alternative splicing: missense mutations would occur in regions that are spliced out in specific organs. Challenging WFS1 expression studies of these organs would be needed to prove or reject this hypothesis.syndrome –23. Eibesyndrome  and Valésyndrome  reportedion p.Gln64Lys in WFS1 mutations have been reported to cause DFNA6/14 [To date, 26 DFNA6/14 –24, an uDFNA6/14 –27. In gDFNA6/14 ,29. BecaDue to a lack of previous audiograms, it was difficult to evaluate progression of hearing loss in II:2 and III:2. It should however be noted that the first available audiograms of II:2 were made at the age of 60 years and that the first available audiograms of III:2 were made at the age of 60 years. Progression of their hearing loss may have occurred before these ages. Interestingly, the first pure-tone audiogram of III:3, done at the age of 38 years, shows a more pronounced loss at the lower frequencies, resembling the hearing loss characteristic of DFNA6/14  that appears to be bigger than the mean deterioration (approximately 1 dB/year) that was reported for this frequency based on analyses in several families with DFNA6/14 . The heaWFS1 remain unclear. Expression studies, however, have assessed the presence of wolframin in retinal ganglion cells and optic nerve glia cells of the cynomolgus monkey [The distribution of wolframin in the mammalian visual system, and the pathogenesis of optic atrophy due to mutations in s monkey . In rodes monkey . Kawano s monkey .WFS1 mutation in the cochlea without any neurodegenerative symptoms that are so common in Wolfram syndrome. It is still not clear why DFNA6/14 patients show a stable low frequency hearing impairment with minor progression and why the affected family members in our study have such severe progression and profound hearing impairment. Interestingly, in our study family with visual impairment by optic neuropathy, there were no signs that the hearing impairment is caused by auditory neuropathy. According to the literature [WFS1, causing DFNA6/14, also have sensorineural hearing loss and no auditory neuropathy. Apparently, this specific missense mutation in WFS1 has a different effect in the inner ear than in the eye. Further studies are needed to elucidate the underlying pathogenetic mechanism [The function of wolframin in the inner ear and the mechanisms by which missense mutations cause hearing loss have not been extensively explored . The expterature , most paechanism .WFS1 that is associated with autosomal dominant optic neuropathy and finally a severe to profound hearing loss with relatively flat audiograms. The present phenotype is similar to the phenotype caused by the only other reported WFS1 missense mutation (p.Gln864Lys) that causes autosomal dominant optic neuropathy and deafness. However, some of those patients also have impaired glucose intolerance [OPA1 mutations causing autosomal dominant optic neuropathy and deafness, however, in general these mutations cause a more extensive phenotype that includes ptosis, ophthalmoplegia, ataxia, axonal sensory-motor polyneuropathy, mitochondrial myopathy, macrocytic anemia, and hypogonadism [WFS1 and OPA1 in cases of autosomal dominant optic neuropathy and deafness.The results of our study suggest that p.Lys836Asn is a novel mutation in olerance , and onegonadism –16. The


DNA damage triggers a network of signaling events that leads to cell cycle arrest or apoptosis. This DNA damage response acts as a mechanism to prevent cancer development. It has been reported that fatty acids (FAs) synthesis is increased in many human tumors while inhibition of fatty acid synthase (FASN) could suppress tumor growth. Here we report that saturated fatty acids (SFAs) play a negative role in DNA damage response. Palmitic acid, as well as stearic acid and myristic acid, compromised the induction of p21 and Bax expression in response to double stranded breaks and ssDNA, while inhibition or knockdown of FASN enhanced these cellular events. SFAs appeared to regulate p21 and Bax expression via Atr-p53 dependent and independent pathways. These effects were only observed in primary mouse embryonic fibroblasts and osteoblasts, but not in immortalized murine NIH3T3, or transformed HCT116 and MCF-7 cell lines. Accordingly, SFAs showed some positive effects on proliferation of MEFs in response to DNA damage. These results suggest that SFAs, by negatively regulating the DNA damage response pathway, might promote cell transformation, and that increased synthesis of SFAs in precancer/cancer cells might contribute to tumor progression and drug resistance. FAs play important roles in energy storage, membrane structure, protein acylation, signal transduction, and regulation of gene transcription Numerous studies have implicated that fatty acids, fat diet, and obesity play a role in cancer development De novo synthesized fatty acids account for more than 90% of the triacylglycerol in tumor cells However, cancer cells, especially of the breast, prostate, colon, ovary, endometrium, and thyroid origin, express very high levels of FASN and this up-regulation is under the control of aberrant MAPK and PI-3K-Akt signaling FASN is now becoming a drug target for cancer therapy. It has been found that cerulenin, a natural fungal inhibitor of FASN, specifically targets and suppresses tumor cell growth, with little effect on the surrounding normal tissues DNA damage is generated by exogenous agents such as ionizing radiation (IR), ultraviolet (UV) light exposure, genotoxic compounds including chemotherapeutic drugs such as adriamycin, and endogenous factors such as reactive oxygen species which are generated by mitochondria in the process of β-oxidation. Depending on the types and the severity of DNA lesions, cells respond to DNA damage by undergoing cell cycle arrest or apoptosis when the damage is beyond repair DNA damage activates multiple signaling cascades. At the center of the signaling network are phosphoinositide-3-kinase-like kinases (PIKKs) that include DNA-PKcs, Atm, and Atr, all of which are exclusive serine/threonine kinases Many of the proteins involved in DNA damage response are found to promote cancer development when mutated de novo fatty acid synthesis, and a substrate for lipid synthesis and protein palmitoylation. Primary mouse embryonic fibroblasts (MEFs) were used since they show good response to DNA damage induced by adriamycin, a chemotherapeutic drug that has been proved to cause both DSBs and ssDNA. Treatment of MEFs with different doses of palmitic acid  revealed that palmitic acid was toxic to the cells at concentrations over 200 µM (data not shown). Therefore, 100 µM of palmitic acid was used throughout this study. We also found that palmitic acid itself slightly down-regulated the basal levels of p21 and Bax in MEFs  and myristic acid (C14), both of which are present in the serum with stearic acid accounting for close to 13 % of the total FAs Another genotoxic stress reagent hydroxyurea (HU), which generates ssDNA and activates Atr, was used to confirm this finding. As expected, HU could induce p21 expression and p53 accumulation in primary MEFs . PretreaIn support of this notion, we also found that adriamycin-induced Atm activation was not significantly altered by pretreatment with palmitic acid in MEFs . Since DTo further substantiate the conclusion that SFAs have an influence on DNA damage response, we inhibited FA synthesis by adding FASN inhibitors and then checked DNA damage response. MEFs were pretreated with 1 µg/ml C75 or cerulenin, which have been shown to inhibit fatty acid synthesis in human or mouse fibroblasts In order to exclude a cell type specific effect, we also tested primary osteoblasts that were freshly isolated from the calvaria of new born pups or fetuses. These cells are of bone marrow mesenchymal stem cell origin. It was found that these cells responded to adriamycin treatment to a similar extent as primary MEFs, justified by increased expression of p21 and accumulation of p53. Similarly, pretreatment of these cells with palmitic acid compromised p21 and Bax induction and p53 phosphorylation , suggestWe also expanded our study to include immortalized and tumor cell lines. NIH3T3 cells appear to be a good match for primary MEFs as they share the same origin. Upon DNA damage, NIH3T3 showed response to DNA damage through induction of p21 and accumulation of p53, even though phosphorylation of p53 was modest . Unlike de novo synthesized FAs.We further examined this on human colon cancer cell line HCT116 that expresses wild type p53. This cell line has been shown to highly express fatty acid synthase and likely to contain high levels of FAs DNA damage is known to induce cell cycle arrest and/or apoptosis. We found that palmitic acid had little effect on the distribution of G1, S, and G2 phases, in the presence or absence of adriamycin, suggesting that its effect on cell cycle checkpoints is minimal (data not shown). On the other hand, several studies have reported that SFAs, especially palmitic acid, could regulate apoptosis. In some cases such as pancreatic cells, palmitic acid induces cell death through mtDNA damage and genomic DNA damage Our studies indicate that SFAs could compromise DNA damage-induced cell response in primary cells but not in immortalized cells. Pretreatment with palmitic acid, myristic acid, or stearic acid leads to reduced accumulation of p53, reduced induction of p21 and Bax, diminished activation of Chk1, and improved cell proliferation. Moreover, inhibition of FAs synthesis, with pharmacological inhibitors or knock-down of FASN, enhanced the induction of p21 and Bax and accumulation of p53. Based upon the fact that the levels of FASN expression and FA synthesis are dramatically increased in the pre-cancer stages, and that DNA damage response occur during the transition from precancerous to cancer, we propose that the increased levels of FAs might modulate cell response to DNA damage, leading to defects in cell cycle checkpoints and apoptosis. Compromised DNA damage will likely result in accumulation of DNA lesions and eventually facilitate tumorigenesis. This concept is also supported by the observation that overexpression of FASN is correlated with high degree of microsatellite instability in colorectal cancer independent of CpG island methylator phenotypes DNA damage response is controlled by signaling pathways started from the DNA damage-induced foci, which are believed to be DNA repair centers. Many proteins were accumulated at the foci including the signaling molecules Atm and Atr, which phosphorylate p53, Mdm2, Chk1/2 and other molecules to regulate cell cycle control and apoptosis. Since SFAs interfered with p53 phosphorylation, p53 accumulation, induction of p53 target genes such as p21 and Bax, and phosphorylation of Chk1, we conclude that SFAs act at a rather early step of the signaling cascade. Since foci formation is not affected, SFA is likely to act at a step affecting Atm/Atr activation, or adaptor proteins that recruit effectors such as p53 and Chk1/2 to the foci, or both. We found that SFAs had a more profound effect on Atr phosphorylation