Ultraviolet photodegradation products from pesticides form both in the field and during water treatment.We evaluated the photolytic breakdown of the organophosphate pesticide chlorpyrifos (CPF) in terms of both the chemical entities generated by low-pressure ultraviolet C irradiation and their potential as developmental neurotoxicants.We separated by-products using high-performance liquid chromatography and characterized them by gas chromatography/mass spectrometry. We assessed neurotoxicity in neuronotypic PC12 cells, both in the undifferentiated state and during differentiation.Photodegradation of CPF in methanol solution generated CPF oxon and trichloropyridinol, products known to retain developmental neurotoxicant actions, as well as a series of related organophosphate and phosphorothionate derivatives. Exposure conditions that led to 50% degradation of CPF thus did not reduce developmental neurotoxicity. The degradation mixture inhibited DNA synthesis in undifferentiated cells to the same extent as native CPF. In differentiating cells, the products likewise retained the full ability to elicit shortfalls in cell number and corresponding effects on cell growth and neurite formation. When the exposure was prolonged to the point where 70% of the CPF was degraded, the adverse effects on PC12 cells were no longer evident; however, these conditions were sufficiently severe to generate toxic products from the methanol vehicle.Our results indicate that field conditions or remediation treatments that degrade a significant proportion of the CPF do not necessarily produce inactive products and, indeed, may elicit formation of even more toxic chemicals that are more water soluble and thus have greater field mobility than CPF itself. The nearly ubiquitous exposure of the human population to organophosphate pesticides has raised increasing concern about their propensity to elicit developmental neurotoxicity at exposures that go undetected because of the absence of systemic signs of intoxication . Chlorpyin vivo  in methanol  and carried out photodegradation experiments in a collimated-beam low-pressure UV bench reactor, configured with four mercury vapor germicidal lamps , emitting monochromatic light at 254 nm. We placed the test solution in a 70 × 50 mm crystallization dish, which was sealed with a quartz cover to prevent evaporation, and exposed it to UVC irradiation. Temperature in the dish was maintained at 24°C by a coiled water cooling system, and the solution was mixed on a stir plate to ensure complete mixing and consistent batch conditions. We determined incident irradiance using a calibrated radiometer  and calculated delivered UVC fluence with a spreadsheet program that included lamp spectrum, solution absorbance, exposure time, and incident irradiance . At specWe measured concentrations of CPF during photolysis with a Varian Pro Star HPLC  equipped with a polychromatic diode array detector and a 4.6 × 150 mm C18 reverse-phase column . We used isocratic elution with a mobile phase of acetonitrile and water (80/20 vol/vol) at a flow rate of 1 mL/min. Under these conditions, the retention time for CPF was 7.9 min. For by-product identification, we used a Shimadzu GC/MS-QP2010 gas chromatograph/mass spectrometer (GC/MS) equipped with a 15-m RTX-5MS column .6 PC12 cells  onto poly-d-lysine–coated plates in RPMI-1640 medium  supplemented with 10% inactivated horse serum , 5% fetal bovine serum (Sigma), and 50 μg/mL penicillin streptomycin (Invitrogen). Cells were then incubated with 7.5% CO2 at 37°C and the medium was changed every 2 days. For studies in the undifferentiated state, the medium was changed 24 hr after seeding to include CPF or CPF photolysis products. Because of its poor water solubility, CPF was dissolved in methanol, the same medium used for the photolysis exposures. The final concentration of methanol in the culture medium ranged from 16 to 47 mM (0.05–0.15%), and the same vehicle was added to the control cultures. By itself, methanol had no effect on proliferation, growth, or differentiation of PC12 cells, as established previously ; we examined each culture under a microscope to verify the subsequent outgrowth of neurites. CPF or CPF photolysis products were added concurrently with the start of NGF treatment, and cultures were maintained for 6 days, with the test agents included with every medium change.For studies in differentiating cells, we seeded 3 × 10We chose the CPF concentrations on the basis of previous work. Given our objective to evaluate the loss of activity upon photolysis, our strategy was to elicit both threshold and robust responses for each of the effects evaluated. Accordingly, we used 10 or 30 μM CPF in undifferentiated cells, concentrations that span the range from low to high effects on DNA synthesis, and 30 μM CPF for studies in differentiating cells .3H]thymidine  along with the continued inclusion of the test substances. After 1 hr, the medium was aspirated and cells were harvested in ice-cold water. Duplicate aliquots of each sample were treated with 10% trichloroacetic acid and sedimented at 1,000 × g for 15 min to precipitate macro molecules. The resulting pellet was washed once with additional trichloro acetic acid and then with 75% ethanol. The final pellet was hydrolyzed with 1 M KOH overnight at 37°C and neutralized with 6 M HCl; the DNA was then precipitated with ice-cold 5% trichloroacetic acid and resedimented. The supernatant solution, comprising solubilized RNA and protein, was discarded. The DNA-containing pellet was hydrolyzed in 5% trichloroacetic acid for 15 min at 90°C and resedimented, and an aliquot of the supernatant solution was counted for radiolabel. We assayed another aliquot for DNA spectrophotometrically by absorbance at 260 nm. Previous work has demonstrated quantitative recovery of DNA by these techniques . Cells were harvested in ice-cold buffer and homogenized , and aliquots were withdrawn for measurements of DNA and protein using dye-binding methods . To prepp< 0.05.We conducted CPF photolysis in three separate sets of samples, with each sample prepared several weeks apart, and then tested them in multiple batches of PC12 cells. Results are reported as mean ± SE. Because of the multiple treatments in each experiment, significant differences were first established by a global analysis of variance (ANOVA) incorporating all treatments, followed by Fisher’s protected least significant difference to evaluate differences between specific treatment groups. The initial test included sample batch and cell batch; however, because the treatment effects were the same, we then combined the results across batches after normalization to account for the differing absolute values for each batch of cells. Significance was assumed at Direct photolysis of CPF can be described as a pseudo-first-order reaction following these two equations whereE mol−1 sec−1); ΦM is the quantum yield; kd is the pseudo-first-order rate constant; [M] is the concentration of target compound; EP0 is incident photon irradiance (10−3E cm−2 sec−1); ɛM is molar absorbance (M−1 cm−1) of the target compound; Z is the solution depth; and a is the light absorbance by the solution. Using a spectro radiometer, we confirmed that the UVC lamp emitted monochromatically at 254 nm, with insignificant contributions from other wavelengths , 3,6-dichloro-2- thiophosphate, and CPF oxon. Whereas photolysis in methanol solution also produced dimethyl ethyl phosphate, water solution produced the similar  product, O,O-diethyl phosphate. Photolysis in water also produced 3,5,6-trichloro-2-pyridinyl acetic acid, which was not found with the methanol solution, although this is obviously quite similar to trichloropyridinol. We found none of these products in untreated CPF solutions.The application of GC/MS made it possible to determine the major photodegradation by-products by comparing mass spectra of the unknowns with a National Institute of Standards and Technology mass spectral library search (NIST 2008) and through analysis of the mass fragmentation patterns . The proIn agreement with earlier work , exposurp < 0.0001; CPF × UV × measure, p < 0.002), reflecting a greater effect of CPF on cell number than on cell growth (When CPF was introduced simultaneously with NGF and differentiation allowed to proceed for 6 days, we observed a substantial reduction in the total number of cells as monitored by DNA content . Short-tl growth . Accordil growth ; longer CPF exposure also reduced membrane protein  but to aThe main finding of these studies is that photodegradation of CPF does not necessarily lead to biological inactivation of adverse effects directed at developing neurons, one of the major concerns for CPF as well as other organophosphates . AlthougAlthough we have already characterized the ability of both CPF oxon and trichloropyridinol to act as developmental neurotoxicants , it is lThere are a number of limitations in the interpretation of our results regarding UV photolysis as it occurs in water treatment or as a result of sunlight exposure in the field. First, we examined the effects of exposure to a fixed wavelength of 254 nm, rather than sunlight or other UV combinations. Notably, however, earlier work with a Suntest apparatus emitting in the range of 300–800 nm, as well as a variety of low-pressure UV light sources, found the same major breakdown products likely to possess biologic activity, including CPF oxon and trichloropyridinol ; accordiThe present findings indicate that photodegradation of CPF, and potentially other environmental toxicants, does not necessarily produce inactive products, and indeed, some of the resultant compounds and mixtures may actually exhibit equal or greater toxicity than the parent chemical. These factors should be taken into account in determining whether the disappearance of an agent in the field or during remediation treatments actually connotes a safer outcome.


