Beauveria bassiana is an important entomopathogenic fungus currently under development as a bio-control agent for a variety of insect pests. Although reported to be non-toxic to vertebrates, the potential allergenicity of Beauveria species has not been widely studied.B. bassiana extracts was measured using intradermal skin testing.IgE-reactivity studies were performed using sera from patients displaying mould hypersensitivity by immunoblot and immunoblot inhibition. Skin reactivity to B. bassiana crude extracts. Proteinase K digestion of extracts resulted in loss of IgE reactive epitopes, whereas EndoH and PNGaseF (glycosidase) treatments resulted in minor changes in IgE reactive banding patterns as determined by Western blots. Immunoblot inhibitions experiments showed complete loss of IgE-binding using self protein, and partial inhibition using extracts from common allergenic fungi including; Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Candida albicans, Epicoccum purpurascens, and Penicillium notatum. Several proteins including a strongly reactive band with an approximate molecular mass of 35 kDa was uninhibited by any of the tested extracts, and may represent B. bassiana specific allergens. Intradermal skin testing confirmed the in vitro results, demonstrating allergenic reactions in a number of individuals, including those who have had occupational exposure to B. bassiana.Immunoblots of fungal extracts with pooled as well as individual sera showed a distribution of IgE reactive proteins present in Beauveria bassiana possesses numerous IgE reactive proteins, some of which are cross-reactive among allergens from other fungi. A strongly reactive potential B. bassiana specific allergen (35 kDa) was identified. Intradermal skin testing confirmed the allergenic potential of B. bassiana. Metarhizium anisopliae, Verticillium lecanii, and Beauveria bassiana are being used as biocontrol agents for a number of crop, livestock, and human nuisance pests [B. bassiana have been licensed for commercial use against whiteflies, aphids, thrips, and numerous other insect and arthropod pests. B. bassiana fungal formulations are being spread onto a range of vegetables, melons, tree fruits and nuts, as well as organic crops. As alternatives to chemical pesticides these agents are natural occurring and are considered to be non-pathogenic to humans, although a few cases of B. bassiana mediated tissue infections have been reported [Microorganisms are currently under intensive study for use as biopesticides -3. Severce pests -7. Straireported ,9.Alternaria, Aspergillus, and Cladosporium genera [Airborne mold spores are widespread, and many have been identified as inhalant allergens eliciting type I hypersensitive reactions in atopic individuals -14. Commm genera -19. The m genera ,20-22, aBeauveria bassiana crude extracts contain numerous allergens capable of being recognized by human serum IgEs. The allergens were proteinaceous in nature, and immunoblot inhibition experiments revealed the presence of shared epitopes between Beauveria and several other common fungal moulds. Potential Beauveria-specific allergens were also identified, including a strongly reactive ~35-kDa protein band. Intradermal skin testing using B. bassiana extracts resulted in allergenic reactions in several individuals, including some who have had occupational exposure to the fungus.In the present study, we demonstrate Beauveria bassiana (ATCC 90517) was grown on Sabouraud dextrose + 0.5–1% yeast extract or Potato dextrose (PD) media on either agar plates or in liquid broth. Plates were incubated at 26°C for 10–12 days and conidia were harvested by flooding the plate with sterile dH2O containing 0.01% Tween-20. Liquid cultures were inoculated with conidia harvested from plates at 0.5–1 × 105 conidia/ml.Alternaria alternata, Aspergillus fumigatus, Candida albicans, Cladosporium herbarum, Epicoccum purpurascens, and Penicillium notatum were acquired from Greer Laboratories inc., . Extracts were resuspended in TE  to a final concentration of 2 mg/ml. Beauveria bassiana was grown in Sabouraud's broth containing 1% yeast extract with aeration at 25°C for 3–5 d. Cellular mass was harvested by centrifugation  and freeze-dried. Cells were resuspended in TE containing 0.1% phenylmethylsulfonyl fluoride (PMSF) and homogenized using a bead-beater apparatus.B. bassiana were subjected to three successive precipitations before use in Western blots.Crude extracts of B. bassiana extracts (50 ml) were mixed with 8 × volume (400 ml) of acetone (kept at -20°C), with rapid stirring, and incubated overnight at -20°C. The precipitate was collected by centrifugation , and the pellet was air dried (10 min) before being resuspended in TE containing 0.1% PMSF.Homogenized Streptomycin sulfate (5 ml of 10% solution) was added dropwise to resuspended acetone precipitated extracts (40 ml) at 4°C with rapid stirring. Samples were incubated for an additional 30 min on ice before being centrifuged  in order to remove the precipitate. Proteins in the resultant supernatant were precipitated using ammonium sulfate.Beauveria extract (40 ml) at 4°C with rapid stirring. The solution was allowed to stir overnight at 4°C and precipitated proteins were harvested by centrifugation . The protein pellet was resuspended in TE containing 0.1% PMSF (40 ml) and extensively dialyzed against the same buffer before use.The proteins present in the streptomycin sulfate treated supernatant were precipitated using ammonium sulfate . Saturated ammonium sulfate (120 ml) was added dropwise to the Protein samples (30–40 μg) were analyzed by sodium-dodecyl-sulphate-polyacrylaminde gel electrophoresis  using standard protocols. Gels were stained with Gelcode blue stain reagent  and subsequently de-stained with dH20.Protein samples were separated under reducing conditions using 10% Bis-tris polyacrylamide gels (Invitrogen Mops system) and transferred to polyvinylidene-fluoride (PVDF) membranes (Invitrogen) as described. Immunoblot experiments were performed using individual and pooled human sera as the primary antibody solution as indicated. Typically, sera were diluted 1:5 with Tris-HCl buffered saline (TBS) containing 5% dry milk + 0.1% Tween-20. IgE-specific reactivity was visualized using a horseradish peroxidase (HRP) conjugated goat anti-human IgE  secondary antibody . Membranes were washed with TBS containing 0.1% Tween-20 and bands were visualized using the Immuno-Star HRP detection system .B. bassiana crude extracts was treated with Proteinase K  following standard protocols. Typically, samples (36 μl) were incubated with 4 μl Proteinase K solution  for 2 hr at 37°C before analysis. Samples were also treated with endoglycosidase-H  and peptide: N-Glycosidase F  according to the manufacturer's recommendations. For EndoH and PNGaseF treatments, samples (36 μl) were denatured in 4 μl 10 × denaturing buffer  at 100°C for 10 min prior to the addition of the EndoH  and PNGaseF reaction buffers (50 mM sodium phosphate pH 7.5) and enzymes (5 μl), respectively. Reactions were incubated at 37°C for 2 h before being analyzed by SDS-PAGE and Western blotting.The ammonium sulfate fraction of B. bassiana proteins were competed with proteins of other fungal extracts. SDS-PAGE resolved B. bassiana proteins were electroblotted to PVDF membranes as described above. Membranes were blocked with TBS containing 5% dry milk + 0.1% Tween-20 and strips were incubated with pooled human sera (1:5 v/v in same buffer) containing 100–500 μg of the indicated fungal crude protein extract.IgE binding to Alternaria tenius, Aspergillus fumigatus, Cephalosporium (Acremonium strictum), Curvularia spp. Bipolaris, Epicoccum nigram, Fusarium spp., Helminthosporium sativum, Hormodendrum horde, Penicillium . Extracts were tested using a 1:10 dilution of the 20,000 PNU/ml stock solution, and skin sensitivity was recorded