and activation of Chk1, implying that SFAs might mainly regulate the activation of Atr and/or the phosphorylation of Atr targets. But how do SFAs execute their function on Atr? In view of this, there are a few possibilities. Firstly, SFAs might alter the synthesis of phospholipids, which are known to regulate signaling events. A recent study showed that a shift of phosphatidylcholine chains from polysaturated fatty acids to polyunsaturated fatty acids activates p53 Ser15 phosphorylation through Atr in vivo condition, where the nutrients SFAs modulate cell response to modest DNA damage generated by endogenous factors such as ROS. These results also suggest that SFAs may play a minimal role in DNA damage caused by high dose of genotoxic agents.One intriguing finding is that while primary cells' response to DNA damage is repressed by SFAs, immortalized/transformed cells show resistance to this effect. One possible explanation is that immortalized/transformed cells have acquired some changes in the gene expression patterns, in order to adapt to the long term presence of elevated levels of FASN and fatty acids. So they will not respond to further increases in SFAs. The second possibility is that the immortalized/transformed cells might have acquired mutations in the proteins that link fatty acids to the DNA damage response and repair pathways, e.g., proteins involved in DNA damage response that can bind or be modified by SFAs. The observation that both primary and transformed cells are responsive to FASN inhibition in genotoxic stress favors the first hypothesis as genetic changes are usually irreversible. Another interesting finding is that SFAs only modulate cell survival and proliferation in response to low dose of adriamycin, which might reflect the Inhibitors of FASN have been found to induce apoptosis in cancer cells that have high levels of FASN. These include breast, prostate, colon, and lung cancers. Based upon our findings that an increase in SFA levels compromises DNA damage response while inhibition of FAs synthesis enhances these cellular events, lowering the cellular level of FAs might reduce the risks of cancer development. In addition, lowering the cellular level of FAs might also improve the efficacy of radiotherapy and chemotherapy with genotoxic drugs. Some chemotherapeutic drugs such as cyclophosphamide, busulfan, cisplatin, and mitomycin cause interstrand and/or intrastrand crosslinks, while some, e.g., irinotecan and dactinomycin, affect DNA unwinding and therefore DNA replication 2 at 37°C.Mouse embryonic fibroblasts were prepared as previously described DNA damage was generated with genotoxic stress. 0.1–1 µM adriamycin, 10 Gy of gamma-radiation, or 5 mM hydroxyurea was used to treat different cells for different periods of time. To test the effect of SFAs or FASN inhibitors on DNA damage response, different concentrations of palmitic acid, myristic acid, stearic acid , or Cerulenin or C75  were added to the cultures before the addition of DNA damage reagents. Briefly, for SFAs treatment, 100 µM of SFAs were included in the culture medium for overnight. The cells were then treated with adriamycin or hydroxyurea for different periods of time before being harvested for further experiments. To inhibit FASN, C75 or Cerulenin were included in the culture medium for 24 hrs. The cells were further treated with adriamycin or hydroxyurea. To knock down FASN, SiGenome ON-TARGET plus SMART pool duplex of FASN were purchased from DHARMACON (Cat# L-040091).Cells were washed with cold PBS and lysed in RIPA buffer containing 1 mM sodium orthovanadate, 1 mM NaF, and protease inhibitors. Protein concentration was determined by the Bio-Rad assay. For immunoblotting, proteins were resolved by SDS-PAGE and were transferred to PVDF membranes (Millipore), followed by incubation with primary and secondary antibodies and detected by ECL kit (Amersham Biosciences) 4/well, and then treated with fatty acids or FASN inhibitors for 24 hrs, followed by adriamycin for different periods of time. Cell proliferation reagent WST-1 (Roche) was added to each well and the cells were further incubated for 3 hour at 37°C. The absorbance was measured against a background control by microplate (ELISA) reader at 430 nm. The reference wavelength is 650 nm.To measure cell death rates, cells were plated in 96 well plates at 1×10Cells were grown overnight on glass cover-slips, subjected to adriamycin or hydroxyurea treatment, and were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 20 min, blocked with 5% BSA for 30 min, and then incubated with anti-H2AX antibodies and FITC-conjugated secondary antibodies. The number of foci was counted under confocal microscope.t-test (STATISTICA).Statistical analysis was performed using Student's unpaired Figure S1(1.96 MB DOC)Click here for additional data file.Figure S2(1.23 MB TIF)Click here for additional data file.Figure S3(0.67 MB TIF)Click here for additional data file.Figure S4(1.56 MB TIF)Click here for additional data file.Figure S5(1.44 MB TIF)Click here for additional data file.Figure S6(0.99 MB TIF)Click here for additional data file.Figure S7(1.00 MB TIF)Click here for additional data file.Figure S8(0.86 MB TIF)Click here for additional data file.Figure S9(0.45 MB TIF)Click here for additional data file.


Currently, the assessment of sperm function in a raw or processed semen sample is not able to reliably predict sperm ability to withstand freezing and thawing procedures and in vivo fertility and/or assisted reproductive biotechnologies (ART) outcome. The aim of the present study was to investigate which parameters among a battery of analyses could predict subsequent spermatozoa in vitro fertilization ability and hence blastocyst output in a goat model. Ejaculates were obtained by artificial vagina from 3 adult goats (Capra hircus) aged 2 years . In order to assess the predictive value of viability, computer assisted sperm analyzer (CASA) motility parameters and ATP intracellular concentration before and after thawing and of DNA integrity after thawing on subsequent embryo output after an in vitro fertility test, a logistic regression analysis was used. Individual differences in semen parameters were evident for semen viability after thawing and DNA integrity. Results of IVF test showed that spermatozoa collected from A and B lead to higher cleavage rates (0 < 0.01) and blastocysts output (p < 0.05) compared with C. Logistic regression analysis model explained a deviance of 72% (p < 0.0001), directly related with the mean percentage of rapid spermatozoa in fresh semen (p < 0.01), semen viability after thawing (p < 0.01), and with two of the three comet parameters considered, i.e tail DNA percentage and comet length (p < 0.0001). DNA integrity alone had a high predictive value on IVF outcome with frozen/thawed semen (deviance explained: 57%). The model proposed here represents one of the many possible ways to explain differences found in embryo output following IVF with different semen donors and may represent a useful tool to select the most suitable donors for semen cryopreservation. After cryopreservation spermatozoa are particularly susceptible to DNA damage since freezing and thawing procedures lead to significant reduction in the level of spermatozoa antioxidant . Therefoin vitro fertilization ability and hence blastocyst output. To carry out our experiments we used Sarda goats as a model, since semen freezing and thawing procedures are well established for this species  and used to quantify DNA damage . The com2 in air at 39°C.Ovaries were recovered at the local slaughterhouse and placed in Dulbecco's PBS at a temperature between 25°C and 35°C. After washing in fresh medium, ovaries were sliced using a micro-blade and the follicle content was released in medium TCM199  supplemented with 25 mmol Hepes, penicillin and streptomycin and 0.1% (w/v) of polyvinyl alcohol (PVA). The cumulus-oocyte complexes (COCs) comprised of 4-10 layers of granulosa cells and oocytes with a uniform cytoplasm, homogenous distribution of lipid droplets in the cytoplasm and with an outer diameter of about 90 μm (mean) were selected for the experimental procedure. The selected COCs, after three washes in the same fresh medium, were in vitro matured in TCM199 supplemented with 10% estrous goat serum, 10 μL/mL of FSH/LH and 100 μm of cysteamine. COCs were put in groups of 30-35, in 600 μL of the maturation medium in a four-well Petri dish , layered with 300 μL mineral oil and cultured for 24 hr in 5% CO2, 5% O2 and 90% N2 atmosphere in four-well Petri dishes (Nunclon). Synthetic oviduct fluid (SOF) containing 3% bovine serum albumin (BSA-fraction V) supplemented with 25 mm Hepes (sperm-SOF) was used for sperm preparation. For IVF, SOF medium was supplemented with 10% estrous goat serum, 20 μg/mL heparin and 1 μg/mL hipotaurine (IVF-SOF). Percoll® gradients were prepared as described by Rosenkrans et al. . For. For56]]Following the introduction of CASA, a wide variety of parameters have been found to be associated with fertilization rates in vitro. Those selected as the best predictors include: linearity in semen and percentage spermatozoa in insemination medium with velocities between 10 and 20 μm/sec , % progrIn the present study we did not found any predictive value of spermatozoa energetic status, as evaluated by ATP intracellular concentration before and after thawing, on IVF outcome. In other studies, sperm oxygen consumption has been correlated with bull fertility and measurement of total ATP formation has been proposed as a test for bull fertilizing ability after freezing and thawing . We repoIn conclusion, the model proposed here represent one of the many possible ways to explain differences found in embryo output following IVF with different goat semen donors. In our system, DNA integrity, as evaluated by a high sensible method such as the comet assay, has a high predictive value on IVF outcome with frozen/thawed semen (deviance explained: 57%). Besides, adding to the model other two factors, such as of rapid spermatozoa in fresh semen and viability after thawing a 72% of deviance in embryo out put could be explained. This model may represent a useful tool to select the most suitable donors for semen cryopreservation.To the authors' best knowledge, no competing interests of any nature arise from the current publication.FB participated in the design of the study, carried out experimental analysis, performed the statistical analysis and drafted the manuscript. MM performed the semen collection and analyses, and helped drafting the manuscript. VP performed the comet assays and the ATP analyses. SS, AS and VS helped in sample collection and in drafting of the manuscript and participated in the design of the study. PM helped performing the comet assays and the ATP analyses. GGL participated in the design of the study and performed the statistical analysis. SN conceived the study, participated in its design and coordination and helped to draft the manuscript. All co-authors provided inputs during final manuscript preparation. All authors read and approved the final manuscript.