Until the end of follow-up (1 January 1996), 397 postmenopausal breast cancer cases were identified and a subcohort of 424 women was then taken from all eligible women. Women using hormones were excluded, leaving 364 breast cancer cases and 382 women in the subcohort for the analyses. Concentrations of oestrone, oestradiol, testosterone, 5α-androstane-3α, 17β-diol and creatinine were measured in first morning urine samples, which had been stored since enrolment at −20°C. A Cox proportional Hazards model was used, with Barlow's adjustment for case–cohort sampling, to estimate breast cancer risk in quartiles of each of the, creatinine corrected, hormone levels, the lowest quartile being the reference group. Women with higher levels of all four of the hormones were at increased risk for postmenopausal breast cancer . In conclusion, women with higher excretion levels of both oestrogens and androgens have an increased risk of breast cancer.To assess the relation between urinary endogenous sex steroid levels and the risk of postmenopausal breast cancer, a nested case–cohort study was conducted within a large cohort (the DOM cohort) in the Netherlands ( Established risk factors for breast cancer, such as early age at menarche, late age at menopause, late age at first full-term pregnancy or nulliparity, indicate that endogenous sex hormones play an important role in breast cancer aetiology .For almost 30 years now, several case–control and cohort studies addressed this topic. Recently, a pooled analysis of nine prospective studies, including 663 breast cancer cases and 1765 controls, showed that postmenopausal women in the highest quintiles of oestrone-, oestradiol- and testosterone concentrations in blood had a statistically significant two-fold increased risk of breast cancer . Howeverα-androstane-3α, 17β-diol) and the risk of postmenopausal breast cancer, we conducted a nested case–cohort study in a large population-based prospective cohort study in the Netherlands.To further evaluate the relation between urinary endogenous sex steroids  between 1975 and 1986 (n=9349). All women were followed until 1st January 1996 for the occurrence of breast cancer through their general practitioners and, from 1986 onwards, through linkage with the regional cancer registry. A nested case–cohort study was designed to study the relation between endogenous sex steroids and the risk of postmenopausal breast cancer. Until the end of follow-up, 397 breast cancer cases were identified and a random sample of 424 women (the subcohort) was taken from all eligible women. A urine sample could not be found for three cases and three women from the subcohort. Women using hormone replacement therapy or oral contraceptives at the time of urine sampling were excluded (30 cases and 38 women from the subcohort). Urine samples were sent to the International Agency for Research on Cancer (IARC), Lyon, France, for hormonal measurements. One sample was lost during the analyses and was excluded, leaving 364 cases and a subcohort of 382 women for the analyses.Women who were naturally postmenopausal at recruitment  and who had no history of breast cancer were eligible for the present study  were measured by radioimmunoassay (RIA) after enzymatic hydrolysis, solid phase extraction and high-performance liquid chromatography (HPLC) purification of the urine samples. Results were expressed in nanogram analyte per litre. The method used in this study has been described in detail elsewhere  and between the second and the third collection was 4.0 years (1.3–7.7 years). Intraclass correlation coefficients for sample replicates were above 0.93 for all four hormone metabolites, indicating good reproducibility from a laboratory error perspective. The intraclass correlation coefficients over time were 0.59 for E1, 0.62 for E2, 0.85 for TST and 0.55 for 3αD, indicating reasonable stability of women's urinary hormone levels over several years , oestradiol (E2), testosterone (TST) and 5Creatinine was measured in each sample by kinetic Jaffé reaction .To correct for between-subject variability in urine dilution, in each sample the hormone concentration (expressed in ng/l) was divided by the creatinine concentration (expressed in mg/l). All analyses included creatinine-adjusted hormone levels.Concentrations of all four hormone-metabolites were logarithmically transformed to produce approximately normal distributions. Geometric mean values and their 95% confidence intervals were then calculated in cases and in the subcohort.αD, based on the distribution of the creatinine-corrected hormone levels in the subcohort. Women with hormone levels below the detection limit  were included in the lowest quartile. Incidence rate ratios (IRR) were calculated in each quartile compared to the first, using a Cox Proportional Hazards Model with Barlow's weighting method  oestrogen- and androgen levels.Women were classified into quartiles of concentrations of E1, E2, TST and 3To evaluate the effect of latent disease at urine collection on breast cancer risk, we studied the association between levels of hormone concentrations and the risk for breast cancer for those women who were free of breast cancer for at least 2 years after urine sample collection. To evaluate the influence of the time interval between urine collection and the diagnosis, analyses were done separately for women who developed breast cancer within the first 9 years of follow-up and after 10 years. Linear trends were tested by including quartile scores 1, 2, 3 and 4 as a continuous variable in the model.Cox Proportional Hazards models were performed using the SAS macro described by The median follow-up in the subcohort was 19 years (range 0–21 years). Compared to the subcohort, women with breast cancer more often reported a positive family history of breast cancer and were slightly heavier (αD adjusted for family history of breast cancer. Compared to the lowest quartiles of oestrogens (E1 and E2), women with higher levels of these metabolites were found to be at higher risk of developing breast cancer . Tests for trend were statistically significant for both oestrogens.αD had an increased risk of breast cancer , with statistically significant tests for trend. Analysis of hormone levels unadjusted for creatinine, showed similar results, but relative risks were slightly smaller and not all statistically significant anymore.Also, women with higher levels of both testosterone and 3Additional adjustment for BMI did not change the risks associated with any of the hormone metabolites . ExcludiThe present prospective study shows that postmenopausal women with higher excretion levels of oestrogens as well as androgens have an increased risk of breast cancer.n=364) and follow-up was long (median time to diagnosis=9.8 years). The largest prospective study so far published included 156 cases and a median time to diagnosis of 2.6 years  in most cases  and urinary androstanediol  (in vitro studies (For elevated levels of the nonaromatisable androgen 3Since adipose women have a higher conversion from androgens into oestrogens in the adipose tissue, it has been hypothesised that BMI might actually be in the aetiological pathway, rather than act as a confounder. This may be the case for the reproductive risk factors as well . AdjustmIn conclusion, this study, the largest prospective study on endogenous hormones and breast cancer risk so far, shows that women with higher levels of oestrogens and androgens after menopause have an increased risk for breast cancer compared to women with lower concentrations.


Salmo salar).Spinal disorders are a major cause of disability for humans and an important health problem for intensively farmed animals. Experiments have shown that vertebral deformities present a complex but comparable etiology across species. However, the underlying molecular mechanisms involved in bone deformities are still far from understood. To further explicate the mechanisms involved, we have examined the fundamental aspects of bone metabolism and pathogenesis of vertebral fusions in Atlantic salmon (in situ hybridization. At early stage in the fusion process, disorganized and proliferating osteoblasts were prominent at the growth zones of the vertebral body endplates. PCNA positive cells further extended along the rims of fusing vertebral bodies. During the developing pathology, the marked border between the osteoblast growth zones and the chondrocytic areas connected to the arches became less distinct, as proliferating cells and chondrocytes blended through an intermediate zone. This cell proliferation appeared to be closely linked to fusion of opposing arch centra. During the fusion process a metaplastic shift appeared in the arch centra where cells in the intermediate zone between osteoblasts and chondrocytes co-expressed mixed signals of chondrogenic and osteogenic markers. A similar shift also occurred in the notochord where proliferating chordoblasts changed transcription profile from chondrogenic to also include osteogenic marker genes. In progressed fusions, arch centra and intervertebral space mineralized.Experimentally, juvenile salmon were subjected to hyperthermic conditions where more than 28% developed fused vertebral bodies. To characterize the fusion process we analyzed an intermediate and a terminal stage of the pathology by using x-ray, histology, immunohistochemistry, real-time quantitative PCR and Loss of cell integrity through cell proliferation and metaplastic shifts seem to be key events in the fusion process. The fusion process involves molecular regulation and cellular changes similar to those found in mammalian deformities, indicating that salmon is suitable for studying general bone development and to be a comparative model for spinal deformities. Salmo salar) compression (platyspondyly) and/or vertebral fusion (ankylosis) accounts for 9 out of 20 recently described vertebral deformities [The vertebral column is the defining character of vertebrates providing the organism with a unique ability of movement, form and function. Obviously, abnormalities to this organ can lead to severe and often painful pathological conditions. Spinal disorders are a major cause of disability for humans and an important health problem for intensively farmed animals. A number of animal models have been used to further explore the pathology and revealed that vertebral deformities present a complex but comparable cross species etiology ,2. Morphormities . Spinal ormities , patholoSkeletogenesis in salmon involves activity from the three main bone and cartilage cell types; chondrocytes, osteoblasts and osteoclasts. Bone formation further occurs via two basic mechanisms; compact bone of the amphicoel and trabeculae is formed directly through intramembranous ossification, whereas the cartilaginous template is replaced by bone in the arch centra through endochondral ossification. Bone formation is brought about by a complex set of highly regulated molecular pathways, involving extracellular matrix (ECM) constituents , signaling molecules (e.g. hedgehogs and bmps) and transcription factors (reviewed -17). Somin situ hybridization (ISH). We found that loss of cell integrity and ectopic bone formation characterizes the development of spinal fusions. During the fusion process a metaplastic shift appeared in the arch centra where cells in the intermediate zone between osteoblasts and chondrocytes co-expressed mixed signals of chondrogenic and osteogenic markers. A similar shift also occurred in the notochord where proliferating chordoblasts changed transcription profile from chondrogenic to also include osteogenic marker genes. We suggest that hyperthermic induced development of spinal fusions involve a metaplastic shift in cells from the chondrocytic lineage. With this work, we bring forward salmon to be an interesting organism to study development of spinal fusions.During the last decade problems with spinal disorders in salmon have been increasingly in focus due to the importance of this species in the aquaculture industry. To further elucidate the mechanisms involved in the development of vertebral deformities, we analyzed an intermediate and terminal stage of the fusion process at a morphological level by using radiography and histology and gene transcriptional changes using quantitative PCR (qPCR) and The elevated temperature regime used in this study induced mainly vertebral deformities of the fusion type. The incidence of complete