on a relative scale from 0–4 reflecting the size of induration or weal (4 representing the highest reactivity) and using histamine (0.1 mg/ml) reaction scored as a 3 if no interference was present.Patients were tested with 9 common fungal extracts for allergy diagnosis using a skin prick assay. The following extracts were obtained from ALA-Abello ; B. bassiana crude extracts were prepared as described above but were extensively dialyzed against 0.15 N NaCl and filtered through a 0.22 μm filter before use. Subjects were given intradermal injections of 0.1 ml crude extract ranging in concentration from 0.01–1 mg/ml. Control injections included saline and histamine (0.1 mg/ml). Allergenic reactions were allowed to develop for 15–30 min before the height and width of the reactions were recorded.B. bassiana proteins was resolved on SDS-PAGE ; with the most prominent bands located around 64, 45, and 35 kDa. Control experiments omitting either the primary or secondary antibody incubation steps resulted in complete loss of signal. Proteinase K digestion of samples also resulted in loss of all signal  were resolved by SDS-PAGE, blotted to PVDF membranes, and the lanes were cut into separate strips. Each strip was treated with a 1:5 dilution of each respective serum as described in the Materials and Methods . Blots probed with one serum (H) resulted in a large smear ranging from ~30 kDa to 55 kDa. The reason for the observed smear is unknown and efforts to distinguish separate bands by manipulating the concentrations of either sera or extract were unsuccessful. A number of bands (based upon molecular mass) appeared to be common to several sera including proteins of approximately 35, 42–48, and 60 kDa. A number of allergens of high molecular weight (~100–200 kDa) were also visible; however the resolution in this range on the Western blots is poor.In order to determine the variation and distribution of serum IgEs reactive to ies Fig. . The reaies Fig. , lane 2.ies Fig.  and reprB. bassiana crude extracts using an intradermal delivery procedure. Data using 1 mg/ml B. bassiana crude extract and histamine controls are presented in Table B. bassiana extracts  were resolved by SDS-PAGE, blotted to PVDF membranes, and lanes were cut into separate strips. Each strip was treated with a 1:5 dilution pooled sera (serum mix-II) as the primary antibody supplemented with concentrations of fungal crude extracts as described in the Materials and Methods. Fig. B. bassiana extracts were competed with: excess crude extracts from Alternaria alternata , Cladosporium herbarum (lanes 7), Epicoccum purpurascens (Lane 8), Penicillium notatum (lane 9), and Candida albicans (lane 10). There was complete loss of all signals using 2-fold excess B. bassiana extract as the competitor (data not shown). These data indicate that while Beauveria possess many epitopes in common with several other fungi, notably Alternaria and Penicillium, a 35-kDa major reactive band was not inhibited by any extract tested.In order to determine the extent of cross-reactivity of Beauveria could barely be detected in airborne samples, and represented less than 0.1% of the airborne fungal "flora", the incidence of allergic skin test reaction to Beauveria was the highest of all fungal species tested [Beauveria bassiana is imperative.Although it is well known that fungi are important triggers of respiratory allergies, the potential allergenicity of entomopathogenic fungi used in biocontrol has largely been untested. Aerobiological surveys of conidial fungi and skin sensitivity tests to fungal extracts performed in the 1980s in the Netherlands revealed that although s tested ,23,24. IB. bassiana directly by intradermal skin testing of individuals and in vitro by revealing the presence of serum IgEs capable of binding allergens present in fungal crude extracts. Over 20 different IgE binding proteins were observed using Western blots probed with sera from patients displaying mould allergies. Results using individual sera revealed a wide variation in IgE-binding proteins between sera, although several common bands, including a protein with an apparent molecular mass of 35 kDa were visible among the sera of several patients.The present study demonstrated the allergenic potential of in vitro observations were confirmed by intradermal skin testing on individuals using B. bassiana extracts. While the testing sample population was small, these results indicated that our extracts were able to elicit allergic reactions in individuals, including some that have had occupational exposure to the fungus. Concentrations of ~1 mg/ml of B. bassiana extracts were required to elicit indurations equivalent to 0.1 mg/ml histamine in most individuals, indicating the possibility of potent allergens in the Beauveria extract. Interestingly, not all individuals specifically exposed to B. bassiana displayed allergic reactions and individuals J, K, and M  exists between the two fungal species, implying that primary sensitization by one organism can result in an allergic reaction when exposed to the homologous allergen of another organism. Competitive immunoblot inhibition experiments revealed significant epitope homology between B. bassiana and several clinically important fungi responsible for IgE-mediated allergic reactions in atopic individuals. Thus, an allergic reaction to Beauveria exposure may arise in patients sensitized to other fungi. Extracts from A. alternata and E. purpurascens almost completely competed with allergens present in the B. bassiana extract with the notable exception of the ~35 kDa allergen. Competition experiments using A. fumigatus, C. herbarum, C. albicans, and P. notatum extracts also indicated the presence of many shared epitopes, although distinct (non-competed) IgE-binding B. bassiana proteins of 35 kDa, 64 kDa, and >200 kDa molecular mass were detectable. These proteins, particularly the 35 kDa allergens may represent B. bassiana specific allergens. Experiments are underway to characterize the 35 kDa allergen, which may lead to a diagnostic assay for B. bassiana sensitization. Finally, our analysis of potential B. bassiana allergens was limited to cell extracts grown under specific conditions and did not include the culture filtrate. Extracellular proteases, an important class of fungal proteins that can elicit allergenic reactions, have been characterized from a number of fungal species [B. bassiana. A careful examination of culture growth conditions is also warranted in order to provide a standardized reagent for testing purposes.Numerous studies have revealed the presence of cross-reactive proteins among fungal species between genera -22,25-27 species -31, and Beauveria holds promise as an arthropod biological control agent, there have been few reports on the allergenic potential of these organisms. Identification of B. bassiana specific allergens can lead diagnostic methods for determining sensitization to this organism and may provide a rational basis for allergen attenuation in order to yield safer biocontrol products. The observed cross-reactivity among proteins of B. bassiana and the fungi tested, highlight the importance of considering the possibility that multiple fungal sensitivity can occur due to exposure to a single fungus. Further testing should be performed to determine the scope, severity, and range of allergenic reactions to B. bassiana.Although The author(s) declare that they have no competing interests.in vitro experiments. SWH performed the clinical experiments and participated in the design of the study. NOK conceived of the study, and participated in its design and coordination, and drafted the manuscript.GSW carried out the immunoassays and other