Background: The pathophysiology of functional abdominal pain (FAP) is unknown. The upright portion of a tilt table test triggers typical symptoms in certain children. Aim: To compare the pathophysiology and treatment response of children with FAP whose gastrointestinal symptoms (GI) were replicated (RGI) by tilt table testing (TTT) to those in whom TTT did not have this effect (NRGI).Methods: An IRB-approved retrospective review of the autonomic laboratory database identified all children tested for GI complaints. We compared results of TTT, Valsalva maneuver, deep breathing and the axon reflex sweat test. Overall treatment response and that specific to fludrocortisone was ranked from 1 to 5, with 1 “much worse,” 3 “neutral,” and 5 “much better.” Results: 32/76 identified children had reproducible symptoms on TTT (RGI) and 44 did not (NRGI). The RGI group was younger, had a shorter duration of symptoms, more postural tachycardia syndrome (POTS) and benefited more from fludrocortisone (73% in RGI vs. 25% in NRGI).Conclusion: Dividing patients with FAP according to the effect of TTT on their symptoms appears to delineate 2 fundamentally different groups, with potentially different pathophysiologies and treatment responses. A prospective study is needed. Functionalgastrointestinal disorders (FGID) affect a large percent of children andeffects quality of life. The exact prevalence of FGID in children is unknown. Onestudy found 10–20% of children suffer from chronic abdominal pain [According to theproposed biopsychosocial conceptualization of functional gastrointestinaldisorders, the autonomic nervous system links the psychosocial factors with thepathophysiological elements . A role  POTS is defined as an increase in heart rate (HR) from supineto standing position of greater than 30 bpm, without sustained drop in bloodpressure. This definition has a stronger clinical implication if the increasein heart rate is associated with symptoms replicating the chief complaint forthe testing, like dizziness, headache, nausea, and so forth. The symptoms of POTS andmany of the symptoms of functional gastrointestinal disorders, particularlydyspepsia, overlap. When Sandroni et al.  describeThis associationalso holds true when explored from the reverse, gastrointestinalperspective. Children with chiefcomplaints of abdominal pain and diagnosed with functional gastrointestinaldisorders have dizziness 58%), headaches (52%), while 10% have prominentfatigue, and 5% have episodes of loss of consciousness %, headac, also deWe have frequentlyobserved the reproduction of gastrointestinal symptoms when the subject isupright during the head-up tilt table test. The clinical implications and associated outcome of such findings havenot been evaluated. The purpose of this study was to determine, through areview of comprehensive autonomic testing performed in children with functionalgastrointestinal disorders if replication of symptoms during tilt test isassociated with a different treatment outcome than when symptoms are notpresent during TTT.n = 76). We excluded children witha known organic gastrointestinal disease or genetic/metabolic abnormality. Mostof the children had been referred to the Autonomic Clinic by their pediatricgastroenterologist or general pediatricians for evaluation of dysautonomia as apossible cause of their gastrointestinal complaints.A retrospective UniversityHospitals Case Medical Center Institutional Review Board approved chart reviewwas conducted from the Pediatric Autonomic Laboratory database containing 419 pediatricrecords between 1994 and 2007. From this group, we selected only those childrenreferred for gastrointestinal (GI) complaints  and one test of sweating, the sudomotor axon reflexresponse. The patients stopped any medications that could affect autonomicfunction for 5 half-lives prior to testing, such as beta-blockers, caffeine,anticholinergic medications, and so forth. TTT consistedof 20-minute supine rest on a motorized tilt table prior to an upright tilt to70°. Continuous BP and HR were monitorednoninvasively in the supine position for 3 minutes, and upright for 10 to 40minutes. The diastolic BP normally decreasesless than 10 mmHg, and the systolic BP, less than 20 mmHg, and HR shouldincrease less than 30 bpm .Deep breathing consists of 6 deepbreaths per minute, with the HR variation calculated from the best6 of 12 cycles . ResultSudomotor axon reflex response tothe iontophoresis of acetylcholine  across the skin of the feet,calves, hands and upper arms, was measured by standard methods . Normal Patients were divided into twogroups on the basis of their symptoms during the TTT: patients withreproducible GI complaints (RGI) and those without (NRGI). The response totreatment was scored from 1 to 5 with 1 “much worse,” 2 “little worse” 3 “neitherbetter nor worse,” 4 “somewhat better” and 5 “much better.” The score of 5 was reserved for patients whosesymptoms resolved completely. Foranalysis purposes, subjects with a “somewhat better” response (rating of 4) andthose with a “much better” (response rating of 5) were collapsed into a singlegroup and compared to all others. Thus a favorable treatment response was ≥ 4 out of 5 on the symptom response scale.Three orthostatic disorders weredefined for this group of patients: POTS required an increase in (HR) ofgreater or equal to 30 bpm in the first 10 minutes of upright position duringtilt test associated with symptoms, and without a drop in blood pressure. While the adult definition also accepts anabsolute heart rate of 120 bpm we did not include this criterion in thispopulation, since 120 bpm may be represent a normal (HR) in youngerchildren. Reflex syncope required an abrupt drop in blood pressure and often(HR) (over 3 minutes or less) leading to syncope or pre-syncope. OH was defined by asustained drop in blood pressure or ≥ 20 mmHg systolic or ≥ 10 mmHgdiastolic in the first 3 minutes of the tilt study. Patients with an orthostatic disorder whoalso had syncope were classified in the group with the orthostatic disorder, asthis was considered to be the underlying process. For example, if a patient had POTS andsyncope, they were placed in the POTS group.t test, or Wilcoxon rank sum test, depending on thedistribution. All analyses were doneusing SAS, v 9.1 . The level of significance was set at 0.05.The study population wasdescribed overall and by group. Nominalvariables were described by frequencies and percents. Continuous variables were described using theappropriate measures of central tendency and dispersion. Groups were compared using two-sample testsof significance. Nominal variables werecompared using Fisher's exact test, and continuous variables were comparedusing either a two-sided P = .17).Of the 76 childrenincluded in the study, 51 were females with a mean age of 13.4 ± 3.6 years. In 32 out of 76 children the tilt table test replicated their GI complaints (RGI) while in 44 it did not (NRGI).p = 0.09, The symptomreplicated during tilt was abdominal pain in 28/32 (87%) of the RGI group, nausea2/32 (6%) and vomiting in 2/32 (6%). By definition, symptoms were notreplicated in the NRGI group . The RGIP = .01. Of patientsreceiving fludrocortisone, patients in the RGI group tended to benefit more,with 11/15 (73%) reporting an improvement in their symptoms versus 2/8 (25%) in the NRGI group  and a subgroupwhere this did not occur (NRGI). Theresults suggest that this classification may make sense from both a physiologicand an outcome perspective. The RGI grouptended to be younger, with a shorter duration of symptoms, a preponderance ofabdominal pain, and a higher incidence of POTS. These children appeared to respond better when treated for theirorthostatic symptoms with fludrocortisone. In contrast, the older NRGI subgrouphad less POTS, and most of the abnormal autonomic reflexes in the entire cohortwere found in this subgroup. From anautonomic perspective, the RGI group was quite homogeneous, as nearly allpatients had some form of orthostatic intolerance on TTT . On the other hand, the NRGI group appears more heterogeneous, with somesubjects entirely normal, some with the same findings as the RGI group, andsome even more abnormal with abnormal cardiovascular reflexes such as a reducedresponse to deep breathing or reduced Valsalva ratio. Such a discrepancybetween the two groups further suggests that symptom replication by tilt study maybe physiologically important.One could imagine severalexplanations for the difference between these groups. Bothsets of children could be suffering from the same disorder, but the RGI groupin whom abdominal pain predominated may have harbored more bothersome symptoms,resulting in an earlier presentation. According to this hypothesis, the disorder might evolve through a phasein which symptoms are more variable depending on patient activity, diet, position,and so forth, (represented by the RGI group in this study) to a final stage in which symptomsbecome “hard-wired,” here represented by the NRGI group. Support for this notion can be drawn from thechronic pain literature, where such a progression is well recognized, forexample, in complex regional pain syndrome, and in chronic failed back syndrome , 14.Alternatively, ourfindings could reflect 3 physiologically distinct groups of children withfunctional abdominal pain. The firstgroup (the entire RGI cohort) has symptom replication on tilt table testing,owing to some type of dynamic alteration in gastrointestinal blood flow duringorthostatic challenge, an abnormality that is more responsive to medicalmanagement, with consequent better outcome.This group might correspond tothe term “gastrointestinal vasomotor dysautonomia.” A second group, perhaps termed“gastrointestinal neuropathic dysautonomia” would constitute the NRGI subgroupwith abnormal autonomic testing, reflecting a static abnormality of autonomicinnervation to the gut, perhaps resulting in abnormal motility, and lessresponsiveness to treatment. The thirdgroup, “gastrointestinal nonautonomic dysfunction” would comprise the NRGIsubgroup with entirely normal autonomic testing, in whom there is neither