fusions was 10.0 (not significant = n.s.), 17.9 (p ≤ 0.001) and 28.1% (p ≤ 0.0001) at 2, 15 and 60 g, respectively  and hypertrophic cartilage ECM production (col10a1) and mineralization . Col2a1 transcription was down-regulated in intermediate while up-regulated in the fused group (n.s.). Osteonectin was up-regulated in both groups. Of genes involved in osteoclast activity, mmp9 showed opposite transcription, being down-regulated in intermediate while up-regulated in fused. Mmp13 and cathepsin K showed similar transcription pattern in the two groups, mmp13 up-regulated and cathepsin K down-regulated (n.s. in fused).Nine out of 11 structural genes had a down-regulated transcription in the intermediate group compared to only five in the fused group Figure . Four geISH analyzes of col1a, col2a, col10a, osteonectin and osteocalcin revealed cells exhibiting characteristics of both osteoblasts and chondrocytes. These findings were more pronounced in fused than intermediate specimens. Col1a was expressed in osteogenic cells along the rims of the vertebral body endplates and in osteoblasts at the lateral surfaces of trabeculae at the intermediate stage  were down-regulated in both intermediate and fused group.All of the regulatory genes Figure  were lesISH, runx2 was never detected in chordocytes, chordoblasts or chondrocytes in non-deformed vertebral bodies. Positive runx2 staining was however detected at the osteoblast growth zone of the vertebral endplate  were upregulated whereas the osteoblast inhibitor (twist) and genes involved in chondrocyte hypertrophy  were downregulated, results also supported by ISH. Deletion of Ihh has been shown to disrupt the normal pattern of various zones of chondrocyte differentiation in the growth plate [runx2 expression, as found in our studies, is further associated with trans-differentiation of chondrocytes into bone cells [col1a, col2a, col10a, osteocalcin and alp) revealed that these transcripts had reduced activity in both intermediate and fused vertebrae. These findings might reflect the reduced radiodensity described in fish reared at elevated temperatures [Several eoblasts  and are eoblasts . Howevereoblasts . Intereseoblasts ,30, a prand col2 . In a reand col2  it was sth plate , whereasth plate . Sustainne cells . On the eratures .cathepsin k had a down-regulated transcription level. In normal developing salmon vertebrae, these areas are modeled through endochondral bone formation, a process requiring invasion of osteoclasts and activity of TRAP, Mmps and Cathepsin K [mmps are up-regulated during IDD [mmp9 and mmp13 were also up-regulated during fusion of vertebral bodies in salmon. Excessive co-activity of mmp9 and mmp13 is linked to development and healing of chronic wounds in rainbow trout [mmp's were up-regulated in fused vertebral bodies as a response to chronic injury rather than bone resorption.To further characterize the pathological bone formation in the chondrocytic areas in the arch centra, we analyzed osteoclast activity. Absence of osteoclasts visualized through TRAP staining was characteristic during the development of vertebral fusions, indicating that normal endochondral ossification was restrained. In addition, hepsin K ,35. Tranring IDD  and compring IDD ,38 in maow trout  and salmow trout . Lack ofOur results suggest that the ossification type during development of spinal fusions and fast growth could be trans-chondroid ossification. A mixed type of intramembraneous and endochondral ossification, as suggested by Yasui et al.  and democol2a. As the pathology progressed, proliferating chordoblasts seemed to occupy most of the intervertebral space and vacuolated chordocytes disappeared. Moreover, cells in the notochord had a transcription profile resembling the trans-differentiating cell at the borders between the osteoblast growth zones and the chondrocytic areas connected to the arches. Transcription of marker genes changed from chondrogenic to also include osteogenic, as mRNA of osteocalcin, runx2, osteonectin and col1a were detected. QPCR further showed up-regulated transcription of both runx2 and sox9 throughout the developing deformity. Comparative to our findings, disc cell proliferation and a switch in the synthesis of ECM components are associated with disc degeneration [ISH revealed that whereas sox9 and col2a was present in chordoblasts from the non-deformed stage, runx2 and col1a was only detected in fused samples, when intervertebral space was severely narrowed. This co-transcription of chondrocytic and osteogenic markers in the notochord supports the hypothesis of a metaplastic shift during vertebral fusions in salmon [The completing step in the fusion process is transformation of notochordal tissue into bone . As inteneration ,42. Howen salmon .The metaplastic shift in the notochord and arch centra may be induced to produce more robust cells, able to withstand increased mechanical load. However, as bone replaced chondrocytic areas throughout the pathology, notochordal tissue did not calcify until the deformity developed into severe fusion. We therefore suggest that metaplasia leads to cell types more suited to the new environment but that changes are related to a threshold of the stimuli, in this case, grade of fusion. A shift in NP cell population coincides with spinal disorders like IDD and changes in the synthesis of matrix molecules differ with the degree of degeneration . A compaVertebral fusions develop through a series of events. Disorganized and proliferating osteoblasts at the growth zones and along the rims of affected vertebral bodies characterized the fusion process. Moreover, loss of cell integrity through cell proliferation was prominent at the border between the osteoblastic growth zone and the chondrocytic areas in the arch centra and in intervertebral space. During the fusion process a metaplastic shift appeared in the arch centra where cells in the intermediate zone between osteoblasts and chondrocytes co-expressed mixed signals of chondrogenic and osteogenic markers. A similar shift also occurred in the notochord where proliferating chordoblasts changed transcription profile from chondrogenic to also include osteogenic marker genes. As the pathology progressed, ectopic bone formation was detected in these areas. Since transcription turned from chondrogenic to osteogenic, our suggestion is that trans-differentiated cells produce the ectopic bone. In complete fusions, all intervertebral tissue was remodeled into bone. The molecular regulation and cellular changes found in salmon vertebral fusions are similar to those found in mammalian deformities, showing that salmon is suitable for studying general bone development and to be a comparative model for spinal deformities. With this work, we bring forward salmon to be an interesting organism to study general pathology of spinal deformities.This trial was performed under the supervision and approval of the veterinarian that has appointed responsibility to approve all fish experiments at the research station in accordance to regulations from the Norwegian authorities regarding the use of animals for research purposes. The experiment was carried out at Nofima Marins research station at Sunndalsøra, Norway, in 2007, as described in Ytteborg et al. . During ISH and histology were fixed in 4% PFA (n = 24) and samples for RNA isolation were snap frozen in liquid nitrogen and stored at -80°C (n = 45).Sampling was directed from radiographs so that the sampled area corresponded to the deformed or normal area. Fish were sedated  and radiographed during the experiment at 2 g, 15 g and 60 g. Fish that were not sampled were put back into oxygenated water to ensure rapid wakening. The x-ray system used was an IMS Giotto mammography system  equipped with a FCR Profect image plate reader and FCR Console . At 15 g size, fish were sampled for histological and gene transcriptional analysis. Samples for All fish were divided into three categories where the first group was non-deformed. These spinal columns had no observable morphological changes in the vertebral bodies or in intervertebral space. We further sampled vertebral areas at two different stages in the pathological development of fusions, termed intermediate and fused. Vertebrae diagnosed as intermediate included various degrees of reduced intervertebral space and compressions. Samples characterized as fused ranged from incomplete fusions to complete fusions.Incidence of fusions were observed through radiography and calculated using a one-way analysis of variance model . Results are represented as means ± standard deviation (st.dev).Statistics for mRNA transcription analysis are described in the real-time PCR chapter.ISH was carried out on five μm Technovit 9100 ®New sections according to the protocol [2O.Histological staining and protocol . Serial 2O in between. Single staining with the two dyes was also performed. All sections were dehydrated in ethanol and mounted with Cytoseal 60  prior to microscopy. To demonstrate osteoclast activity, TRAP was visualized with the Acid phosphatase leukocyte kit No. 387 (Sigma-Aldrich) was applied according to the manufacturer's protocol, with the exception of a 2 h incubation at 37°C. Subsequently, slides were rinsed in dH2O and counterstained with Mayers hematoxylin (Sigma-Aldrich) for 30 s. Cell proliferation and apoptosis were assessed by immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and cleaved Caspase 3, respectively [2O2 in methanol. The sections were washed 3× in PBS and incubated with a mouse anti PCNA monoclonal antibody  or Cleaved Caspase 3 , following the manufacturer's instructions. Slides were washed 3× 5 min in PBS-Tween 20 before counterstained with Mayer's hematoxylin for 2 min, washed in water, dehydrated in a graded series of ethanol solutions, cleared with xylene, and mounted with Cytoseal60 (Electron Microscopy Science). Controls were incubated without substrate. Microscopic analyses were performed by the stereomicroscope Zeiss Axio Observer Z1 using brightfield illumination and digitized images obtained with an AxioCam MRc5 camera using AxioVision software .Bone and cartilage formation in the spinal columns were assayed by Alizarin Red S/Toluidine Blue  staining. Sections were stained for 5 min in Alizarin red (pH 4.2) and for 2 min in 0.1% Toluidine blue (pH 2.3), with a brief rinse in dH ectively -51. SlidPrimers Table  for tran® (Invitrogen). Brief, tissue was homogenized in a mortar with liquid nitrogen and total RNA was extracted using Trizol reagent and Micro to Midi Kit® (Invitrogen) before DNase treatment . The quality of the RNA was assessed spectrophotometrically  1 μg RNA was reverse transcribed to cDNA using oligo(dT) primer and the Taqman Gold RT-PCR kit (Applied Biosystems). The cDNA synthesis was performed with 10 min primer incubation at 25°C, 1 h RT step at 48°C and 5 min RT inactivation at 95°C. All reactions were performed in accordance to the manufacturer's protocol.Tissue homogenization from 15 replicates from each group was achieved in a mortar with liquid nitrogen. RNA was extracted using Trizol reagent and Micro to Midi Kitef1a), we used the standard curve method. Relative target gene mRNA was normalized to relative ef1a mRNA levels for all sample, as recommended by Olsvik et al. [© [Real-time qPCR was conducted using the Light cycler 480 and SYBR Green chemistry  at the following thermal cycling conditions: 95°C for 10 min, followed by 45 cycles at 95°C for 15 s, 60 ± 1°C for 15 s and 72°C for 15 s. Further, specificity was assessed by the melting curves, determined post PCR . To determine the efficiency of target genes and reference gene , using 250 ng of SP6 and T7 tailed PCR fragments as template. escribed , and micp-toluidine salt/nitro blue tetrazolium chloride; Bmp: Bone morphogenetic proteins; Col1a1: collagen type 1a1; Col2a1: collagen type 2a1; Col10a1: collagen type 10a1; Ef1a: Elongation factor 1; Ihh: Indian hedge hog; IVD: intervertebral disk degeneration; IDD: Intervertebral disc disease; ISH: In situ hybridization; Mef2c: myocyte enhancer factor 2c; Mmp: Matrix metalloproteinase; PBS: phosphate-buffered saline; PFA: paraformaldehyde; qPCR: quantitative polymerase chain reaction; Runx2: runt-related transcription factor 2; Shh: Sonic hedge hog; Sox9: (sex determining region Y) box 9; TRAP: Tartrate resistant acid phosphatase.Alp: alkaline phosphatase; BCIP/NBT: 5-bromo-4-chloro-3 indolyl phosphate ISH experiments, sequence alignment and probe design, microscopic analyzes and acquisition and interpretation of data. GB carried out the radiological diagnostics, accompanied in statistical analysis and participated in the experimental design. HT conceived the study and its experimental and molecular design, coordinated the sampling, and participated in acquisition and interpretation of data and in the drafting of the manuscript. All authors read and approved the final manuscript.EY participated in sampling, carried out the molecular studies, microscopic analyzes and acquisition and interpretation of data and drafted the manuscript. JT participated in the design of the Sense probes. No staining was detected for ISH with sense probes. nc, notochord; ns, notochordal sheath, eb, endbone; tb, trabecular bone. Scale bar = 100 μm.Click here for file