Coccidioides in Southwest US and Central America. Primary pulmonary infection is initiated by inhalation of air-borne arthroconidia. Since, lung is the first organ that encounters arthroconidia, different components of the pulmonary innate immune system may be involved in the regulation of host defense. Pulmonary surfactant proteins (SP)-A and SP-D have been recognized to play an important role in binding and phagocytosis of various microorganisms, but their roles in Coccidioides infection are not known.Coccidioidomycosis or Valley Fever is caused by Coccidioides posadasii infection, and binding of SP-A and SP-D to Coccidioidal antigens. Mice were challenged intranasally with a lethal dose of C. posadasii (n = 30 arthroconidia) and bronchoalveolar lavage fluid (BALF) samples were collected on day 10, post infection. In another group of animals, mice were immunized with protective formalin killed spherule (FKS) vaccine prior to infection. The concentrations of BALF SP-A, SP-D, total phospholipid were measured using enzyme linked immunosorbent assay and biochemical assays.In this study, we studied the changes in amounts of pulmonary SP-A, SP-D and phospholipid in murine model of C. posadasii infected mice, the concentrations of total phospholipid, SP-A and SP-D were 17 % , 38 %  and 4 %  of those in lavage fluid samples of non-infected control mice, respectively. However, the concentrations of SP-A and SP-D remained unchanged in BALF samples of C. posadasii protected mice after immunization with FKS vaccine. Also, we found that both SP-A and SP-D bind to Coccidiodal antigens.We found that in lavage fluid samples of C. posadasii infection perturbs the pulmonary SP-A, SP-D, and phospholipids, potentially enabling the disease progression and promoting fungal dissemination.Our results suggest that the  Coccidioides immitis or posadasii [C. posadasii or C. immitis, are the most virulent fungal pathogens enlisted in Select Agent list and pose a risk for bioterrorism [Coccidioides [Coccidioides.Coccidioidomycosis or Valley Fever is a fungal disease caused by the biphasic, highly virulent, soil-fungus osadasii . It is eosadasii . C. posaerrorism . The prierrorism -4. Previidioides ,6. HowevStreptococci [Pseudomonas aeruginosa [At alveolar level in the lung, the innate immune system is composed of many cell types and chemical mediators, including surfactant. The pulmonary surfactant is a complex mixture of lipids 88–90%) and proteins 10–12%), synthesized by type II epithelial cells and Clara cells. It lines the alveoli, and helps in maintaining normal lung function . Among f8–90% and–12%, synptococci , Pseudomruginosa . Also, iruginosa . Both SPruginosa . Recent ruginosa . HoweverC. posadasii and C. posadasii protected mice after immunization with protective formalin killed spherule (FKS) vaccine, and binding of pulmonary collectins to Coccidioidal antigens.This study focuses on analyzing the changes in amounts of the SP-A and SP-D in the bronchoalveolar lavage fluid (BALF) samples from mice infected with lethal dose of C. posadasii infection. The BALB/c mice were used to study the changes in pulmonary surfactant after intranasal challenge with C. posadasii. And, the C57BL6 mice were used to study the changes in pulmonary surfactant after vaccination with protective FKS vaccine. Mice were housed in Biosafety Level-3 animal facility at UTHSCSA and provided with food and water ad libitum. All experimental animal care and treatment protocols were reviewed and approved by Institutional Animal Care and Use Committee.BALB/c and C57BL6 mice  from Jackson Laboratory  were used in this study. Both mouse strains are susceptible to C. immitis (now posadasii) Silveira strain, cultured on 1 % glucose-0.5 % yeast extract agar (GYE), was used for infecting the mice [C. posadasii were carried out in Biosafety Level-3 facility at UTHSCSA.C. posadasii arthroconidia) suspended in endotoxin-free 0.15 M NaCl  using sterile pyrogen-free microtip. Mice were held in an upright position for 1–2 min to resume normal breathing after injection. Control mice were challenged with equal volume of endotoxin-free 0.15 M NaCl.Mice were anaesthetized after intramuscular injection of ketamine- xylazine (75 μg/g body weight ketamine and 10 μg/g body weight xylazine) and were then challenged intranasally with a lethal dose of arthroconidia arthroconidia were used to prepare FKS as described earlier [2 incubator. The spherules were collected from the harvested culture, washed in endotoxin-free water and killed with 1 % formalin. FKS preparation was checked for sterility and lyophilized. C57BL6 female mice (age 6 weeks old) were immunized intramuscularly twice and subcutaneously once at one week interval with FKS (0.7 mg/dose each time). The mice in FKS immunized, infected group were then challenged intranasally with 30 C. posadasii arthroconidia, 15 days after last immunization, as described above. earlier . BrieflyMice were anaesthetized as mentioned above, prior to sacrifice on day 10, post intranasal infection. This standard procedure was used for intranasal injection since it does not cause respiratory depression during anaesthesia. The lung and spleen tissues were collected in sterile 0.15 M NaCl for studying fungal load.The fungal burden was studied by plating ten fold dilutions of lung and spleen homogenates in 0.15 M saline on Mycosel agar plates  and incubating for 72 h at 30°C. The cfu counts were recorded and normalized with organ weight.At the time of necropsy, we collected BALF by injecting 1 ml endotoxin-free 0.15 M NaCl solution  three times, via an angiocatheter  placed in the trachea. The volume of the input solution was kept constant  and approximately, 90–95 % of the solution was recovered consistently. The BALF was centrifuged at 500 rpm for 10 min at 4°C to remove cells. The cell free BALF supernatant was filtered through 0.2 μm syringe filters  and stored at -80°C for further analysis.The total protein concentration was measured in BALF specimens using micro bicinchonic acid protein assay kit  against bovine serum albumin (BSA) standard protein. The total phospholipid content in lipid extracts of BALF specimens was determined using the method of Stewart, against Dipalmitoyl-phosphatidylcholine  standard solutions ,18. BrieThe BALF and lung tissue homogenate samples  were run on 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) running gel and transferred on nitrocellulose membranes  overnight at 15 mA current. The nonspecific sites on the membrane with transferred proteins were blocked by 15% nonfat milk in Tris-buffered saline containing 0.05% tween 20 (TBST). The membrane was washed and incubated for 1 h with diluted (1:500) primary anti-human SP-A polyclonal antibody raised in rabbit  or anti-mouse SP-D antibody . After washing the membrane with TBST, the membrane was incubated for 1 h with 1:10,000 diluted alkaline phosphatase conjugated anti-rabbit IgG raised in goat . The immunoreactive bands were observed by alkaline phosphatase conjugate system . Purified human SP-A  and recombinant human SP-D  were run with the samples.C. posadasii mycelia , prepared as a toluene-induced lysate of young nio, TX, ) were al3, pH 9.6. The wells were washed three times with deionized water, and nonspecific sites were blocked with a buffer containing 0.25% BSA, 0.05% tween 20, 0.17 M boric acid and 0.12 M NaCl, pH 8.5. The wells were washed and incubated for 2 h with rabbit anti-human SP-A or rabbit anti-mouse SP-D antibody. After washing the wells, the horseradish peroxidase conjugated anti-rabbit IgG antibody  was added. After incubation for 2 h, the wells were washed again and incubated with tetramethylbenzidine substrate reagent . The reaction was stopped by adding 50 μl of 2 N H2SO4 and read at 450 nm spectrophotometrically. The regression coefficient for a least-square linear fit to the standard curve of SP-A and SP-D was 0.99. The limits of detection for SP-A and SP-D were 2 ng/ml.The concentrations of SP-A and SP-D were measured in BALF samples as described earlier . The