adynamic blood flow abnormality, nor a static loss of innervation withdysmotility, and where the abdominal pain appears to bear no relation to anyautonomic abnormality.The concept of agastrointestinal vasomotor disturbance accounting for symptomology in the RGIgroup dovetails well with some recent POTS investigations, emphasizingmesenteric hyperemia as a major finding in certain POTS subgroups . As posClinically, theclassification into 3 groups may have some utility. The “gastrointestinal vasomotor dysautonomia”subgroup is slightly younger, have had symptoms for less time, and respondbetter to treatment. The“gastrointestinal neuropathic dysautonomia” and “gastrointestinal non-autonomicdysfunction” groups, both contained in the NRGI group in this study, may welldiffer in characteristics, but will require a larger prospectively gatheredcohort for a better comparison of their features and outcomes. Utilizing this putative classification toclassify patients prospectively will also likely determine which treatmentmodalities provide optimal benefit in each group, and further delineate thephenotype of these children.A majority ofpatients, regardless of subgroup, demonstrated POTSas the primary abnormality on autonomic testing. The pathophysiology underlying this disorderremains elusive to-date, although, akin to the proposed classification abovefor functional gastrointestinal disorders, one can group patients into thosewho have abnormal autonomic reflexes and those who do not . POTS reThis study hasseveral additional limitations. The large number of children with functionalabdominal pain who had POTS 77%,  could re


Mimulus guttatusA set of experiments demonstrates the involvement of a chromosomal inversion in the adaptive transition between annual and perennial ecotypes of the yellow monkeyflower,  Mimulus guttatus, revealed that alternative chromosomal inversion arrangements are associated with life-history divergence over thousands of kilometers across North America. The inversion polymorphism affected adaptive flowering time divergence and other morphological traits in all replicated crosses between four pairs of annual and perennial populations. To determine if the inversion contributes to adaptation and reproductive isolation in natural populations, we conducted a novel reciprocal transplant experiment involving outbred lines, where alternative arrangements of the inversion were reciprocally introgressed into the genetic backgrounds of each ecotype. Our results demonstrate for the first time in nature the contribution of an inversion to adaptation, an annual/perennial life-history shift, and multiple reproductive isolating barriers. These results are consistent with the local adaptation mechanism being responsible for the distribution of the two inversion arrangements across the geographic range of M. guttatus and that locally adaptive inversion effects contribute directly to reproductive isolation. Such a mechanism may be partially responsible for the observation that closely related species often differ by multiple chromosomal rearrangements.The role of chromosomal inversions in adaptation and speciation is controversial. Historically, inversions were thought to contribute to these processes either by directly causing hybrid sterility or by facilitating the maintenance of co-adapted gene complexes. Because inversions suppress recombination when heterozygous, a recently proposed local adaptation mechanism predicts that they will spread if they capture alleles at multiple loci involved in divergent adaptation to contrasting environments. Many empirical studies have found inversion polymorphisms linked to putatively adaptive phenotypes or distributed along environmental clines. However, direct involvement of an inversion in local adaptation and consequent ecological reproductive isolation has not to our knowledge been demonstrated in nature. In this study, we discovered that a chromosomal inversion polymorphism is geographically widespread, and we test the extent to which it contributes to adaptation and reproductive isolation under natural field conditions. Replicated crosses between the prezygotically reproductively isolated annual and perennial ecotypes of the yellow monkeyflower,  Mimulus guttatus. One arrangement of the inverted region is found in an annual ecotype of this species that lives in Mediterranean habitats characterized by reduced soil water availability in the summer. The other arrangement appears in a perennial ecotype that lives in habitats with high year-round soil moisture. The inversion was observed to influence morphological and flowering time differences between these ecotypes across most of western North America. To test whether the inversion polymorphism contributes to adaptation and reproductive isolation, we conducted a field experiment by breeding plants to reciprocally swap the alternative chromosomal arrangements between the annual and perennial genetic backgrounds. We demonstrated that this inversion polymorphism contributes to local adaptation, the annual/perennial life-history transition, and three reproductive isolating barriers. These results are consistent with the theory that adaptation to local environments can drive the spread of chromosomal inversions and promote speciation.Genome rearrangements that change the order of genes along a chromosome are known as inversions and have long been hypothesized to be involved in the origin of species. Yet the way inversions contribute to adaptation and speciation remains mysterious. In this study, we identified a geographically widespread adaptive inversion polymorphism in the yellow monkeyflower,  Closely related species frequently differ by chromosomal rearrangements such as inversions However, these theories, which emphasize the resistance of inversions to homogenization by hybridization, or the differential accumulation in inversions of hybrid incompatibility factors, cannot readily explain how inversions initially establish or why they often appear linked to loci involved in climatic or habitat adaptation (reviewed in In(3R)Payne inversion in Drosophilia melanogaster that is latitudinally distributed along clines around the world and has recently shifted its distribution in response to global climate change Numerous studies have found associations between putatively adaptive phenotypes and inversions  mapping population derived from the IM  and DUN  populations of central Oregon Previously, we observed substantially suppressed recombination among markers tightly linked to a large-effect, pleiotropic QTL on linkage group eight In contrast, substantial recombination was observed among these markers in four crosses  among siM. nasutus (SF population), thought to be derived from M. guttatusWe observed recombination in the inverted region in the cross between the annual IM population and two other inland annual populations, LMC and MED . The marwww.mimulusevolution.org). Markers from within the inversion are located on two genome sequence scaffolds (11 and 233) of the recently released draft M. guttatus genome assembly v1.0 (sequence data available at www.phytozome.net). While the inversion breakpoints are as yet unknown, the inversion encompasses at least 2.22 Mbp, including at 68.6% of scaffold 11 (2.98 Mbp) and 48.8% of scaffold 233 (0.37 Mbp), or less than 1% of the 450 Mbp estimated genome size of Mimulus. This region appears to contain 362 genes identified by the current v1.0 genome annotation.Markers in the inverted region, which are completely linked in crosses between ecotypes, span a genetic map region between the most distant markers e178 and e299 of at least 33 cM in previous M. guttatus. While many inland populations of M. guttatus are annual, numerous inland perennial populations are found in areas of year-round soil moisture, such as on the edge of lakes or in rivers, hot springs, and alpine habitats M. guttatus populations have many traits in common Perennial life-history is not limited to coastal perennial populations of To determine if inland perennial populations have the same chromosomal inversion arrangement (PE) as coastal perennial populations, we conducted independent crosses between the DUN coastal perennial populations and four inland perennial populations, from as far as 1,000 km from the Pacific Ocean . PatternTo determine if the inversion contributes to the divergence of morphology and flowering time over the range of the annual and perennial ecotypes, we conducted replicated QTL mapping experiments using an outbred breeding design. Progeny resembling annual and perennial parental types were observed in all four inland annual×coastal perennial mapping populations in the F2 generation . However8,322F = 7.808, p<0.0001), LMC×SWB , RGR×OPB , and SAM×OSW  mapping populations. The inversion also explained a large percentage of the parental divergence (21%–45%) and F2 variance (2R = 0.06–0.15) in flowering time in all four of these mapping populations  in the CAN×BCB  of the F2 variance in flowering time in this inland annual×perennial cross .Given that both the AN and PE arrangements of the inversion are found in inland regions, we hypothesized that the inversion would also have an effect on flowering time divergence between inland annual and inland perennial populations. To test this, we scored flowering time in a F2 population created through a cross between lines from the inland annual LMC and inland perennial BOG populations. The inversion significantly explained 43% of the parental divergence and nearly a quarter . In each backcross population, markers in the inverted region were genotyped to identify a single inversion heterozygote to be used in an additional generation of backcrossing to the recurrent parental inbred line. This procedure was continued for a total of four backcross generations.After the fourth backcross generation, a single inversion heterozygote in each of the six backcross populations was self-fertilized and the resulting progeny were genotyped to identify a single homozygote of each arrangement. For each of the two genetic backgrounds, we then intercrossed