MicroRNAs (miRs) are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE) tissue.vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p < 0.001), when examining all miRs, regardless of RNA extraction method used. Examining correlation coefficients between FFPE and fresh-frozen samples in terms of miR abundance reveals correlation coefficients of up to 0.32 (low abundance), 0.70 (medium abundance) and up to 0.97 (high abundance).Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access) platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility  and outlines the optimal performance conditions of this platform using clinical FFPE samples. We also outline a method of data analysis looking at differences in miR abundance between FFPE and fresh-frozen samples. By dividing the profiled miR into abundance strata of high (Ct<30), medium (30≤Ct≤35), and low (Ct>35), we show that reproducibility between technical replicates, equivalent dilutions, and FFPE Our study thus demonstrates the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies. MicroRNAs (miRs) are small, non-coding RNA molecules of 17-27 nucleotides in length, involved in gene regulation at the post-transcriptional level . TherefoAnatomical pathology laboratories worldwide contain a vast stock of samples that can potentially be used for analysis of disease states. These are in the form of formalin-fixed, paraffin-embedded (FFPE) samples that are stored for up to 20 years and possibly longer depending on professional or governmental guidelines. Given the length of the storage period for these samples, extensive retrospective analyses with significant periods of clinicopathological follow-up for patient studies can be carried out.Embedding of samples in paraffin after formalin fixation is a standard of practice . This poDespite these challenges, extraction of miRs from FFPE tissue is possible, as the small size contributes to their stability during fixation and processing . miRs maRNA from a FFPE benign, reactive lymph node was reverse transcribed and the resultant cDNA applied to the TaqMan array. Varying RNA concentrations  were used to assess the sensitivity of the TaqMan array platform to detect small quantities of RNA. All reverse transcription reaction products were diluted 15× prior to adding TaqMan 2× Universal PCR Master Mix, No AmpErase UNG and undergoing qRT-PCR. Duplicate plates were run to examine the reproducibility of the method. Ct values were determined for each individual miR for each sample. The Pearson correlation coefficients for each pair of samples were: 0.92 (10 and 25 ng/μL), 0.94 (50 and 100 ng/μL) and 0.95 (200 ng/μL). cDNA dilution factors in this analysis were kept constant at 15× for each plate.Mixed model regression analysis was applied in order to adjust for repeated measures of the same sample and potential dilution effects. Using this analysis, the adjusted Pearson correlation coefficient was 0.95 (p < 0.00001). These results showed that RNA from FFPE tissues can be reliably amplified by qRT-PCR, and the quantification measures are reproducible between duplicates. The correlation coefficients obtained between duplicate pairs of input RNA amounts were high, ranging from 0.92 to 0.95, increasing with higher RNA concentrations.In qRT-PCR, accurate and reproducible data are dependent on the Ct value. Low Ct values indicate the presence of higher template abundance, and are usually associated with a higher reproducibility and lower variability . We thusBoxplots outlining the summary statistics for the difference in Ct values between duplicate plates at each input RNA concentration were developed to determine which input RNA concentration has the least variation between duplicate plates Figure . From thIn order to assess the amplification efficiency when using this TaqMan array platform, we counted the number of wells that did not amplify in the plates (defined as well failures). Since the qRT-PCR reactions were set up to run for 40 cycles, a well was marked as a "failure" when, for the same miR, a Ct = 40 was recorded for one plate and a Ct < 40 was recorded for the duplicate. When both wells had a Ct = 40, Ct values for that miR were compared to the Ct values obtained at other concentrations, for the same sample, and also compared to the positive control, Universal RNA. If Ct values < 40 were obtained at other concentrations or on the plates containing Universal RNA for that miR, both wells were deemed to be "failures". The total number of well failures were assessed, for each plate, at each RNA concentration, and averaged among duplicate plates. The percentage of well failures was calculated as follows: Average percentage of well failures = Average well failure between duplicate plates/Number of miRs expressed*100 , 30×, and 15×, respectively. Well failures were assessed as previously outlined. Since we observed high correlation coefficients in the previous experiments, demonstrating robustness of the platform, only one plate per cDNA dilution was run to assess well failure. In this case, we define a well failure when a Ct value of 40 was obtained for any given miR known to be expressed in that same sample.Our analysis showed that the percentage of well failures increases with cDNA dilution, and well failures are higher with lower input RNA concentration at the same cDNA dilution factor  and the percentage of well failures (assessed as described earlier) between the samples was similar, ranging from 4-7% .2 = 3.3, df = 6, p = 0.77)  are seen when comparing FFPE to fresh-frozen samples, regardless of extraction method used, and a correlation of 0.92 (p < 0.0001) is seen on comparison of fresh-frozen tissues extracted with different methods. A comparison of the summary statistics using mean Ct values obtained for each extraction method between tumour and control samples was also performed  for tumours and normal samples, taking into account the extraction method used. Figures Unsupervised hierarchical clustering analysis was performed ; mirVana and FFPE ], but not between TRIzol and mirVana . This difference may be due to reduced RNA yields in FFPE samples, which is known to occur and has been reported in the literature . In our laboratory, we have optimized the experiments to use an input RNA concentration of 200 ng/μL with a 15× cDNA dilution factor. This gives us the flexibility to use input RNA concentrations as low as 60 ng/μL with a 5× cDNA dilution factor, which is the lowest cDNA dilution possible according to the manufacturer's protocol, in order to maintain equivalent miR profiles between samples.Well failures are also less common as input RNA concentrations increase or cDNA dilutions decrease. Interestingly, dilution of cDNA can be used in order to compensate for low input RNA amounts, a scenario which can occur should RNA yield be low. This can be observed when comparing an input RNA concentration of 200 ng/μL with a 15× cDNA dilution factor and an input RNA concentration of 66.7 ng/μL with a 5× cDNA dilution factor. Our results show the consistency of Ct values obtained with different cDNA dilution factors (Pearson correlation 0.95). This was also shown to be true when examining correlation coefficients relative to miR abundance. There was also no statistically significant shift in the proportion of miRs in each abundance stratum between equivalent combinations of input RNA concentration and cDNA dilution  is a 384-well microfluidics card containing 365 primer-probe sets for individual miRs as well as duplicates of 3 primer-probe sets for endogenous small nucleolar RNAs and duplicate negative control blank wells. Reverse transcription reaction products were diluted  prior to adding an equivalent volume of TaqMan 2× Universal PCR Master Mix, No AmpErase UNG. A minimum of 5× cDNA dilution is required to obtain the necessary volume for application to the TaqMan Human MicroRNA Array. The entire mixture was then added to individual ports of the TaqMan Human MicroRNA Array v1.0 (Early Access); the array was centrifuged at 1,200 rpm twice for 1 minute each, and then run on an Applied Biosystems 7900HT Fast Real-Time PCR system for 40 cycles. Raw microRNA data (GEO series record GSE22264) are available for download at ©2009 SAS Institute Inc. SAS and all other SAS Institute Inc. product or service names are registered trademarks or trademarks of SAS Institute Inc., Cary, NC, USA [® software, version 6.4 Copyright © 2009 Partek Inc., St. Louis, MO, USA [Mixed model regression analysis was used to adjust for repeated measures of the same sample and the potential dilution effects. This, as well as bar plots, were generated using SAS software, Version 9.1 of the SAS System, Copyright  NC, USA . 3-D pri MO, USA .All other statistical analysis and plotting were performed using the R statistical environment  , on the The authors declare that they have no competing interests.RSG designed the study, helped collect and extract samples, ran the TLDA assays, helped with data analysis and drafted the manuscript. LW performed the data analysis and helped draft the manuscript. JM and NKC helped with sample extraction and data analysis. WX performed statistical analysis and helped draft the manuscript. PPR helped with study design, data interpretation and helped draft the manuscript. DJB contributed with sample collection and pathological diagnosis. IJ and MRC helped coordinate the study. SKR helped design and supervised the study and gave final approval of the manuscript. All authors read and approved the manuscript.Figure S1. Absolute value of the difference between duplicate measurements as a function of the duplicate mean for all input RNA concentrations. Measurements where at least one of the duplicate Ct values equals 40 are removed. The red line shows a cubic spline fit using default settings for the smooth spline function in the R statistics package . The apparent decrease in variability for mean Ct>37 is an artifact caused by the absence of Ct values greater or equal to 40.Click here for fileFigure S2. A) Graph of average percentage of well failures according to input RNA concentrations. Note that cDNA dilution factors were kept constant (15×). B) Percentage of well failures as they relate to input RNA concentration and cDNA dilution factor. cDNA dilution factors are shown on the x-axis and percentage of well failures are shown on the y-axis.Click here for fileTable S1. Table of Pearson correlation coefficients between equivalent input RNA concentration/cDNA dilution factor combinations .Click here for fileTable S2. Table depicting number of miRs in each abundance stratum for equivalent dilutions. No significant difference exists in the abundance distributions of the columns by chi-square analysis .Click here for file