ant3 buffer, pH 9.6) or CDN-filtrate  or BSA  at room temperature. The nonspecific binding was blocked with phosphate buffered saline (pH 7.4) containing 0.1% triton-X 100 and 3% nonfat milk (buffer A). The purified human SP-A and recombinant human SP-D diluted in 20 mM Tris (pH 7.4) containing 0.15 M NaCl, 5 mM CaCl2 and 1 mg/ml BSA were then added to the wells and incubated for 3 h at 37°C. The wells were then washed with buffer A and incubated for 1 h at room temperature with diluted (1:1000 in buffer A) anti-SP-A and anti-SP-D antibodies. After washing the wells, the horseradish peroxidase conjugated anti-rabbit IgG antibody  was added. After incubation for 2 h, the wells were washed again and incubated with tetramethylbenzidine substrate reagent . The reaction was stopped by adding 2 N H2SO4 and read at 405 nm spectrophotometrically.A microtiter well based method  was usedThe coating of Coccidioidal antigens (CDN-lysate and CDN-filtrate) to the plates was confirmed using a positive control antibody that recognizes Coccidioidal antigens as described earlier . The alkStatistical analyses of the data (t-test or ANOVA) were done using Prism Software . The p value <0.05 was considered significant.C. posadasii infected mice survived till the day of sacrifice (day 10 post infection). However, the mice were lethargic and lost body weight (Table C. posadasii infected mice.All of the C. posadasii infected mice (58 μg) when compared to non-infected saline injected controls .The mean protein content of BALF samples from infected mice was 788 μg versus 326 μg protein in BALF samples from non-infected saline injected control mice, after 10 days of intranasal infection  .The anti-human-SP-A and anti-mouse SP-D antibodies recognized 34 kDa and 43 kDa monomer bands of SP-A and SP-D in BALF and lung tissue samples Fig . The upp001) Fig . No signC. posadasii were recovered from both lung and spleens of infected animals indicating the presence of active infection  C57BL6 mice when compared to FKS immunized, non-infected mice  versus FKS immunized, non-infected mice (538.4 μg).No significant changes were seen in SP-A or SP-D levels in BALF samples of protected  coated onto microtiter wells Fig . Both SPC. posadasii infected mice, but were intact in lungs of C. posadasii protected mice after immunization with protective FKS vaccine. Furthermore, our results suggest that both SP-A and SP-D bind to Coccidioidal antigens. This is the first study where the amounts of SP-A and SP-D were measured in BALF samples from mice infected with lethal dose of C. posadasii and the binding of pulmonary collectins to Coccidioidal antigens was assessed. Since lung is the first organ of the body that comes into contact with air-borne C. posadasii arthroconidia, we hypothesized that the pulmonary surfactant may play an important role in regulating the immune response against C. posadasii. Among four surfactant proteins, SP-A and SP-D, interact with most of the clinically important fungal pathogens including Pneumocystis carinii [Cryptococcus neoformans [Aspergillus fumigatus [Candida albicans [C. albicans and directly inhibit growth by aggregation of the organism without involvement of macrophage dependent phagocytosis [A. fumigatus conidia and C. neoformans by alveolar macrophages and neutrophils [A. fumigatus [C. posadasii infection versus the unaltered amounts in the lungs of protected mice and binding of collectins to Coccidioidal antigens indicate that pulmonary collectins may be involved in uptake/phagocytosis of C. posadasii by antigen presenting cells and downstream immune regulation.In the present study we found that the levels of pulmonary surfactant collectins were altered in the lungs of  carinii Cryptococoformans , Aspergiumigatus  and Candalbicans . SP-D haocytosis . On the trophils ,29. In sumigatus . The decC. posadasii infected mice  and growth factors are released during infection and inflammation. These cytokines and growth factors affect the synthesis and secretion of pulmonary surfactant by pulmonary epithelial cells [C. posadasii protected mice, but not of SP-A (Fig Besides changes in amounts of SP-A and SP-D, a decrease in the amount of BALF phospholipids was also observed in ce Table . Earlier carinii ,30. To d carinii . Likewisal cells ,33. In tSP-A Fig . We specC. posadasii infected patients with disseminated disease is not very effective [Coccidioides infection in murine model of coccidioidomycosis, but failed in humans [C. posadasii and C. immitis are highly virulent organisms, cause endemic infection, and pose a risk for bioterrorism, there is an urgent need for discovery of improved therapeutic drugs and regimens or preventive vaccines [At present, the treatment of ffective . Treatmen humans . More prvaccines ,35.in vivo administration of artificial or natural surfactant in C. posadasii infected mice may be useful in determining their therapeutic usefulness. We speculate that the findings from our present study would initiate similar studies to understand the role of pulmonary innate immune components in infectious diseases caused by C. posadasii or other virulent respiratory pathogens.In future, clearance experiments after SA designed and co-ordinated the study, performed assays and statistical analysis and drafted the manuscript. JJC assessed lung pathology. DMM prepared and immunized mice with FKS. All authors read and approved the final manuscript.


Cathepsin K is a cysteine protease that plays an essential role in osteoclast function and in the degradation of protein components of the bone matrix by cleaving proteins such as collagen type I, collagen type II and osteonectin. Cathepsin K therefore plays a role in bone remodelling and resorption in diseases such as osteoporosis, osteolytic bone metastasis and rheumatoid arthritis. We examined cathepsin K in the serum of 100 patients with active longstanding rheumatoid arthritis. We found increased levels of cathepsin K compared with a healthy control group and found a significant correlation with radiological destruction, measured by the Larsen score. Inhibition of cathepsin K may therefore be a new target for preventing bone erosion and joint destruction in rheumatoid arthritis. However, further studies have to be performed to prove that cathepsin K is a valuable parameter for bone metabolism in patients with early rheumatoid arthritis. Progressive bone and cartilage destruction in arthritic joints leads to irreversible joint destruction, and subsequently to functional declines and work disability ,2. New bCathepsin K is a cysteine protease that plays an essential role in osteoclast function and in the degradation of protein components of the bone matrix. It is produced by bone resorbing macrophages and synovial fibroblasts, and it cleaves proteins such as collagen type I, collagen type II and osteonectin . CathepsCathepsin K is a tissue-specific protease associated with pycnodysostosis, a rare genetic disorder that manifests itself in bone abnormalities such as short stature, acroosteolysis of distal phalanges and skull deformities ,15. CathThe aim of this study was to measure serum levels of cathepsin K in RA and to prove that cathepsin K is a parameter of bone remodelling and resorption in a nonselected cohort of patients with longstanding RA. This patient group shows a variation of age, inflammatory level and Larsen score. We divided this cohort into different groups, according to age, inflammatory level, disease-modifying antirheumatic drug (DMARD) therapy, radiological progression and disease activity, to verify cathepsin K as an age-independent and laboratory inflammatory parameter-independent protease.n = 50; 21 female, 29 male) aged 18–65 years.Serum levels of cathepsin K were measured in the sera of 100 patients suffering from RA according to the criteria of the American Rheumatism Association . ClinicaMost of the patients received DMARDs. The most frequently used DMARD was methotrexate, followed by leflunomide, sulfasalzopyrine and gold. Furthermore, azathioprine and chloroquine but no biological therapy were prescribed Table .Each examination consisted of a full interview, the assessment of functional disability and a standardised physical examination, which included a joint examination for tenderness (Ritchie score), pain on motion, soft tissue swelling, 44-swollen joint count and swollen proximal interphalangeal score ,19.The disease activity of RA was measured by the disease activity score . The radiological progression in RA was calculated by the Larsen score .