the three AN homozygotes to each other, and the three PE homozygotes to each other, to generate plants that had one of the four combinations of genetic background and homozygosity for a particular chromosomal arrangement but were outbred throughout the genome. To generate outbred parental lines, the three annual inbred parental lines were intercrossed to each other, as were the three perennial inbred parental inbred lines . Finallyp<0.0001; p<0.0001; p<0.0001; p>0.05).As in previous studies 1,445F = 23.10; p<0.0001). Later flowering plants produced fewer flowers before the summer drought made further survival impossible  and coastal (19.39%) genetic backgrounds . The effpossible . This efpossible ; Table 21,192F = 8.34; p = 0.0043), where plants with the AN arrangement flowered earlier  and inland (p = 0.0124) field sites  was near complete, with very little overlap in flowering time between the ecotypes across habitats (As in a previous study habitats . The invhabitats .IRI) was very strong in both inland and coastal habitats . The inversion had less effect on the difference in fitness between inversion arrangements for migrants of the inland annual genetic background to coastal habitat, for an individual locus contribution of I,InversionRI = 0.079.Ecological reproductive isolation due to selection against immigrants (habitats . CompariTSRI) was high in coastal perennial habitat but low in inland annual habitat  to temporal reproductive isolation in coastal perennial habitat. The inversion had little effect  on sympatric temporal isolating barrier in inland annual habitat  through the comparisons of flower production between arrangements of the genetic background native to that habitat. Previously EP,InversionRI = 0.216) but negative in coastal habitat (RI = −0.241). However, extrinsic postzygotic isolation of the inversion based on multiseason survival in coastal perennial habitat was strong .Finally, we quantified the effect of the inversion on extrinsic postzygotic isolation .Markers from within and on both sides of the presumed inverted region  were genPopulations used in crosses for this experiment were collected in the summer of 2005 To be more confident that inversion effects are robust to differences in genetic background and effects were not due to rare alleles, a highly outbred breeding design was employed. For the 2006 experiment, each F2 mapping population was derived from 8–10 parental plants and involved eight different crosses to produce F1s. Due to difficulties with following multiple parental alleles in crosses in the 2006 experiment, the 2007 F2 mapping populations were only derived from four parental plants, where F1 progeny were intercrossed. This outbred design, with multiple parentals from each population, contrasts with many QTL studies where only two inbred lines are used in the initial cross. However, only one pair of parental lines was used for the LMC×BOG mapping population. For each population pair we grew 19–24 of each parental type, 17–25 F1 progeny, and 126–172 F2s. Differences in samples sizes were due to a combination of number of seeds available, germination rate, and space availability.TGATAATTTGTCCAATTGCGT, R = TCCAAATCAATAATCAAATCCC) were designed using Primer3 (rodo.wi.mit.edu/primer3/) targeted to a microsatellite on a sequenced bacterial artificial chromosome (GenBank accession number 154350257), which was incorporated as part scaffold 11 of the M. guttatus genome assembly v1.0, within the known inverted region (www.phytozome.net).Multiple markers were screened in the region of the inversion. Only one marker, Micro6046, was divergent among parentals and polymorphic in all five F2 mapping populations. Micro6046 primers  with a MANOVA for each population pair. To test for an association between the Micro6046 and individual traits, we conducted separate one-way ANOVAs. All analyses were carried out in JMP 7.0.1 .Crosses between three independent sets of LMC  and SWB  inbred lines were used to initiate backcross introgression line breeding . These pN = 3 backcross lines with introgressed region and 3 backcross lines where introgression was selected against in last generation per two genetic backgrounds = 12 crosses; see To facilitate the introgression of the inversion into each genetic background, two flanking makers  were genotyped outside of the inversion and one marker (e173) was genotyped in the middle of the inversion. Each generation, 32 backcross individuals of each type were genotyped for these markers. Individuals heterozygous for the three markers were then backcrossed to each inbred parental line. Fourth generation backcrosses were then self-fertilized and progeny were used for crosses to create the final generation. In the penultimate generation, plants homozygous for either the AN or PE arrangements were selected through genotyping to create the final generation. To eliminate effects of inbreeding depression, the final generation of breeding involved intercrosses within independently derived lines of the same genetic background and introgression type  and inland (N = 16) field sites. Therefore, separate analyses were conducted for each field site to test the effects of the inversion on survival.To test for effects of genetic background and the inversion on survival over the course of the season, survival analysis was conducted using ASTER, where survivorship for each field census was modeled as Bernoulli. The dates of censuses as well as the total number of censuses differed between the coastal  were conducted within each field site, with the inversion and genetic background as factors. The least square means for inversion alleles were used as a quantification of the magnitude of its effect on flowering time.InversionRI = ForeignRI−NativeRI, where ForeignRI is reproductive isolation between the native population and the backcross line with the foreign inversion arrangement and NativeRI is reproductive isolation between the native population and the backcross line with the native inversion arrangement.The overall strength of ecological reproductive isolating barriers was quantified using previously developed methods TBH,InversionRI = TBH,f,f,fRI−TBH,n,f,fRI, where TBH,f,f,fRI is the temporal reproductive isolation of the native parent relative to a pollen donor backcross line with the foreign arrangement in the foreign genetic background at the foreign field site, and TBH,n,f,fRI is the temporal reproductive isolation of the native parent relative to the backcross line with the native arrangement in the foreign genetic background at the foreign field site.Temporal flowering time isolation between habitats was calculated from the flowering distribution of LMC plants at the inland field site and SWB plants at the coastal field site, with calculations made relative to each other. All quantifications of temporal isolation were calculated for each native population (LMC or SWB) as the pollen recipient relative to a foreign pollen donor at the foreign field site. The individual contribution of the inversion to between habitat temporal isolation was calculated as Reproductive isolation due to selection against immigrants was quantified as Here, TS,InversionRI = TS,f,f,nRI−TS,n,f,nRI, where TS,f,f,nRI is the temporal reproductive isolation in sympatry of the native parent relative to a pollen donor with the foreign arrangement in the foreign genetic background at the native field site, and TBH,n,f,nRI is the temporal reproductive isolation of the native parent relative to a pollen donor with the native arrangement in the foreign genetic background at the native field site.Temporal flowering time reproductive isolation in sympatry was calculated for the native parental (SWB or LMC) relative to the foreign parental within each field site. The contribution of the inversion was calculated as The strength of extrinsic postzygotic reproductive isolation was calculated with data from a previous reciprocal transplant experiment that incorporated F1 progeny Figure S1Annual rainfall and temperatures. Thirty year (1961–1990) average monthly data from the closest weather stations  to the field sites of the reciprocal transplant experiments. (A) Rainfall (mm) in coast (blue) and inland (orange) habitats. (B) Average high  and low  temperatures.(0.70 MB TIF)Click here for additional data file.Figure S2Histogram of F2 flowering time. Distribution of days to first flower under greenhouse conditions for progeny of crosses between (A) CAN and BCB, (B) LMC and SWB, (C) RGR and OPB, and (D) SAM and OSW. Orange and blue arrows indicate the mean flowering time for the inland annual and coastal perennial parental types, respectively.(0.55 MB TIF)Click here for additional data file.Figure S3Histogram of F2 stem thickness. Distribution of second internode thickness under greenhouse conditions for progeny of crosses between (A) CAN and BCB, (B) LMC and SWB, (C) RGR and OPB, and (D) SAM and OSW. Orange and blue arrows indicate the mean stem thickness for the inland annual and coastal perennial parental types, respectively.(0.39 MB TIF)Click here for additional data file.Figure S4Photos documenting onset of drought at inland field site. View of the inland annual field site  from same perspective over the course of the spring on (A) March 3, (B) May 7, and (C) June 12, 2009.(3.65 MB TIF)Click here for additional data file.Figure S5Photo of the coastal perennial field site. Located near Manchester, CA in a seep on a cliff at the edge of a coastal terrace formation.(5.73 MB TIF)Click here for additional data file.Table S1Geographic locations of populations used in this study.(0.02 MB XLS)Click here for additional data file.Table S2Geographic distribution of chromosomal inversion arrangement as determined by crosses.(0.02 MB XLS)Click here for additional data file.Table S3Morphological trait variation in crosses between ecotypes measured in the greenhouse.(0.02 MB XLS)Click here for additional data file.Table S4Fitness analysis of coastal perennial and inland annual parental ecotypes from the field experiment with ASTER.(0.02 MB XLS)Click here for additional data file.