Electrospinning is a non-mechanical processing strategy that can be used to process a variety of native and synthetic polymers into highly porous materials composed of nano-scale to micron-scale diameter fibers. By nature, electrospun materials exhibit an extensive surface area and highly interconnected pore spaces. In this study we adopted a biological engineering approach to ask how the specific unique advantages of the electrospinning process might be exploited to produce a new class of research/diagnostic tools.The electrospinning properties of nitrocellulose, charged nylon and blends of these materials are characterized.Nitrocellulose electrospun from a starting concentration of < 110 mg/ml acetone deposited as 4–8 μm diameter beads; at 110 mg/ml-to-140 mg/ml starting concentrations, this polymer deposited as 100–4000 nm diameter fibers. Nylon formed fibers when electrospun from 60–140 mg/ml HFIP, fibers ranged from 120 nm-6000 nm in diameter. Electrospun nitrocellulose exhibited superior protein retention and increased sensitivity in slot blot experiments with respect to the parent nitrocellulose material. Western immunoblot experiments using fibronectin as a model protein demonstrated that electrospun nylon exhibits increased protein binding and increased dynamic range in the chemiluminescence detection of antigens than sheets of the parent starting material. Composites of electrospun nitrocellulose and electrospun nylon exhibit high protein binding activity and provide increased sensitivity for the immuno-detection of antigens.The flexibility afforded by electrospinning process makes it possible to tailor blotting membranes to specific applications. Electrospinning has a variety of potential applications in the clinical diagnostic field of use. The art and technology of electrospinning has generated considerable interest in the field of tissue engineering. Studies describing various aspects and applications of the electrospinning process and patent filings for intellectual property concerning this rapidly evolving technology have undergone a remarkable expansion from 1995 to 2007. Relevant to the biological sciences and the tissue engineering fields, this technology can be used to process a variety of native  and syntThe physical, biochemical, and biological properties of electrospun materials can be regulated at several sites in the production process. For many polymers, physical properties, including fiber diameter, fiber alignment and pore dimension ,9, can bThe flexibility inherent to the electrospinning process makes this fabrication strategy adaptive to a variety of different fields of use. Notably, in biological applications, electrospinning shows great potential as a gateway to the development and fabrication of physiologically relevant tissue engineering scaffolds ,12, hemoi.e. Western blot) and/or nucleic acid detection (i.e. Northern blot and Southern blot). In conventional protein and nucleic acid blotting experiments, a charged sheet of nitrocellulose or nylon is used as a solid phase support [Electrospun materials, by nature, exhibit an extensive surface area. The sequential deposition of the discreet, individual fibers that are formed in this process also results in a unique and complex interconnected network of pores. In this study we report that it is possible to exploit these characteristic to fabricate solid phase platforms designed for protein  unless noted. Nitrocellulose  was suspended and agitated for 24 hr at varying concentrations in acetone. Charged nylon  was suspended and agitated for 24 hr in 1,1,1,3,3,3-hexaxafluoro-2-propanol (HFIP). A Brookfield RVDV-III Ultra programmable rheometer was used to measure solution viscosity.Nitrocellulose was dissolved at various concentrations  in acetone under agitation for 24 hr. Electrospinning suspensions were loaded into a 20 ml Becton Davis syringe capped with an 18 gauge blunt tipped needle. The negative lead of a high voltage supply  was attached by an alligator clip to the blunt tipped needle. This polarity was found to reduce drying of the acetone/nitrocellulose solution at the tip of the syringe and functioned to stabilize the Taylor cone. A 22 kV accelerating voltage was used in the electrospinning process. A Harvard perfusion pump was used to meter the delivery of the nitrocellulose solution to the electric field, the rate of delivery was set to the maximal rate that did not induce dripping from the tip of the syringe or the introduction of solvent defects in the resulting membrane .Charged nylon was dissolved at various concentrations 60, 80, 100, 120 mg/ml in HFIP for 24 hr. Suspensions were electrospun from a 20 ml Becton Davis syringe capped with an 18 gauge blunt tipped needle. The negative lead was attached to the syringe and charged to 25 kV. The air gap distance between the source suspension and the grounded mandrel was set to 20 cm. A Harvard perfusion was used as described, the rate of solvent/polymer delivery was set at the maximal rate that did not induce dripping from the tip of the syringe (10–14 ml/hr). For all solutions, a stainless steel cylindrical mandrel (10 cm × 4.0 cm) was used as a grounded target. Each finished sheet had approximate dimensions of 15 cm × 4.0 cm.Membranes were sputter coated and imaged with a Zeiss EVO 50 XVP scanning electron microscope equipped with digital image acquisition. Average fiber diameter was determined from representative samples using NIH ImageTool (UTHSCSA version 3). All measurements were taken perpendicular to the long axis of electrospun fibers. Measurements were calibrated from size bars incorporated into the SEM images at the time of capture. NIH Image J software was used to conduct fiber measurements ,9.post hoc analysis of these data sets. Statistical significance was determined at P < 0.05.Fiber diameter data sets were screened by One-way ANOVA to determine the effects of starting conditions on fiber diameter. A Tukey test was used in the Slot blot experiments were conducted to characterize the performance of electrospun materials. Sheets of commercially available nitrocellulose and nitrocellulose electrospun from this parent material were wet in transfer solution and mounted into a slot blot apparatus . Each well was rinsed 3× in transfer buffer. A series of serial dilutions of human fibronectin (Fn) was prepared in PBS and added sequentially to the blotting wells . After 5 minutes, the Fn solution was drawn through the blot apparatus as per slot blot manufacture's directions. The wells were each rinsed and blocked 3× with 250 μl of PBS supplemented with 1% BSA plus 0.1% Tween 20 (subsequently referred to as blocking buffer). Control lanes were incubated with blocking buffer for 5 minutes and rinsed as described.Primary antibody (Sigma F3648) against human Fn was diluted 1:1000 in blocking buffer and applied to each lane and allowed to incubate for 5 minutes. At the conclusion of this incubation lanes were rinsed in blocking buffer 3×. Secondary antibodies  tagged with HRP were prepared at a 1:10,000 dilution in blocking buffer and allowed to incubate for 5 minutes, the wells were re-rinsed 5× with 250 μl of blocking buffer. Samples were removed from the blot apparatus, rinsed in PBS and processed for chemiluminescence detection as per the manufacture's instructions . Images were captured on Kodak Blue XB-1 film (Kodak). Gel images were captured with a BioRad Gel Doc 2000 system, relative optical density calculated from digital images with BioRad Gel Doc 2000 software.Serial dilutions of human Fn  were prepared in Laemmli buffer and separated by SDS gel electrophoresis . Separated samples were transferred overnight  onto blotting membranes. Benchmark MW Standards were used in these experiments .Membranes were blocked in 5.0% non-fat milk prepared in PBS plus 0.1% Tween 20 for 30 minutes at room temperature (this formulation referred to as Western blocking buffer). Primary antibody (Sigma F3648) against human Fn was diluted 1:1000 in Western blocking buffer and applied to the membranes for 1 hr at room temperature. Membranes were rinsed 30 minutes in Western Blocking buffer under agitation using 4–5× complete changes of buffer. Secondary antibodies  were prepared at a 1:10,000 dilution in Western blocking buffer and allowed to incubate for 1 hr at room temperature. Membranes were once again rinsed 4–5× in fresh Western blocking buffer. Samples were rinsed in PBS and then processed for chemiluminescence as described for slot blotting. Images were captured on Kodak Blue XB-1 film (Kodak).We examined the structural characteristics of electrospun nitrocellulose as a function of starting conditions. Samples electrospun from the 60, 80, and 100 mg/ml solutions were very similar in nature. At 60 mg/ml the bulk of the material deposited as 4–8 μm diameter beads, at 80 mg/ml foci of small diameter fibers were observed interspersed with these beads . Plotting average fiber diameter as a function of starting solution viscosity and conducting the regression analysis with a 1st order equation generated an R2 = 0.437   and used as a backing material for both constructs. Next, nitrocellulose was electrospun onto the nylon backing from a starting concentration of 110 mg/ml (average fiber diameter 398 nm) or 60 mg/ml (4–8 μm diameter beads). Representative SEM images of these composites are illustrated in Figure The nylon/nitrocellulose fiber composite exhibited high signal detection but, provided low band resolution Figure . As withThis study demonstrates the feasibility of using electrospinning to process nitrocellulose and nylon-based materials into unique membranes designed for Western (Northern and Southern) Blotting applications. We were able to generate a variety of physical states for these materials by manipulating the starting concentrations of the electrospinning solutions. For example, at low starting concentrations, nitrocellulose underwent electrospraying and deposited as 4–8 μm diameter beads, at higher starting concentrations, this polymer formed discreet sub-micron-to-micron diameter fibers. Charged nylon formed fibers over a wide range of starting concentrations, although bead formation can undoubtedly be induced by driving the initial source solution concentration below the electrospinning threshold.For nitrocellulose, changes in average fiber diameter were most closely associated with the changes in the starting concentrations of the polymer used at the onset of electrospinning Figure  and 2A. st order equation engineer the physical properties and performance characteristics of nitrocellulose and charged nylon in Western blot applications. The electrospinning process imparted gross physical features that provided an extensive surface area for protein binding and a highly interconnected pore space that made these materials readily permeable to staining and wash solutions. There may be added, and entirely un-expected, advantages provided by the nano-structure of these electrospun materials. A protein bound to a fibril of electrospun nitrocellulose or electrospun nylon may adopt a very different conformation than a protein that has been immobilized to the surface of the parent starting materials.Similar to scaffolds designed for tissue engineering, a variety of electrospinning and post-processing techniques might be applied to our system to further modulate and customize membrane performance. For example, membranes composed of polymer fibers of varying diameters and/or varying compositions can be prepared by simultaneously electrospinning from separate source solutions. In this type of construct the small diameter fibers might be used to more effectively capture small molecular weight materials, domains with larger diameter fibers can be used to capture large molecular weight materials. In more exotic applications, for example in the analysis of proteases, it may be possible to incorporate a small concentration of a specific protein, such as collagen, into an electrospun blotting platform. A sample of interest could be separated by SDS gel electrophoresis and then transferred onto the hybrid platform. Theoretically, the bound proteases would attack and degrade the incorporated protein substrate. Upon staining, much like a zymogram, the sites where active proteases were bound would appear as a clear lytic band. As an added advantage, this type of electrospun membrane could be subsequently processed for Western blot to verify enzyme identity and/or to measure enzyme content. In the clinical arena, the flexibility afforded by the electrospinning process could be exploited to produce diagnostic or research grade materials targeted to broad classes of patients, or even to specific individuals.BSA: Bovine Serum AlbuminFn: FibronectinHFIP: 1,1,1,3,3,3-hexaxafluoro-2-propanolHr: hournm: nanometerPBS: Phosphate Buffered SalineSEM: scanning electron microscopyμm: micronThe authors, Manis, Bowlin, Bowman and Simpson have U.S. and International Patents Issued and Pending concerning aspects of the electrospinning process and the fabrication of blotting platforms.The 4 authors of this manuscript each contributed equally to the work described in this manuscript, each has read and approved the final publication. AEM, GLB and DGS conducted electrospinning experiments, JRB conducted Western and Slot blotting experiments. Image analysis and viscosity measurements by DGS and AEM.