® RF; Dade Behring, Liederbach, Germany), antinuclear antibodies  and C-reactive protein (CRP) .The blood examination at each visit consisted of the determination of cathepsin K, the erythrocyte sedimentation rate, the haemoglobin level, the thrombocyte count, the serum rheumatoid factor  was used. The Cathepsin K test kit is an enzyme immunoassay designed to determine cathepsin K directly in biological fluids . The ELISA used in this study is based on antibodies specific for amino acids 1–20 and amino acids 196–210 of the mature enzyme. The antibodies were produced by immunisation of sheep with peptides of that amino acid sequence coupled to Keyhole Limpet Hämocyanine . Antisera were purified using the biotinylated peptides coupled to streptavidine sepharose . A synthetic cathepsin K  was used as the calibrator. Signal generation was accomplished by labelling with horseradish peroxidase.2SO4. The yellow colour that is directly proportional to the amount of cathepsin K present in the sample was measured on a standard microplate reader at 450 nm with 620 nm as the reference. The detection limit of the assay was calculated as 1.1 pmol/l (0 standard + 5 × standard deviation).Briefly, the assay procedure consisted of incubating 50 μl sample with 200 μl horseradish peroxidase-labelled detection antibody on capture antibody precoated plates overnight at room temperature. After a washing step to remove unbound detection antibody, tetramethyl benzidine was added as the substrate. The reaction was stopped after 30 min by adding 50 μl of 0.9% HNo cross-reactivity to cathepsin E, cathepsin D, cathepsin B and cathepsin L or rheumatoid factors was detected.P < 5% was considered statistically significant.Statistical methods included Spearman correlation analysis, the Wilcoxon two-sample test the Kruskal–Wallis test and analysis of variance, if appropriate. P = 0.0003)  compared with the healthy control group  were significantly elevated (P = 0.004). The highest levels of cathepsin K were observed in patients with the highest Larsen scores. We divided the cohort into three Larsen groups with equal numbers of patients . Cathepsin K levels showed an increase with the augmentation of radiological destruction (P = 0.035) . The Spearman rank correlation showed a statistically significant correlation between cathepsin K and the Larsen score (P = 0.27), but weak correlations were found with the erythrocyte sedimentation rate (P = 0.03) and the disease activity score of the whole cohort (P = 0.04). However, the division of the disease activity score into three groups with low activity, medium activity and high activity did not show any difference. We could not find any correlation with sex and age , whereby the two groups were comparable in disease activity (3.53 versus 3.12), laboratory parameters , clinical score  and radiological score .Cathepsin K seems to be independent or only weakly correlated with laboratory inflammation parameters. It was not associated with CRP (n = 42), followed by leflunomide (n = 10) and sulfasalzine (n = 10). Twenty-two patients had no DMARD at the time of examination (Table The most frequently used DMARD was methotrexate (on Table . The lowIn vivo the activation of cathepsin K occurs intracellularly, before secretion into the resorbing lacunae and the onset of bone resorption, whereby local factors may regulate the processing of procathepsin K to mature cathepsin K [Bone resorption and formation is a well-balanced system and is mediated by osteoclasts. Cathepsin K is essential for bone resorption, which depends on the production of cathepsin K by osteoclasts and its secretion into the extracellular department. This leads to a degradation of the organic matrix between the osteoclasts and the bone surface . In vivohepsin K . In accohepsin K . Increashepsin K Proinflammatory cytokines such as IL-1β and tumour necrosis factor alpha influence the expression of cathepsin K. Its overexpression in the rheumatoid synovium, induced by IL-1β and tumour necrosis factor alpha due to the increase of cathepsin K-expressing cells, proves this protease to be a valuable tool for bone research, and cathepsin K also may become a new and highly specific biomarker for RA .in vitro. This inhibitor leads to a significantly reduced bone loss [Votta and colleagues demonstrated high levels of cathepsin K expression in osteoclasts at sites of extensive bone loss. According to this, they developed a peptide aldehyde inhibitor of cathepsin K that inhibits osteoclast-mediated bone resorption in foetal rat long bone organ cultures and even in a human osteoclast-mediated assay one loss . Furtherone loss .Wittrant and colleagues demonstrated osteoprotegerin to be an inhibitor of cathepsin K. Osteoprotegerin is an osteoblast-secreted decoy receptor that inhibits osteoclast differentiation and activation. Human osteoprotegerin inhibits cathepsin K and tartrate-resistant acid phosphatase, both osteoclast markers, but stimulates the expression of tissue inhibitor of metalloproteinases-1 . These r+ macrophage-like synoviocytes. Highly interesting is the expression of cathepsin K by fibroblasts and giant cells at sites of cartilage erosions. This was two to five times higher compared with osteoarthritic synovium. In normal synovium, cathepsin K expression was not increased and was restricted to fibroblast like cells [In the synovium of RA, the cathepsin K protein was localised in synovial fibroblasts, stromal multinucleated giant cells and CD68ke cells ,30-32. TTo our knowledge, no study has previously investigated the serum levels of cathepsin K in RA. Our results demonstrate that cathepsin K is elevated in the serum of patients with RA compared with that of a healthy control group Table . The uprFurther studies should investigate whether elevated cathepsin K levels precede osseous destruction or whether they occur as result of them. In the first case, determination of cathepsin K could be an important additional tool to decide on aggressive forms of disease-modifying antirheumatic therapies.This is the first study that demonstrates increased cathepsin K levels in the serum of patients with RA. As could be shown in the synovia of RA, the elevated serum levels of this protease are significantly correlated with the joint destruction, which in this study was assessed by the Larsen score. Cathepsin K seems to be a valuable parameter for the assessment of bone metabolism in patients with established RA and its measurement will probably contribute to developing targeted therapies for the prevention of further bone destruction. However, more studies need to be performed to verify the presence of cathepsin K in patients with early RA and its value as a prognostic factor for bone destruction in RACRP = C-reactive protein; DMARD = disease-modifying antirheumatic drug; ELISA = enzyme-linked immunosorbent assay; IL = interleukin; NF = nuclear factor; RA = rheumatoid arthritis.Dr G Hawa and Prof. W Woloszczuk are members of BIOMEDICA who developed the Cathepinsin K kit, but they did not receive any financial benefits.MS is the corresponding author, and GH and GK are coauthors of the manuscript. GH and WW developed the cathepsin K ELISA kit. AK performed the statistical analysis.