To be really useful, a vaccine must be highly efficacious, safe, and must be accepted by and administered to the majority of the target population. Current prophylactic vaccines against human papillomavirus (HPV), based on L1 virus-like particles, have been extensively evaluated for both efficacy against severe precancerous lesions of the cervix uteri and safety  were such agents . AlthougInformation from over 30 000 invasive cervical cancers from five continents has now accumulated, and has not only confirmed the predominance of HPV16 (57%) and HPV18 (16%) in every studied population, but has also showed the similarity in the relative importance of other oncogenic types  . Immunisation against HPV16/18 in England has, therefore, the potential to prevent 56–65% of CIN3 and 73–77% of invasive cervical cancer. Finally, cross-protection against oncogenic types other than HPV16/18 may prevent an additional 3–4% of invasive cervical cancer.Howell-Jones et al also highlighted the high frequency of infection with oncogenic HPV types among women in England. Prevalence was 16% among all screened women, but rose to 29% among women aged 25–29 years, which is as high as that found in countries like Colombia  they are expensive vaccines; (2) they target adolescent girls for whom no delivery platform is readily available in any country; and (3) they are meant to prevent a cancer for which an effective secondary prevention strategy exists . Also imHigh-vaccine uptake was also reported in similar school-based programmes in Scotland , Wales , and Aus


Most human cytomegalovirus (HCMV) genes are highly conserved in sequence among strains, but some exhibit a substantial degree of variation. Two of these genes are UL146, which encodes a CXC chemokine, and UL139, which is predicted to encode a membrane glycoprotein. The sequences of these genes were determined from a collection of 184 HCMV samples obtained from Africa, Australia, Asia, Europe, and North America. UL146 is hypervariable throughout, whereas variation in UL139 is concentrated in a sequence encoding a potentially highly glycosylated region. The UL146 sequences fell into 14 genotypes, as did all previously reported sequences. The UL139 sequences grouped into 8 genotypes, and all previously reported sequences fell into a subset of these. There were minor differences among continents in genotypic frequencies for UL146 and UL139, but no clear geographical separation, and identical nucleotide sequences were represented among communities distant from each other. The frequent detection of multiple genotypes indicated that mixed infections are common. For both genes, the degree of divergence was sufficient to preclude reliable sequence alignments between genotypes in the most variable regions, and the mode of evolution involved in generating the genotypes could not be discerned. Within genotypes, constraint appears to have been the predominant mode, and positive selection was detected marginally at best. No evidence was found for linkage disequilibrium. The emerging scenario is that the HCMV genotypes developed in early human populations (or even earlier), becoming established via founder or bottleneck effects, and have spread, recombined and mixed worldwide in more recent times. Herpesviridae, subfamily Betaherpesvirinae, genus Cytomegalovirus, species Human herpesvirus 5) is ubiquitous and host–specific. Infection is asymptomatic for most people, but can result in serious disease in immunocompromised individuals and congenitally infected newborns. The minimally passaged Merlin strain is considered best to represent wild–type HCMV, and has a 236 kbp genome that is predicted to contain approximately 165 protein–encoding genes .Human cytomegalovirus . Details of the 171 samples in the collection that yielded sequence data are available on request, and include the age, sex, and pathology of the patient, the clinical source of the sample, and the UL146 and UL139 genotypes determined. The samples numbered 18 from Australia, 10 from Hong Kong, 6 from Germany, 13 from England, 18 from The Gambia, 24 from Hungary, 7 from Italy, 6 from The Netherlands, 41 from Scotland, 5 from the USA, 8 from Wales, and 15 from South Africa. A minority of strains (40) had been passaged in human fibroblast cell culture, either as routine diagnostic specimens or as laboratory strains. DNA was extracted by standard methods from body tissues, urine, saliva or infected cells. The South African samples were obtained from the saliva of mothers (10 of whom tested HIV-negative) attending rural clinics in KwaZulu/Natal . Since tUL146 and UL139 were amplified separately by single round or nested PCR, using primers in conserved regions . Single For the single (and first) round, 1µl ofDNAwas added to the PCR reaction mixture, which consisted of 40µl of water, 5µl of buffer, 1µl of10µM dNTPs, 1µl of each the two primers (10µM) and 1µl (1U) of DNA polymerase . The conditions for amplification were 95°Cfor 2 min followed by 35 cycles of 95°Cfor 2 min, 60°Cfor 30 sec and 68°Cfor 1 min. Second round PCR utilized 1µl of first round PCR products as template amplified under the same conditions. PCR reactions were set up in a dedicated, PCR product–free room. Approximately one–third of the samples were tested on three separate occasions to assess reproducibility.PCR products were separated by agarose gel electrophoresis. Appropriate DNA fragments were excised, purified using a Geneclean turbo kit , and eluted using 100µl of nucleasefree water. The single round or second round primers were used for direct sequencing.E. coli TOP 10 cells, 5 recombinant colonies were picked and grown overnight at 37°C in 2YT-broth containing 100°g/ml ampicillin. Plasmid DNA was purified using a QIAprep Spin miniprep kit (Qiagen). Plasmid inserts were sequenced using universal forward and reverse primers. Sequencing was carried out on both DNA strands using a BigDye terminator kit  in an ABI 3730 instrument. Samples containing multiple strains were identified by the derivation of plasmids representing different genotypes of UL146 or UL139.In some cases, including those where direct sequencing indicated the presence of more than one genotype of UL146 or UL139, fragments were cloned using a pGEM–T kit . Following ligation and transformation into chemically competent Sequence chromatograms were viewed using Editview (Applied Biosystems) and analyzed using Pregap4 and Gap4 . NucleotKall et al., 2004]Sample origin was divided into four regions  for assessment of the geographical distribution of genotypes. Chi-square tests were used to assess the significance of variability of genotype frequencies among regions. Yates' correction for continuity was applied to chi–square tests in cases where the expected values fell below 5. Similarly, Chisquare tests with Yates' correction were applied to 60 samples where single genotypes were detected for both UL146 and UL139, in order to test for linkage disequilibrium. Samples containing mixed infections were excluded from this analysis.The UL146 and UL139 genotypes in 184 samples were investigated by PCR and sequencing using primers in conserved regions. UL146 was amplified from 159 samples and sequences were determined from 134, and UL139 was amplified from 168 samples and sequences determined from 131.Atotal of 13 samples failed to yield products from either gene. Since some samples contained more than one virus strain, totals of 182 UL146 sequences and 183 UL139 sequences were obtained. Alignment and phylogenetic analyses involved the 350 UL146 sequences and 300 UL139 sequences derived from the present study or reported by others in the literature  or publiThe UL146 coding sequences range in length from 114 to 126 codons, and phylogenetic analyses indicated that all fall into the 14 genotypes defined previously and designated G1–G14 . Amino aDifferences in overall genotypic frequencies were observed . For exaThe UL139 coding sequences range in length from 124 to 148 codons, and phylogenetic analyses indicated that all fall into 8 genotypes designated G1–G8. The protein encoded by each HCMV UL139 genotype contains a putative signal peptide sequence and a transmembrane region. Variation is concentrated in the N–terminal portion of the protein. Amino acid sequence variation between genotypes is high (p=0.275), whereas within each genotype it is low (p=0.007–0.095 with a mean of 0.025) . VariatiDifferences in overall genotypic frequencies were observed . For exaIn order to assess positive selection , the dN/dS ratio was calculated for each UL146 and UL139 genotype  and III.The sequence data derived in the present work were divided into four groups representing strains obtained from Africa, Asia, Australia, and Europe. Insufficient sample numbers were obtained from America to war–rant inclusion. Observation of frequencies initially suggested no significant differences in the distribution of UL146 and UL139 genotypes among continents  and V. HIdentical nucleotide sequences were frequently obtained from geographically distant and presumably epidemiologically unrelated patients. For example, certain samples from The Gambia, Scotland, and Hungary contained identical UL146 G12 sequences. Also, UL139 G2, which was identified in 27% of samples, was represented by identical sequences from Hungary, The UK, and The Gambia.Potential linkage disequilibrium was investigated in 60 strains for which single genotypes of both UL146 and UL139 were obtained. Of 112 possible genotype pairs, 41 were observed at least once . No statMultiple genotypes in one or both genes were detected in at least 14% of samples upon first analysis (rising to 29% when repeat experiments were included), distributed among immunocompetent and immunocompromised individuals. More than one genotype was detected in 11% of European samples, 16% of Gambian samples, 47% of South African samples, and 10% of Hong Kong samples .This study focused on the genotype definitions, frequencies, occurrence in mixed infections, geographical distribution and evolution (in terms of linkage disequilibrium and mode of selection) of two hypervariable HCMV genes, UL146 and UL139. Totals of 182 UL146 and 183 UL139 sequences were obtained from a large panel of clinical isolates collected from Africa (South Africa and The Gambia), Asia (Hong Kong), Australia and Europe (various countries). These were used in all analyses, and were supplemented by 168 previously published UL146 and 117 UL139 sequences in analyses of genotype definitions, frequencies and mode of selection.The UL146 sequences fell into the 14 genotypes described previously , and no The UL139 sequences grouped into eight genotypes.A recent analysis of 26 clinical samples  describeA region of sequence identity . Therefore, the absence of a genotype from a particular sample cannot be assured. If anyUL146 or UL139 genotypes have escaped recognition, they may emerge from future studies involving different primers or from whole genome sequencing exercises.As found in previous studies [reviewed in The occurrence of mixed infections is being recognized increasingly as potentially significant to the biology of HCMV. This feature adds to the limitations inherent in studies of whether particular genotypes are associated with disease outcome; other features include the number, origin and pathological categorization of samples, the choice of gene, the absence of linkage disequilibrium, and host factors. In light of these limitations, our opinion is that robust evidence in favor of any association between genotype and pathology has proved elusive in the literature. Further work utilizing genotype–specific approaches is required to explore the true frequency of mixed infections, both to validate studies of this type and to determine whether mixed infections have geographical or biological correlates.Similar to the conclusions drawn from a study on UL73 (encoding gN) , no statThe extensive divergence between genotypes and the consequent inability to produce reliable sequence alignments for both UL146 and UL139 in the hypervariable regions compromised assessments of the role of positive selection in generating the genotypes. In contrast, variation within genotypes is low, and identical nucleotide sequences were obtained from geographically distant individuals. The analysis suggests that constraint has been the predominant factor in evolution within genotypes, with positive selection detected only marginally. A previous study  involvin