The transformation event is characterized by a dramatic cellular deformation process which includes: condensation of certain cellular materials into a much smaller tubular structure, synthesis of a tubular wall and growth of filamentous extensions. This study continues the characterization of straw cells in blood, as well as the mechanisms of tubular transformation in response to stress; with specific emphasis placed on investigating whether tubular transformation shares the same signaling pathway as apoptosis.Mammalian cells transform into individual tubular straw cells naturally in tissues and in response to desiccation related stress 3/hr), respectively. Studies using apoptosis inhibitors and a tubular transformation inhibitor in CACO2 cells and in mice suggested apoptosis produced apoptotic bodies are mediated differently than tubular transformation produced straw cells. A single dose of 0.01 mg/kg/day of p38 MAPK inhibitor in wild type mice results in a 30% reduction in the SBC. In 9 domestic animals SBC appears to correlate inversely with an animal's average lifespan (R2 = 0.7).There are approximately 100 billion, unconventional, tubular straw cells in human blood at any given time. The straw blood cell count (SBC) is 45 million/ml, which accounts for 6.9% of the bloods dry weight. Straw cells originating from the lungs, liver and lymphocytes have varying nodules, hairiness and dimensions. Lipid profiling reveals severe disruption of the plasma membrane in CACO cells during transformation. The growth rates for the elongation of filaments and enlargement of rabbit straw cells is 0.6~1.1 (μm/hr) and 3.8 . Tubular transformation is a programmed cell survival process that diverges from apoptosis. SBCs may be an important indicator of intrinsic aging-related stress. We have observed mammalian cells, in a variety of tissues, transform into individual tubular straw cells in response to desiccation related stress and also occur naturally in live blood and tissues . The traCertain features of the tubular transformation resemble events in apoptosis (the process of programmed cell death that has been well documented -4). The Caspases constitute a large protein family that is highly conserved among multicellular organisms. They are constitutively expressed in most cell types as inactive zymogens that are proteolytically processed before they gain full activity. Because caspases exist as zymogens, their activity is thought to be regulated primarily post-translationally. Inhibition of apoptosis to treat diseases has shown some success -11 usingThe production of straw cells from diseased or stressed tissues could conceivably be associated with loss of organ function. The accumulation of a filamentous network of straw cells could also potentially interfere with aspects of the circulatory system. Therefore we undertook a study to more fully characterize straw cells in blood, as well as the mechanisms of tubular transformation in response to stress. Specifically, we wanted to investigate if tubular transformation shares the same signaling pathway as apoptosis. More importantly, the unknown regulation and the number of molecular players in the tubular transformation signaling pathway provides the potential for subsequent development of therapeutics to modulate a variety of human diseases. In fact, we have tested leading small molecule candidates in a screening program to inhibit this type of transformation in both cell lines and in mouse models to help shed light on possible approaches.At least five different types of straw cells can be grouped according to their size and morphology. These types include: large straw cells Figure , middle,6/ml. In human adult male blood, we calculated a SBC of (4.5 ± 1.5) × 107/ml  × 10.4 × 106/ml Table . TherefoStraw cells have different properties than the rest of the cells in blood. These cells are extremely hydrophilic and stay intact in a hypotonic buffer. They have filamentous extensions that are often covered with numerous microscopic hairs. Depending on their size and the degree of filamentous extensions, they can be separated from the blood matrix by differential centrifugation and filtration. Straw blood cells from chickens, dogs, rats, and cattle have a large amount of filaments compared to those from sheep, pigs, and horses. Straw cells from humans, mice, and rabbits are the least filamentous. When centrifuged, the hydrophilic interaction with surrounding water keeps straw cells floating at 10,000 rpm. Naked tubular centers (straw cells without filaments) precipitate to the bottom at 5,000 rpm . The amount of straw cells in blood by weight was estimated for 9 mammals and one bird  and vortexed vigorously typically lost their fragile filamentous extensions. When viewed under light microscopy these straw cells contained only the tubular center with dimensions just less than 1 μm Figure . Straw c3/hr over five days. In two weeks, spherical shaped cells derived from synchronized SBCs without extensions were observed to occupy the bottom of the 4-well chambers  showed a filament elongation rate of 0.6 μm/hr for the floating straw cells and an elongation rate of 1.1 μm/hr for those straw cells that were attached to the bottom of the plate Figure . The enls Figure , and 1M.Minute tubes can be seen aggregating around places where synthesis of new filaments may be happening Figure . In the In vitro, transformed straw cells in solution have a typical apoptosis phenotype in the early stage with the observed membrane blebbing  and FA 18:0 (283.26 [M-H]-1) which were absent from the outer leaflet in controls, were detected on the transforming cell's surface. However, changes of phosphatidylserine species, such as PS 36:1 (811.534 m/z), were inconclusive between treatment and controls.The exposure of phosphatidylserine (PS) on the cell surface is a general marker of apoptotic cells . Cell suin vitro/vivo either alone or in combinations with other apoptosis inhibitors. At 2 mg/kg body weight, it had no inhibitory activity against tubular transformation and the number of straw cells in vivo was not changed compared to controls  no DNA ladder was observed Figure , right. m Figure , Table 22 = 0.7  SBCs are found to correlate with the animal's average lifespan in an inverse relationship with the coefficient of correlation R.7 Table . Animalsin vivo and 10 ppm in vitro, of a pyridinyl imidazole (SB 202190), which inhibits an inducible P38 MAPK, reduced mice SBC by 30% (p = 0.003) in double blind, triplicate experiments with both the test and control using over 20 individual mice. The effects of treatment by three small molecular inhibitors together with controls on SBC are given in Figure P38 MAPK inhibitor interrupts an environmental stress signal from the cell surface to the nucleus via inhibition of P38 kinase activity. In addition to its promising anti-inflammatory usage , we founazole SB 02190, whde novo peptide sequencing using a data-dependent acquisition method from digestions of samples with trypsin and chymotrypsin as well as peptide mapping using tryptic peptides and high stringency scores . Using these methods we have tentatively identified a match to a human 57 KD protein known to be a component of the walls of ciliary and flagellar microtubules . DuringMammalian blood contains three visible layers when centrifuged. The plasma forms at the top, accounting for ~55% of the volume, then a thin cream-colored layer forms below the plasma consisting of white blood cells and platelets, followed by a final layer consisting of red blood cells, forming the heavy bottom portion of the separated mixture, which accounts for ~45% of the volume . We founWe compared two methods in straw cell counting: (1) use a Hemacytometer and count the number of dark dots around 1 μm in a defined volume in solution without any distortion to the blood samples and (2) count tubular straw cells from 1 μ of dried blood droplet on cover glass slide directly. We found the numbers derived from two different methods (in solution vs. in air) are in surprisingly good agreement to each other. This agreement suggests that no artifacts were introduced in the counting process and SBCs reflect their actual blood content.Straw cells occur naturally and at a constant rate in the humans and animals we tested. SBC from nine domestic animals correlates directly to the animal's average expected life span, suggesting a simple connection between tubular transformation and the amount of cells needed to sustain life. In the animals tested, there was an inverse relationship between SBC and the average lifespan of those animals Figure . HoweverIn vitro, straw cells originating from both normal cells and cancer cells germinate and grow back into vegetative cells within a few days to a few weeks. In general, after the remission of the original cancer, the recurrence of tumors in cancer patients frequently occurs within 2~3 years after initial treatments[Straw cells may be involved in a variety of human aging-related diseases; for example, invasive malignant tumors contain no distinct boundary between healthy tissue and irregularly shaped cancer cells. Current cancer treatments such as chemotherapy and radiotherapy enhance tumor apoptosis, causing these cancerous cells to transform into apoptotic bodies -23. The reatments,27.in vivo.We hypothesize that vast numbers of free circulating cancerous straw cells in blood are capable of regenerating back into actively growing tumor cells at new locations. These circulating straw cells provide a possible mechanism for the establishment of new sites in the body for tumorgenesis. The possibility for straw cells to colonize and grow into tumors may be very small, depending upon the availability of nutrients, DNA mutations, and interactions with the host immune system. However, due to the large number of straw cells present within the blood circulatory system, metastasis could still very well be mediated through straw cells that are produced from increasing physiochemical stresses within a fast growing tumor mass. The straw cells robust cell wall  may alloSince apoptosis was coined in the 1950s, it is believed that programmed cell death is important in keeping cell homeostasis, organ development, and maintaining proper cell population. Although diseases are attributed to abnormal cell death, targeting cancer