CDT2 and SCFSkp2, are redundantly required for Cdt1 degradation. The two E3 complexes use distinct mechanisms to target Cdt1 ubiquitination. Current data suggests that CUL4-DDB1CDT2-mediated degradation of Cdt1 is S-phase specific, while SCFSkp2-mediated Cdt1 degradation occurs throughout the cell cycle. The degradation of Cdt1 by the CUL4-DDB1CDT2 E3 complex is an evolutionarily ancient pathway that is active in fungi and metazoa. In contrast, SCFSkp2-mediated Cdt1 degradation appears to have arisen relatively recently. A role for Skp2 in Cdt1 degradation has only been demonstrated in humans, and the pathway is not conserved in yeast, invertebrates, or even among other vertebrates.In eukaryotes, DNA replication is strictly regulated so that it occurs only once per cell cycle. The mechanisms that prevent excessive DNA replication are focused on preventing replication origins from being reused within the same cell cycle. This regulation involves the temporal separation of the formation of the pre-replicative complex (pre-RC) from the initiation of DNA replication. The replication licensing factors Cdt1 and Cdc6 recruit the presumptive replicative helicase, the Mcm2-7 complex, to replication origins in late M or G1 phase to form pre-RCs. In fission yeast and metazoa, the Cdt1 licensing factor is degraded at the start of S phase by ubiquitin-mediated proteolysis to prevent the reassembly of pre-RCs. In humans, two E3 complexes, CUL4-DDB1 Dbf4-dependent kinase) activity. This phosphorylation allows the recruitment of essential replication factors, including Cdc45, Mcm10, RPA, proliferating cell nuclear antigen (PCNA), and DNA polymerases α and δ.To maintain genome integrity, DNA replication must be strictly regulated to occur only once per cell cycle. Replication is, therefore, tightly regulated to prevent the re-initiation of DNA replication within the same S phase. A failure to restrict DNA replication results in 're-replication', in which the genome is over-replicated within the same cell cycle via origin re-firing. In eukaryotes, the extent of DNA replication is controlled by temporally restricting the assembly of the pre-replicative complex (pre-RC) through a process termed 'replication licensing' . Pre-Cdt1 and Cdc6 are essential loading factors for the Mcm2-7 complex, and they are negatively regulated during S phase to ensure that the Mcm2-7 complex cannot re-bind to origins that have already fired. In budding yeast, Cdt1 is exported from the nucleus during S phase . In contDrosophila and vertebrates, Cdt1 activity is redundantly regulated by its degradation and the binding of a Cdt1-inhibitor called Geminin [Drosophila and in certain human cell lines but not in others [In  Geminin . Loss ofn others -11. In hn others . HeLa cen others .C. elegans and Xenopus embryos, loss of Geminin is not associated with re-replication [C. elegans is associated with re-replication [Xenopus egg extract [In both lication -16. In clication . Likewis extract .Cdt1 is degraded by the ubiquitin-proteasome system. In this pathway, ubiquitin ligases (E3s) provide the specificity for the degradation because they bind specific substrates and then facilitate the transfer of ubiquitin from the ubiquitin conjugating enzyme (E2) to the substrate . The covCDT2 and SCFSkp2, have been reported to target Cdt1 for ubiquitin-mediated degradation. Both of these E3s are members of the cullin-RING ligase (CRL) class of ubiquitin ligases. The two E3 complexes utilize distinct mechanisms for targeting Cdt1 ubiquitination. In this review, we will focus on the regulation of Cdt1 degradation in different species and explore the conservation of pathway components and mechanisms across species and phyla.In humans, two distinct E3 complexes, CUL4-DDB1C. elegans first suggested the involvement of CUL4 in Cdt1 degradation. The inactivation of the C. elegans cul-4 gene by RNAi causes proliferating cells to arrest in S phase and undergo massive levels of DNA re-replication [C. elegans, as in vertebrates and fission yeast, CDT-1 is degraded as cells enter S phase [cul-4 is inactivated, CDT-1 is not degraded in S phase, but instead accumulates in the re-replicating cells [cul-4 RNAi cells, indicating that CDT-1 accumulation is a critical factor in causing the re-replication. This work showed that CUL-4 negatively regulates CDT-1 levels, but did not address whether CDT-1 is a direct target of the CUL-4 complex. It was subsequently shown in humans, Xenopus, fission yeast, and C. elegans that the CUL4 ubiquitin ligase directly mediates Cdt1 degradation during S phase [Studies in lication . The DNA S phase . Howeverng cells . Reducti S phase ,16,21,22Drosophila, Cdt1 is rapidly degraded in response to DNA damage induced by UV- or γ-irradiation, presumably to prevent DNA replication until the DNA damage can be repaired [In humans and repaired . CUL4 isrepaired ,24. The repaired . The CULrepaired ,22-24. GThe cullin-RING ubiquitin ligase (CRL) complexes represent the largest super-family of multisubunit E3s in eukaryotes . The prodamaged DNA-binding protein) [C. elegans, humans, and fission yeast [CUL4 complexes contain the RING finger protein Rbx1 and the adaptor DDB1 (protein)  ; CDW (CUL4 and DDB1-associated WD40 repeat proteins); and DWD (DDB1-binding and WD40 repeat proteins) [The crystal structure of the CUL4-DDB1 E3 complex has recently been determined ,28. DDB1roteins) ,33-35.CSA targets the degradation of the nucleotide excision repair protein CSB [hDET1-hCOP1, which includes a dimeric SRS consisting of hDET1 and hCOP1, targets the degradation of the transcription factor c-jun [VprBp is known to be hijacked by the Vpr protein of the human immunodeficiency virus (HIV) to induce cell cycle arrest [DDB2 mediates stable ubiquitin modifications of histones H2A, H3 and H4 and the nucleotide excision repair protein XPC [CDT2 has been implicated in the degradation of mammalian p53, fission yeast Spd1, and Cdt1 (described below), [The DCAF family is predicted to contain ~90 members in mammals, of which at least 49 have been shown to physically interact with CUL4 or DDB1, mainly by co-expression/co-IP in mammalian cells ,33-35. Ftein CSB . CUL4-DDor c-jun . CUL4-DDe arrest . CUL4-DDtein XPC -40. Fina below), ,33,41,42in vivo [Xenopus, zebrafish, and fission yeast, CDT2 is required for Cdt1 degradation during S phase and in response to DNA damage [Xenopus egg extract, CDT2 is required to load the CUL4-DDB1CDT2 complex onto chromatin in response to Cdt1 binding to PCNA, suggesting that the interaction of CDT2 with Cdt1 mediates the localization [The DCAF protein CDT2 physically interacts with CUL4 and DDB1 in vivo ,33,41. IA damage ,26,33,41in vitro translated C. elegans DDB-1 made with a wheat germ extract binds to bacterially-produced recombinant GST-CDT-1 [C. elegans experiments, however, do not rule out the possibility that a plant protein from the wheat germ extract bridges the interaction between DDB-1 and CDT-1. Additional experiments will be required to clarify exactly how the CUL4-DDB1CDT2 complex binds to Cdt1.There is also evidence that DDB1 may function directly as the SRS for Cdt1 binding. It has been reported that purified human Cdt1 can bind directly to purified DDB1 . FurtherST-CDT-1 . The C. Xenopus egg extract, the degradation of Cdt1 by CUL4-DDB1CDT2 in S phase requires the interaction between Cdt1 and PCNA [PCNA-interacting protein (PIP) box motif in the Cdt1 N-terminus [C. elegans [Drosophila, and fission yeast [In and PCNA ,33  motif within Cdt1 [Skp2 regulates Cdt1 levels in response to CDK-phosphorylation  [Analysis of published results suggests that SCFDB1CDT2) . This imDB1CDT2) .C. elegans. Inactivation of C. elegans cul-4 or ddb-1 fully stabilizes CDT-1 during S phase [C. elegans Skp2 homolog, skpt-1, does not contribute to CDT-1 degradation or re-replication even in a sensitized ddb-1 mutant background [skpt-1 null mutant homozygotes are completely viable and appear overtly wild-type with the exception of a low-penetrance gonad migration defect, indicating that the gene is not required for any essential functions [The roles of Skp2 and CUL4 in degrading Cdt1 have also been explicitly compared in  S phase ,17. In cckground . skpt-1 unctions .Xenopus egg extract, CDK-phosphorylation of Cdt1 is not required for Cdt1 degradation [Skp2 is not required for Cdt1 degradation because CDK-phosphorylation of Cdt1 is a prerequisite for recognition by human Skp2 [Skp2 substrates must be phosphorylated to be recognized [Xenopus egg extract, with DDB1 depletion blocking Cdt1 degradation during S phase [CDT2 is the predominant E3 for Cdt1 degradation in Xenopus, and that SCFSkp2 either has no role or has only a minor, subservient role in Cdt1 degradation.In radation . This imman Skp2 ,48, and cognized . In cont S phase . These rDrosophila, mutation of all of the N-terminal CDK-phosphorylation sites of Cdt1 is not able to block S-phase degradation, although it does provide a limited increase in overall stability [Skp2) either is not involved or is redundant for Cdt1 degradation during S phase in Drosophila. There are currently no reports on the function of the fly Skp2 homolog.In tability . This inCDT2 complex. Fission yeast CUL4-DDB1CDT2 is essential for the degradation of Cdt1 during S phase and in response to DNA damage, indicating that it is the dominant pathway for regulating Cdt1 levels [Skp2-mediated degradation of Cdt1 is not conserved in non-mammalian species  do not have elevated levels of Cdt1 [-/- knockout mice have elevated Cdt1 levels in proliferating tissues [-/- MEFs [The studies described above suggest that SCF of Cdt1 ,50. Howe of Cdt1 ,52. In c tissues . Further-/- MEFs . These r-/- knock-out mice are completely viable and fertile [Kip1, p21Cip1, cyclin E, cyclin D, cyclin A, c-Myc, b-Myb, p130/pRb2, E2F-1, p57Kip2, MKP-1, RAG-2, FOXO1, and Cdk9 [-/- mice are viable, they exhibit a minor defect of polyploidy and extra centrosomes in the cells of a few tissues [Kip1, suggesting that the inability to degrade p27Kip1 causes the defect [CDT2 is the predominant ubiquitin ligase to mediate Cdt1 degradation in mice, and that SCFSkp2 either does not target Cdt1 for degradation or does so only as a minor pathway that cannot compensate for loss of CUL4-DDB1CDT2.It is interesting that Skp2 fertile . This isand Cdk9 ,50,54-73 tissues . Signifie defect . The lacCDT2 and SCFSkp2 complexes arose during evolution, we analyzed divergent species using reciprocal BLAST searches [To determine when the genes for the CUL4-DDB1searches . We limisearches . The adaCDT2 complex, is more ancient than Skp2 and the SCFSkp2 complex.The substrate-specific components CDT2 and Skp2 appear to have arisen at different points in eukaryotic evolution. CDT2 is observed in all animals analyzed, and a majority of fungi and plants, but is not observed in protists  does not contain CUL4, DDB1, or CDT2  declare that they have no competing interests.