Urine proteins were determined using Bradford's method and urinary peptide levels were determined by subtracting Bradford's value from the Lowry value of the same sample. There was a significant decrease in the levels of urinary peptides in group III compared to group I (P < 0.01), however, there was no difference in peptides between groups I and II. The percentage of urinary peptides was decreased in both groups II and III compared to group I (P < 0.01), and there was a significant difference in % urinary peptide content in group II compared to group III (P < 0.01). On correlation, % urinary peptides correlated negatively with urinary proteins/g creatinine  and positively with urinary peptides/g creatinine . Our data suggest that there is a marked decrease in urinary peptide levels with an increase in proteinuria. This may suggest impaired tubular protein reabsorption and degradation capacity of renal tubules.Although considered useful in the diagnosis and prognosis of renal diseases, proteinuria can only be detected after significant renal paranchymal changes. There is considerable interest in the estimation of urinary peptides as an early marker of renal disease. In the current study, we have estimated urinary peptides in patients with different grades of proteinuria. Twenty-four hour urine samples were collected from 138 subjects and classified into three groups based on the urine protein excreted: group I (normoproteinuria, 0–150 mg/day,  The restrictive permeability of the glomerular filter to macromolecules has been attributed to exclusion based on their size, configuration, and electrical charge.ments > 20 Da in net al. have reported increased quantities of highly degraded, tritiated albumin (150–500 Da), but no detectable intact (> 1500 Da), tritiated albumin in the urine of healthy control subjects. This was in contrast with a predominant large molecular peak of presumably undegraded, tritiated albumin with greatly diminished quantities of small fragments in a macroalbuminuric patient with diabetes.10Recently, Strong Urinary peptides < 10 kDa) can be detected with the use of radioactive tracers.1214 kDa can In the current study, we have used a modified chemical assay designed to measure urinary peptides using the Biuret principle, and also, to determine the levels of urinary peptides in normoproteinuria cases in comparison with microproteinuria and macroproteinuria cases.One hundred and thirty-eighty subjects were assigned to three groups based on their daily urine protein content. Subjects with urine protein content of 1–150 mg were categorized as group I, 150–300 mg as group II, and > 300 mg as group III. Twenty-four hour urine samples from 37 group I, 31 group II, and 70 group III cases were collected in brown bottles containing toluene as a preservative. The urine sample bottles were stored at 4°C during the period of collection. Samples were centrifuged at 3000 rpm for 10 min and analyzed immediately after the collection period. Informed consent was taken from the subjects involved in the study followed by ethical clearance from the Institutional Review Board.® Chemicals, St Louis, MO, USA. All other reagents were of analytical grade.Special chemicals such as bovine serum albumin (BSA) were obtained from SigmaProtein stock: BSA was dissolved in phosphate buffered saline (PBS). Standard curves were prepared by dissolving BSA to get the following final concentrations; for Bradford assay: 2, 4, 6, 8, and 10 μg/mL; for Lowry assay: 50, 100, 150, 200, and 250 μg/mL.1 : 1% sodium potassium tartarate, reagent B2 : 0.5% CuSO4 in reagent B1, reagent C: 50 mL reagent A + 1 mL reagent B2, and reagent D: 1N Folin-Ciocalteau reagent. Composition of Folin-Ciocalteau reagent is sodium tungstate, sodium molybdate, orthophosphoric acid, concentrated hydrochloric acid, lithium sulphate, bromine water and sodium hydroxide.For Lowry assay: we used a modified Lowry's assay for determining levels of total urinary proteins; the reagents were prepared as follows: reagent A: 2% sodium carbonate, reagent BProtein and peptide levels in urine were measured using a Genesys 10UV spectrophotometer whereas urine creatinine levels were determined by a Clinical Chemistry Automated Analyzer (Hitachi 912).Both Lowry and Bradford assays were performed after diluting the urine samples suitably. Clear-cut dilution principles were not available in the literature to dilute urine samples for the protein and peptide assays using Lowry and Bradford's methods. Hence, we propose here a range of dilution factors based on the protein content per deciliter of the urine sample .Urinary proteins, together with urinary peptides, were measured using the Lowry assay,For Lowry estimation, 0.2 mL of the diluted urine sample was taken in two sets of eppendorf tubes (sets 1 and 2) while 0.2 mL of 145 mM NaCl was taken in another tube and labeled as reagent blank (RB). To RB and to set 1, 1 mL of reagent C was added while 1 mL of reagent A was added to set 2 tubes. The tubes were shaken vigorously and incubated for 10 min at room temperature. Reagent D was added to all the tubes at the end of 10 min and the tubes were vortexed; this step is crucial for color development. The tubes were incubated at room temperature for 30 min and the absorbance was read at 600 nm.After correcting for respective blanks, absorbance values of set 2 samples were subtracted from their counterparts in set 1. The difference in the readings arises because of the copper pretreatment of set 1 samples. The total protein contents were calculated from the calibration curves, and after multiplying with the dilution factor, the total protein content (including peptides) was expressed in gram per milliliter (g/mL) and g/g of urine creatinine.For the Bradford assay, 1 mL of diluted urine sample was added to 1 mL of Bradford reagent. The reagent blank consisted of 1 mL of PBS and 1 mL of Bradford reagent. The contents were mixed and incubated at room temperature for 30 min and the absorbance read at 595 nm. The protein content in the sample was calculated using a calibration curve, and after multiplying with the dilution factor, values were expressed in g/mL and g/g of urine creatinine. The total urinary peptide content in the sample was calculated by subtracting the Bradford value (protein content) from the Lowry value , and the peptide content in the urine was expressed as g/mL and g/g of urine creatinine.P < 0.05 was considered statistically significant. Microsoft Office Excel 2 was used to prepare correlation figures.Statistical analysis was done using the Statistical Package for Social Sciences (SPSS), version 10. Analysis of variance (ANOVA) followed by multiple comparison by post-hoc test was done to compare the mean values. Pearson's correlation was used to correlate the parameters. P < 0.01), however, there was no such difference between groups I and II. The percentage of urinary peptides was decreased in both group II and III cases compared to group I (P < 0.01), and there was a significant difference in % urinary peptide content in group II compared to group III (P < 0.01). On correlation, the % urinary peptides correlated negatively with urinary proteins/g creatinine  /Lowry × 100) against proteinuria. We have found significant decreases in % urinary peptides in microproteinuria and macroproteinuria cases. Determination and expression of peptides as % urinary peptides have shown significant differences among the three groups when compared to determining and expressing peptide as g peptides/g of urine creatinine. There was no difference in g of urinary peptides/g of urine creatinine between micro- and macroproteinuria cases in our study, but when the filtered load of urinary proteins was taken into consideration using the above formula, we found significant differences in % peptides that were excreted in these two groups .Previous authors have reported similar decreases in % urinary peptides in proteinuria cases and the decrease in % urinary peptides was found to correlate negatively with urine proteins/gm creatinine, indicating a possible relation between % urinary peptides that are excreted in urine and renal function. Significant decreases in lysosomal enzyme levels have been found in the urine of micro- and macroproteinuric patients compared to healthy controls, indicating decreased activity of tubular lysosomal enzymes.We have compared clinical chemistry automated analyzer method  with Bradford's method, and have found no significant difference between the protein values obtained by these two methods; and urine protein values in both methods correlated positively. Since Bradford reagent in Bradford assay reacted only with proteins larger than 13 kDaIn conclusion, our study is consistent with previous studies in that there is a significant decrease in urinary peptide levels with impairment of renal function, and serial monitoring of renal function can be done by a simple determination of urine peptides. Any fall in the levels of urinary peptides from the normal range could be interpreted to indicate renal pathology at an early stage, rather than waiting for significant proteinuria. Although advanced radiological imaging techniques are available for early detection of renal injury, they are still not available in most hospitals. If sensitivity and specificity of this serial monitoring can be proved by further studies, we believe that simple serial monitoring of urinary peptides in the early detection of renal injury will be a noninvasive and cost-effective method that will be very useful in the early detection of renal injury.