cells with promoters of apoptosis is an active route of treatment. It is possible that current approaches to cancer treatment involving chemotherapy may contribute to metastasis. Although numerous chemotherapy treatments have been implemented  and they have saved many lives, the delay of a major breakthrough over controlling the disease may be due to the misleading idea that cells die through apoptosis. Where in fact, a portion of the targeted cells enters into a straw cell survival state through "apoptosis" associated stresses to survive and propagate.We counted the number of straw cells derived from lymphocytes (T and/or B cells) in human blood with characteristic short filamentous extensions to be consistent with the number of lymphocytes that die per day through apoptosis (~50 billion). The SBC in humans is around 45,000/μl, 40% of which are smaller straw cells characteristic of lymphocytes. With a half-life of 20 hr in blood circulation, an estimated 100 billion of all cells are transformed into straw cells every day. Injection of 0.3 μg subcutaneously of a specific protein kinase inhibitor to a 30-gram mouse  resulted in a 30% reduction in SBC in the tail artery over 48 hrs. It is unclear if a 30% SBC reduction in mice could be translated to Humans; more studies are needed to make the distinction. The important questions that remain to be answered are: (1) Characterization of the upstream regulatory pathways that promote straw cell formation, 2) Can treatments associated with straw cell reduction significantly improves overall health and longevity? 3) Can straw cell treatment reduction also be an effective way to reduce the spread of tumors in conjunction with other types of treatments?in vitro and 0.01 mg/kg BW/day in vivo by inhibiting a stress inducible protein kinase, which may be an early event in the signal transduction.We have identified one protein involved in the biogenesis of straw cells from the cleavage of purified tubular extensions. This protein can then be targeted for straw cell inhibition, for example, pyridinyl imidazole inhibits the tubular transformation at 10 ppm Mammalian cells transform into individual tubular straw cells naturally in live blood and tissues in animals and humans, and also in response to increased stresses in CACO2 cells. The transformation event in CACO2 cell is characterized by a dramatic cellular deformation process which includes: nuclei fragmentation and packaging of chromatin into a much smaller tubular structure, synthesis of a tubular wall, growth of filamentous extensions, and the interconnection of tubes to form a tubular network. This tubular transformation occurs constantly and ubiquitously in every tissue we examined, suggesting that this "distinctive trait" involves multiple conserved pathways. Certain features of the tubular transformation resemble events in apoptosis suggesting straw cells may have been studied for decades as products of programmed cell death while in fact they are the products of "programmed cell survival". SBCs may be an important indicator of intrinsic aging-related stress.Pyridinyl imidazole (SB 202190) was purchased from Sigma  Ceramide antagonist, sphingosine 1-phosphate was purchased from Fisher Scientific . Caspase inhibitor, Q-VD-OPh was purchased from MP biomedical . DNA ladder assay kit was purchased from Roche-Applied-Science .2 incubator at 37°C. Chemicals used were from Sigma-Aldrich Company .Blood samples were drawn from young and healthy animals. Female wild type mice, Balb/c, Swiss Webster, between 31–34 g at 120-day-old were purchased from Harlan , Sprague-Dawley rat weighing between 300 – 350 grams at 5.5-month-old were purchased from Harlan . SC). Fresh blood samples from Chicken , Sheep , Quarter Horse  were collected from animal farm via Godley-Snell Research Center , Pig  and Cattle  from the meat processing lab , Dog  from Clemson animal hospital , human  from Oconee memorial hospital . Rabbit sera were prepared by Cocalica Biologicals, Inc. . Bovine liver was purchased from Bi-Lo grocery store . All blood samples were from healthy animals and humans. Cell culturing used DEME medium supplemented with 20% fetal bovine serum  in culture flask in a CODetermining SBC used both an in-solution and an in-air method. In solution, a Hemacytometer  was used to determine SBC straw cells; visible dark dots with diameters around 1 μm were counted and five random squares were used to average the counts. In the in-air method, 1 μl of dried aliquot placed on a glass slide was used to estimate the SBC count. The numbers from both counts were expressed in SBC/ml. They typically agree with each other within a 10% variation. Fresh mouse and human blood samples, along with ten domestic animals were collected and their subsequent SBC straw cells were immediately determined. Blood samples (264) from 88 mice were counted SBC and averaged. Blood samples from the human frontal arm peripheral veins were used. For other domestic animals, multiple samples  were analyzed using 1 μl of blood sample as is, or diluted in 1:10 distilled water to avoid red blood cells in the field. Duplicate readings from each blood sample were averaged and reported.4OH was added to a final 1% solution. For total lipids, cells at room temperature (22°C) were treated with chloroform: methanol: H2O = 1:1:1 (200 μl), sonicated and centrifuged. The bottom chloroform phase was collected, combined, and concentrated down to 50 μl. NH4OH was added to make a final 1% solution. Acquisition of lipid species used 1 μl sample injected directly to ESI-MS in negative ion mode with a mass scanning range from 200 – 1200 m/z.Lipid profiling by ESI mass spectrometry in negative ion mode uses published protocol  Nano-eleBlood and purified SBC straw cells  were estimated for total dry weight by drying out each sample. One ml of sample was dried under a vacuum concentrator  until a constant weight was reached. Percentage of straw cell dry weight was estimated by both extraction and filtration methods. First, the sample was centrifuged at 13,000 rpm for 3 min and the supernatant was vortexed twice at 1 min each to break the filaments from the tubular center. The resulting straw cells were centrifuged at 13,000 rpm for 3 min again. Pellets were washed and combined with blood that was extracted twice, using a 1:1:1 solution of chloroform: methanol: water. Next, the top water layer containing straw cells was passed through a 100 KD microconcentrator  at 5000 rpm for 40 min, twice. Finally, the retained fraction was then loaded on a C18 spin column  to remove remaining soluble material.n. Theoretically, the amount of soluble material retained equals 0.02% of starting material when n = 6, which becomes negligible when calculating the dry weight. The n = 8 retained fraction was dried to completion and washed in 80%, cold ethanol (4°C) twice to remove remaining proteins. The resulting dry matter, calibrated by OD 280 nm for protein content was used to estimate the straw cell content. Most rabbit straw cells flow through 0.1 μm membrane .In the filtration method, straw cells were purified using a nitrocellulose membrane. First, whole blood aliquots of 0.5 ml were diluted to 2 ml in distilled water to lyse the regular cells. The sample was then centrifuged to remove cell debris. When blood samples were centrifuged at 10,000 rpm (9530 g) for 3 min, the filamentous tubes moved to the bottom and separated from the naked tubes on top. The tubes without extensions were the densest cellular material, pelleting to the bottom at 5,000 rpm. The aggregated pellet was then washed three times with distilled water. The resulting supernatant, containing straw cells, was combined with previous straw cell fractions and placed in a syringe and pushed through a 0.025 μm nitrocellulose membrane filter . The amount of soluble material remaining in each passage is governed by (1/4)3/hr. The experiments were repeated three times. The half-life of straw cells is defined as the loss of the filamentous structure in vitro incubated at 37°C.Straw cells were synchronized by removing filaments from the tubular center when vortexed in 30% methanol. The straw cells without extensions were collected and placed in 4-well chambers and incubated in 0.1× medium at 37°C. Measurement of newly elongated filaments under light microscope was calculated from ten randomly selected straw cells and measured length in 24 hr intervals over 72 hrs. The results were averaged by total number of hours to give the growth rate in μm/hr. Growth of CACO2 straw cells to regular spherical cells used a synchronized tubular center and measured cell diameters in a 5-day time frame. The results were averaged by the total number of hours to give the growth rate in μmCompounds mostly insoluble in water were dissolved in medium with help of DMSO, palmitic acid and phosphotidyl choline (PC 36:0) through lipid vesicle hydration methodology ,30. SoniInjection vehicles were prepared using protocols from the literature ,31. TestEach blood drawing sample containing 5 μl was immediately placed in a collection tube coated with anti-coagulant and diluted in distilled water to 50 μl. Aliquots of 1 μl were placed on a glass slide, dried and counted. The results were analyzed by a Student's T-test , where means between test and control groups were compared for significance.Cell imaging uses protocol described in the reference .® homogeneous caspase-3/7 assay, Promega .DNA extraction used a protocol described by the manufacturer . The final preparation was digested with RNase  for 30 min at 37°C to remove RNAs bound to the DNA filter. The eluted fraction was concentrated to 30 μl and used 6 μl for DNA electrophoresis. Caspase-3/7 activity was measured according to the manufacturer's menu  and separated in 10% acrylamide gel at 220 volts. The gel bands were processed by the in-gel tryptic digestion procedure and micro-sequenced by the DDA method . Peptide mapping used tryptic peptides by GPMAW software.Protein purification and characterization used the protocol described in the reference . SynchroYW designed and performed research, wrote the paper, and analyzed data; DCH performed the DNA ladder analysis; KH performed part of the experiment and edited the paper; JPT designed experiment and edited the paper, C–YK prepared cells. All authors read and approved the final manuscript.