Exposure to short periods of very loud noise can cause tinnitus—a persistent ringing or buzzing in the ears that cannot be blocked out. Tinnitus may affect around 10%–15% of the population; severe tinnitus is very debilitating (1%–2% of the population). Previous work has shown that tinnitus has a neurophysiological basis, but precisely which parts of the brain and the auditory circuits are involved is not yet understood.The human ear is essentially a very sensitive vibration sensor, one that is able to receive the minute longitudinal vibrations in air that make up sound waves. It can detect sounds from 20 Hertz (Hz) (very low pitch) to 20,000 Hz (very high pitch) but is particularly sensitive to sounds in the range of 500–5,000 Hz—the so-called speech frequencies. However, the ear, and in particular the cochlea, or inner ear, can be damaged by exposure to excess noise, leading to permanent damage to the ear, i.e., deafness.Some studies in both animals and humans have suggested that tinnitus and hearing loss may be related. These studies have found that neurons in regions of the auditory cortex that have been deprived of stimuli because of hearing loss change their receptive field and may develop enhanced spontaneous activity. Other studies, such as some involving neuroimaging using positron emission tomography, have suggested that parts of the brain involved in attention and emotional regulation might be involved in the production of tinnitus.One of the key research targets in tinnitus has been investigation of cortical activity, especially in animal models of tinnitus, but studies in humans have been rare. Previous studies have identified temporal and frontal temporal changes in individuals whose tinnitus is severely disabling; however, there have been no group studies comparing abnormalities of ongoing, spontaneous neuronal activity in people with and without tinnitus.PLoS Medicine, Nathan Weisz and colleagues studied 17 patients with chronic tinnitus and hearing loss and 16 control individuals with normal hearing. Patients were asked to fill in a questionnaire about the impact of tinnitus on their lives and had their levels of tinnitus assessed.In this month's The team's methods differed from previous work in that the team chose to examine the power spectrum of neuromagnetic oscillatory activity during rest, whereas previous studies had focused on measuring neurophysiological responses following sounds.Normally in awake and healthy subjects a certain rhythm of brain activity at 8–12 Hz—the so-called alpha rhythm—is dominant. Finding enhanced slow-wave, or delta, activity (<4 Hz) in awake subjects is usually a sign of a dysfunctional neuronal network, as these waves can be observed in various neurological and psychiatric disorders. Weisz and colleagues' analysis of the frequency spectrum of recorded magnetic fields revealed that the energy in the alpha band was strongly reduced and that of the delta band enhanced in the group with tinnitus compared with the individuals with normal hearing. This pattern was particularly pronounced in the temporal regions, and overall the effects were stronger for the alpha than for the delta frequency band.This is the first study to show these changes in delta and alpha spontaneous cortical activity, say the authors. But they concede it is still unclear whether the enhancement of delta activity compared with alpha is the abnormal activity perceived as tinnitus. However, the fact that regions that show slow-wave activity during slow-wave sleep are also regions of low alpha activity supports the idea that changes in cortical activity might be mediated by sensory deprivation, in this case that partial hearing loss might be involved in producing tinnitus.Tinnitus-related distress as assessed by the questionnaire was strongly associated with this abnormal spontaneous activity, especially in the right temporal and left frontal areas, thus pinpointing a possible tinnitus-related cortical network.A limitation of this study was that the tinnitus group also had high-frequency hearing loss, whereas the control group did not; the ideal control group would have been patients with the same sort of hearing loss but no tinnitus.In discussing their findings, the authors suggest that their study supports previous work indicating that the prefrontal cortex is a candidate region for integration of the sensory and emotional aspects of tinnitus. Further studies should focus on frontal areas, which could allow identification of interactions and modulating influences that higher-order psychological processes  may have on the generation of tinnitus in the auditory cortex.