Clear cell renal cell carcinoma (ccRCC) is the most frequently encountered tumor in the adult kidney. Many factors are known to take part in the development and progression of this tumor. Nuclear factor kappa B (NF-κB) is a family of the genes that includes five members acting in events such as inflammation and apoptosis. In this study, the role of NF-κB (p50 subunit) in ccRCC and its relation to angiogenesis and apoptosis were investigated.Formalin-fixed and paraffin embedded tissue blocks from 40 patients with ccRCC were studied. Expressions of NF-κB (p50), VEGF, EGFR, bc1-2 and p53 were detected immunohistochemically. The relationship of NF-κB with these markers and clinicopathological findings were evaluated.The expression of NF-κB was detected in 35 (85%), VEGF in 37 (92.5%), EGFR in 38 (95%), bc1-2 in 33 (82.5%) and p53 in 13 (32.5%) of 40 ccRCC patients. Statistical analyses revealed a significant relation between NF-κB expression and VEGF (p = 0.001), EGFR (p = 0.004), bc1-2 (p = 0.010) and p53 (p = 0.037). There was no significant correlation between NF-κB and such parameters as tumor grade, stage, age and sex.The results of this study indicated that in ccRCC cases NF-κB was associated with markers of angiogenesis and apoptosis such as VEGF, EGFR, bc1-2 and p53. In addition, the results did not only suggest a close relationship between NF-κB and VEGF, EGFR, bc1-2 and p53 in ccRCC, but also indicate that NF-κB was a potential therapeutic target in the treatment of ccRCC resistant to chemotherapy. Renal cell carcinoma (RCC) is the most common malignant tumor of the adult kidney, accounting for 3% of all adult malignancies . Clear cThe aim of this study was to investigate the relation between the p50 subunit of NF-κB and the factors like apoptosis and angiogenesis, which affect tumor development and progression in the frequently encountered adult renal tumors, ccRCCs. Detection of a relation between important growth factors and various genes like VEGF, EGFR, p53 and bc1-2 and NF-κB activation will help to enhance the effectiveness of the available therapies and to develop new treatment modalities in the course of curative efforts for RCCs, which are considered as chemoresistant tumors ,32. NF-κForty patients who underwent radical or partial nephrectomy for ccRCC at Adnan Menderes University Hospital from 2002 to 2007 were included in the study. Data about sex, age, tumor size, TNM stage and Fuhrman's nuclear grade were obtained. None of the patients received treatment for cancer before surgery. All specimens were re-evaluated before the current study by two pathologists. Paraffin-embedded specimens that contained normal renal tissue together with the tumoral tissue were selected for immunohistochemical analysis.First, 4 μm thick slices taken from the selected paraffin embedded blocks were placed on positively charged slides and deparafinized with xylene and rehydrated with ethanol. Second, all were washed with 0.1% Tris-buffered saline (TBS) three times for 5 minutes. They were kept in 0.3% hydrogen peroxidase for 20 minutes for the blockage of endogenous peroxidase activity. Third, the slices were rinsed with TBS three times for 5 minutes. For antigen retrieval, the slides were placed in a sodium citrate buffer and left in the microwave oven at 700-W for 10 minutes. Fourth, they were cooled to room temperature and washed with TBS three times for 5 minutes. Fifth, non-specific binding was blocked with 10% normal rabbit serum for 1 hour. Sixth, the slices were incubated with primary antibodies rabbit polyclonal NF-κB/p50 , VEGF , EGFR , p53  and bcl-2  for 1 hour. After the slides were rinsed with TBS three times for 5 minutes, they were exposed to biotin free horseradish peroxidase (HRP) enzyme-labeled polymer  for 30 minutes. Seventh, the slides were rinsed again in TBS three times for 5 minutes. Eight, 3,3'-diaminobenzidine (DAKO) chromogen substrate was added. Last, the slides were rinsed under tap water and countered with Mayer's haematoxyline, dehydrated and mounted. For the negative control, the slides were incubated with TBS without the primary antibody. The slides known to be positively immunostained were used as positive controls.Firstly, the slides were examined at low magnification (×100) and areas containing the highest density of stained markers were chosen as "hot spot" areas and were evaluated at high magnification (×200). The staining cell ratio was determined by counting at least 200 cells. A staining percentage of 10% or above was accepted to be positive for all the markers. The scoring was accomplished according to the degree of staining in different magnifications as in the following: "weak" staining, visible at × 200; "moderate" staining, visible at × 100 and "strong" staining visible at × 40.Chi-square test for trend was used to assess the differential expression of markers in malignant and non-malignant kidney tissues. Spearman's rank correlation coefficient was used to determine the correlation between ink patterns of markers in RCC. P < 0.05 was considered significant. All statistical analyses were performed with SPSS .Clinicopathologic characteristics of the patients are summarized in Table NF-κB staining was detected within the cytoplasm and nuclei of the tumoral cells. The degree of nuclear staining decreased towards the central areas of the tumor whereas the most prominent nuclear staining was seen in the areas adjacent to the non-tumoral renal tissue Figure . There wVEGF staining was present within the cytoplasm of the tumor cells Figure , 2C. TheImmunohistochemical staining properties of NF-κB, EGFR, VEGF, bcl-2 and p53 are shown in Table There was a significant positive correlation between the staining degree of NF-κB and VEGF , EGFR , bcl-2  and p53 . No correlation between the NF-κB staining degree and age, sex, tumor size, TNM stage and Fuhrmans' grade was established.NF-κB activation plays a role in the regulation of more than 400 gene product expressions associated with inflammation, cell survival, proliferation, invasion and angiogenesis ,7. The NNF-κB has an effect on apoptosis during tumor development and progression in many ways . HoweverAngiogenesis plays a key role in the development and progression of tumors. Angiogenesis in tumors has been shown to be associated with the chemokines of the macrophages, neutrophils and other inflammatory cells  and the growth factors . NF-κB activation plays a role in the regulation of these angiogenesis inducing products ,7. VEGF,EGFR expression is frequently associated with proliferation, invasiveness and angiogenesis in many tumors and also considered an indicator for poor prognosis. It has been reported that the overexpression of the EGFR in RCCs plays a role in the development and progression of the tumor and is frequently accompanied by high grade tumors ,42. TherIn some studies exploring the association between the NF-κB activation and the tumors, a correlation between the prognostic indicators like tumor grade and stage and NF-κB have been demonstrated. Oya et al. found that NF-κB activity increased in advanced stage RCCs and that NF-κB played a role in the progression of the tumors at advanced stages . In the In this study we used immunohistochemistry, which is a simple reproducible method for the pathologists to conduct an investigation or use routinely in the investigation of or the estimation of NF-κB, VEGF and EGFR in the future. The review of the literature revealed only a couple of studies concerning the evaluation of NF-κB with immunohistochemical methods. NF-κB signaling pathway activation results in translocation of NF-κB subunit in the nucleus. Therefore, NF-κB activation can be identified by positive nuclear immunostaining of various NF-κB subunits. Nuclear immunostaining has been evaluated in many studies aiming to determine NF-κB subunits. Using immunochemical methods, Levidou et al. in their study on patients with gastric carcinoma evaluated both nuclear and cytoplasmic expression of NF-κB p50 subunit . They foRecent studies have focused on new therapeutic agents and treatment alternatives with better antitumour effectiveness on advanced RCCs ,47. AngiWe found that NF-κB was associated with numerous factors in the development and progression of tumors in ccRCCs and that increased NF-κB activity was accompanied by increased bc1-2, p53, VEGF and EGFR expressions. To conclude, in cancer cells, NF-κB activity is correlated with events like proliferation, apoptosis, angiogenesis, chemo-radioresistance and has a diagnostic and prognostic importance. It is very important to develop new target molecules and to establish new chemotherapic strategies in malignancies like RCCs, which are resistant to chemotherapy. For the antiangiogenic and EGFR inhibitor drugs, which are lately being introduced to the therapeutic line in the treatment of many malignancies, knowing the effects and interactions of the molecules like NF-κB in order to be able to predict and increase their impact, is quite important. Moreover, NF-κB itself will be a target on its own for the chemotherapeutic agents. Therefore, we need to know the interaction of the various subunits of NF-κB in different tumors and their relation with different molecules. Establishing the subunits of NF-κB with the simple methods as we used in this study will enable new studies to be performed on the issue.The authors declare that they have no competing interests.IM conceived of the study and wrote the manuscript. IHE collected the samples and patient's clinical data. ME and NM participated in the design of the study and helped write the paper. SB reviewed the manuscript and wrote the final version. All authors read and approved the final manuscript.