Investigations were performed using a VEGF-secreting human head and neck tumour cell line, CAL33, with a high EGFR content, injected as orthotopic xenografts into the mouth floor of nude mice. Three days after tumour cell injection, bevacizumab , erlotinib  and irradiation  were administered alone and in combination for 10 days. As compared with the control, concomitant administration of drugs produced a marked and significant supra-additive decrease in tumour mass; the addition of irradiation almost completely abolished tumour growth. The drug association markedly reduced the number of metastatic nodes and the triple combination significantly reduced the total number of pathologically positive lymph nodes as compared with controls. The RT-induced proliferation, reflected by Ki67 labelling, was reduced to control level with the triple combination. Radiotherapy induced a strong and very significant increase in tumour angiogenesis, which was no longer observed when combined with erlotinib and bevacizumab. The efficacy of the combination of bevacizumab+erlotinib and RT may be of clinical importance in the management of head and neck cancer patients.Clinical benefit has been demonstrated in patients with head and neck tumours receiving an anti-epidermal growth factor receptor (EGFR) agent in combination with radiotherapy (RT). Recent preclinical and clinical studies suggest beneficial effects from combining anti-angiogenic drugs with RT. To investigate the effect of combining these approaches, we evaluated  Agents that target epidermal growth factor receptor (EGFR) potentially exert anti-tumour effects by inhibiting tumour cell proliferation and survival, as well as reducing the secretion of proangiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor that stimulate tumour neoangiogenesis  in nude mice. The median time to reach a tumour volume of 1000 mm3 was significantly increased for AZD2171 or gefitinib administered alone compared with the control. Greater inhibition of tumour growth was seen with the combination AZD2171+gefitinib compared with either drug alone, and the triple combination compared with either AZD2171+gefitinib or RT alone.Tumour vasculature is a key target in the treatment of solid tumours, particularly in head and neck cancer . In a reThe aim of this study was to examine the anti-tumour effects of the clinically representative anti-angiogenic monoclonal antibody bevacizumab combined with the anti-EGFR tyrosine kinase inhibitor erlotinib and RT. To this end, we adopted an orthotopic head and neck cancer xenograft model consisting in injecting CAL33, a human head and neck cancer cell line, into the mouth floor of nude mice. This model was used to enable examination of the effects of treatment on the primary tumour itself and also, for a more clinically relevant condition, on disease extension to the lymph nodes in the neck. Molecular markers were also examined according to the applied treatments. Ki67 expression was considered as a relevant and classical parameter related to tumour proliferation. The key VEGF receptor present on endothelial cells is VEGFR2. In addition, the presence of VEGFR2 has been reported on tumour cells themselves, particularly in head and neck cancer (−1) was suspended in 0.9% NaCl and 0.01% Tween 80, and bevacizumab (750 mg l−1) was diluted in 0.9% NaCl. For both drugs, the concentrations were adjusted so as to include the daily dose in 0.2 ml of drug suspension. Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin and glutamine were purchased from Whittaker . Fetal bovine serum (FBS) was obtained from Dutscher .Erlotinib was kindly provided by Roche  and bevacizumab by our institution's pharmacy. Working solutions were prepared as follows: erlotinib . This cell line exhibits high EGFR levels  at 37°C in an atmosphere containing 8% CO2. Batches of 15 × 106 cells were frozen in FBS supplemented with 5% dimethyl sulphoxide v/v in advance for injection into mice. Shortly before injection, cells were thawed and suspended in Ringer lactate.The cell line was maintained as monolayer culture in DMEM supplemented with 10% FBS v/v, 2 m6 cells suspended in 200 μl of Ringer lactate (n=10 per treatment condition). There were five animals per cage with food and water available ad libitum; animals were killed and tumours collected at day 10 after cell injection by cervical disruption . As no signs of suffering appeared during the experiment , no animal had to be killed before the end of the experiment. Animal experiments were performed in accordance with the regulations of our institution's ethics commission and guidelines of the United Kingdom Co-ordinating Committee on Cancer Research . They received, as intra-muscular orthotopic xenograft in the mouth floor as described by −1, 5 days a week, 0.2 ml p.o.), bevacizumab  and irradiation  given alone or in combination; when co-administered, erlotinib and bevacizumab were given simultaneously (given the short duration of treatment − 7 days − five doses of each drug and four doses of irradiation were delivered); the doses of erlotinib and bevacizumab were chosen according to preliminary experiments , performed on tumour of this same cell line xenografted in the flank of animals, so that each drug given alone exerted only partial effects on tumour growth.Three days after cell injection, the mice were treated each week with relevant vehicle (controls), erlotinib (50 mg kgπ/6 × length × width2) and weighed, and bilateral radical neck dissection was performed . The effects of erlotinib, bevacizumab and RT, alone or in combination, on tumour growth were evaluated by measuring, for all groups, the mean tumour volume (MTV). Combination ratios (CR), as described previously If CR>1, there were supra-additive effects and if CR<1, infra-additive ones. Strictly additive effects were observed if CR=1.The total number of pathologically positive lymph nodes, having signs of inflammation either invaded or not, having a visible diameter (greater than 1 mm), were recorded after node chain dissection after tumour removal.All analyses of tumour markers were performed on the primary tumours, which were collected 10 days after tumour cell injection.The expression of the microvessel marker VEGFR2 and of the proliferation marker Ki67 was evaluated using immunohistochemistry . The analysis of VEGFR2 involved vessel loss and reduction in vessel area, which were evaluated both at the tumour centre and tumour periphery, and also a more diffuse labelling of tumour cells; in this case, both the intensity  of labelling and the proportion of tumour cells were taken into account. The analysis of Ki67 took into account the proportion of labelled cells; labelling of each microscopic field was attributed to three labelling groups: group 1 with 15–50% of labelled cell nuclei; group 2 with 50–70% of labelled cell nuclei; and group 3 with more than 70% of labelled cell nuclei. For each treatment group, the relative proportion of each of the three labelling groups  was pooled and represented as a histogram.Forty × 10 magnification images of regions of high vascular density within the tumour were analysed to quantify both tumour angiogenesis and tumour cell diffuse labelling (VEGFR2). Whatever the studied marker (Ki67 or VEGFR2), the final score was the result of the examination of a maximum of 10, a minimum of 2 and a mean of 5 fields per tumour, which, due to the small volume of the tumour, allowed a thorough examination; 10 tumours were investigated for each treatment group.Comparison of primary tumour growth, node number, state of invasion and the effects of treatment on VEGFR-2 and Ki67 between different treatment groups were evaluated using the non-parametric ANOVA .P>0.05) (P=0.028 vs controls) and showed supra-additive effects (CR=2).Treatments given alone had no significant impact on primary tumour mass, with erlotinib and RT slightly decreasing tumour growth  . The comP<0.0004 vs controls; P<0.0001 vs RT; P=0.02 vs bevacizumab+erlotinib). The effects of this triple combination were supra-additive (CR=2.3).Finally, the triple combination bevacizumab+erlotinib+RT gave the highest tumour growth inhibition . However, the drug combination had no effect on the total number of pathologically positive lymph nodes. In contrast, the bevacizumab+erlotinib+RT triple combination produced a very significant decrease in the total number of pathologically positive lymph nodes (P<0.001 vs controls) although invaded nodes were still present among these markedly reduced pathologically positive lymph nodes.In contrast, and corroborating the observations on the primary tumour mass, the combination erlotinib+bevacizumab totally prevented node invasion (P>0.05 vs controls) (P=0.007 vs controls). This RT-related effect on tumour cell proliferation was diminished by the presence of erlotinib+bevacizumab to a level similar to the controls .Neither erlotinib  nor bevacizumab  administered alone had a significant impact on tumour cell proliferation (ontrols) . In contP<0.001) increase in tumour angiogenesis. Of note, this RT-related effect on tumour vessels was no longer observed when RT was combined with erlotinib and bevacizumab. The results of the examination of peripheral tumour vessels were found to be superimposable with those of the central part of the tumour (data not shown).Bevacizumab given alone induced a decrease in endothelial cell VEGFR2 labelling . NeverthAll targeted treatments were found to diminish VEGFR2 expression on tumour cells . The comThere are recent preclinical background  or chemoper se and that erlotinib exhibited moderate anti-tumour effects as a single drug (Doses of individual agents were chosen to generate relatively limited effects and thus allow detection of potential interactions. This is for this reason that bevacizumab had no evident impact on tumour growth gle drug . Interesgle drug .As recently underlined by This study was also specifically designed to examine the impact of treatment not only on the primary tumour itself but also on the local metastatic invasion in nodes, thus creating a condition of clinical relevance. This was made possible by the adoption of a head and neck orthotopic model . Of clinIn total, this study provides another strong rationale for a combination between irradiation and EGFR pathway targeting plus anti-angiogenic treatment  in which