Skp2-mediated proteolytic regulation through phosphorylation by Cdks; and (2) replication-coupled proteolysis mediated by the Cullin4-DDB1Cdt2 ubiquitin ligase and PCNA, an eukaryotic sliding clamp stimulating replicative DNA polymerases. The tight regulation implies that Cdt1 control is especially critical for the regulation of DNA replication in mammalian cells. Indeed, Cdt1 overexpression evokes chromosomal damage even without re-replication. Furthermore, deregulated Cdt1 induces chromosomal instability in normal human cells. Since Cdt1 is overexpressed in cancer cells, this could be a new molecular mechanism leading to carcinogenesis. In this review, recent insights into Cdt1 function and regulation in mammalian cells are discussed.In eukaryotic cells, replication of genomic DNA initiates from multiple replication origins distributed on multiple chromosomes. To ensure that each origin is activated precisely only once during each S phase, a system has evolved which features periodic assembly and disassembly of essential pre-replication complexes (pre-RCs) at replication origins. The pre-RC assembly reaction involves the loading of a presumptive replicative helicase, the MCM2-7 complexes, onto chromatin by the origin recognition complex (ORC) and two essential factors, CDC6 and Cdt1. The eukaryotic cell cycle is driven by the periodic activation and inactivation of cyclin-dependent kinases (Cdks) and assembly of pre-RCs can only occur during the low Cdk activity period from late mitosis through G1 phase, with inappropriate re-assembly suppressed during S, G2, and M phases. It was originally suggested that inhibition of Cdt1 function after S phase in vertebrate cells is due to geminin binding and that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance induces re-replication. However, recent progress has revealed that Cdt1 activity is more strictly regulated by two other mechanisms in addition to geminin: (1) functional and SCF It is now clear that the "once and only once replication per single cell cycle" is achieved by the periodic assembly and disassembly of essential pre-replication complexes (pre-RCs) at replication origins . Fir. Fir2]. Drosophila tissue culture cells [Skp2 [To prevent re-replication, the re-establishment of pre-RC, in other words re-binding of MCM, needs to be suppressed during the S, G2 and M phases of the cell cycle Figure . Cdks plre cells . Cdks prre cells -3. One ire cells  while inre cells -39. Suchre cells -42. In hls [Skp2 ,43. It hls [Skp2 ,44. In bls [Skp2 . Also inls [Skp2 ,37,38,46Xenopus, and found to inhibit pre-RC formation through prevention of the loading of MCM complexes [geminin is driven by the E2F transcription factor [Geminin was originally identified as a novel APC/C substrate in omplexes . Subsequomplexes ,8,47. Sin factor  and gemin factor -8.Xenopus egg extracts, immunodepletion of geminin does not induce extensive re-replication [Drosophila tissue culture cells, in contrast to cyclin A depletion, geminin depletion results in only partial re-replication [e.g. even when Cdk activity is down-regulated by the checkpoint mechanism in cells undergoing DNA damage, re-formation of pre-RC can be prevented by geminin.At least in certain cancer-derived cell lines, depletion of geminin by siRNA induces some re-replication ,11. Therlication -15. Alsolication . TherefoRecently, new functions of geminin, independent of Cdt1 binding and related to transcriptional regulation, have been successively uncovered -52. ConsSkp2 recognition, showed only partial resistance to degradation during S phase in Rat-1 cells [Skp2 binding are still degraded with comparable efficiency to the wild type [in vitro DNA binding activity [Although Cdt1 function is suppressed by geminin after S phase, Cdt1 might also be regulated by Cdks. Indeed, it was found that Cdt1 is phosphorylated by cyclin A-dependent kinases (cyclin A/Cdk1 and cyclin A/Cdk2) dependent on the RXL-type cyclin-binding motif (Cy motif) ,17. Cdk -1 cells . Also inild type . These factivity . In cellactivity . Cdk phoDrosophila [Unscheduled Cdt1 hyperfunction induces re-replication  and chroosophila .Cdh1 ubiquitin ligase and Cdks restrains this ligase by phosphorylating Cdh1 subunit [In mammalian cells, Cdk1 inactivation in G2 phase promotes inappropriate re-licensing . The que subunit ,24. Ther subunit . A more Recently, Cul4-based ubiquitin ligase- and PCNA-mediated proteolysis has emerged as a third mechanism regulating Cdt1 during the S phase Figure . Four grC. elegans, in which ablation of Cul4 by RNAi induces re-replication which is in turn suppressed by removal of one genome copy of Cdt1 [C. elegans Cdt1 protein is decreased as cells enter S phase, and this decrease disappears with Cul4 depletion. Thus, the Cul4 ubiquitin ligase appears to regulate Cdt1 stability during S phase, albeit by a mechanism which remains to be elucidated. Subsequently, it was found that in human cells, Cdt1 is rapidly targeted for proteasome-mediated degradation after DNA damage by UV irradiation and that this proteolysis involves ubiquitination of Cdt1 by Cul4-DDB1 ligase [in vitro and that DDB1 can directly bind to Cdt1 [Cul4 is a member of the Cullin ubiquitin ligase family ,58, toge of Cdt1 . The lev1 ligase ,62. It h to Cdt1 ,62. HoweC. elegans to mammalian cells [Skp2- and Cul4-DDB1-mediated ubiquitination. Indeed, co-inhibition of both pathways by siRNA can stabilize wild type human Cdt1 in S phase [in vitro DNA replication system using Xenopus egg extracts although the Cdk-SCFSkp2 system does not seem to operate in this case [C. elegans, Cul4-mediated degradation of Cdt1 may be of prime importance in this organism although it remains possible that Cul4 is also involved in other systems associated with replication control (see below). Interestingly, Cdt1 proteolytic regulation involving PCNA and Cul4 is also observed in fission yeast [Recent progress has resolved this question. Cdt1 has a PCNA-interaction protein motif  ,64 in than cells -21. As man cells ,21. Silean cells ,21. Thus S phase ,21. Essehis case . Consideon yeast . We can E. coli also employs a similar strategy to prevent re-initiation of DNA replication, where an initiation factor, DnaA is inactivated via a prokaryotic sliding clamp, the β clamp [PCNA, a eukaryotic sliding clamp, is present in the nuclei throughout the cell cycle and loaded onto the primer ends synthesized on unwound DNA during S phase. Then, it recruits replicative DNA polymerases and stimulates their activity ,64. PCNA β clamp ,66. Thus β clamp -21. The Xenopus p27Xic1 Cdk inhibitor is also targeted to degradation in a PCNA-dependent manner, although involvement of Cul4 has not been examined [Several other important points also need to be resolved. Cul4-DDB1 ubiquitin ligase may recognize an interface generated by Cdt1-PCNA interaction, but it is unclear how this ligase only recognizes Cdt1-PCNA complexes formed on chromatin. One simple explanation is that functional Cul4-DDB1 could be always tethered to chromatin. Since there are many PCNA-binding proteins, it is of interest why Cdt1 (and possibly some other targets) is ubiquitinated by Cul4 and brought to proteolysis while others are not. In this context, it should be noted that the examined . Presumaexamined -59. In texamined ,17. So, examined ,69. It sexamined . Finallyexamined ,71. TherXenopus egg extract system [The fact that Cdt1 activity is controlled by multiple mechanisms in human cells suggests that its deregulation may induce more deleterious insult than with other pre-RC components such as ORC1 and CDC6. Indeed, it is reported that overexpression of Cdt1 can induce overt re-replication in cancer-derived cell lines, with activation of ATM/ATR checkpoint pathways . This cot system -15, a syt system . Under pt system . It has t system . On the t system ,11. The In non-transformed cultured cells, Cdt1 overexpression at pathophysiological levels appears not to induce re-replication but nevertheless leads to chromosomal damage, as revealed by activation of the ATM-Chk2 DNA damage checkpoint pathway . FurtherCdt2 by RNAi [Xenopus egg extract system, PIP motif-mutated Cdt1 is stabilized and induces more re-replication than the wild type [There is evidence that silencing of geminin induces re-replication, suggested to be attributable to Cdt1 deregulation ,11. Howe by RNAi ,68,75, i by RNAi ,54. On tild type . This coIt has been reported that Cdt1 overexpression endows murine NIH3T3 cells with the capacity to form tumors in nude mice . HoweverCdt1 transcription is driven by E2F transcription factor [Importantly, Cdt1 protein is actually overexpressed in human cancer cells ,79,22. Cn factor , which iSkp2-mediated proteolysis, and by replication-coupled proteolysis involving Cul4-DDB1Cdt2 and PCNA. Details of these multiple regulations have only recently been uncovered and many questions remain, including how Cdt2 specifically recognizes interfaces between Cdt1 and PCNA on chromatin and how Cul4-DDB1 ubiquitin ligase coordinates several related chromatin transactions. As expected from the strict regulation, deregulation of Cdt1 is a deleterious insult, leading to re-replication and/or chromosomal damage. The induced chromosomal instability may eventually lead to carcinogenesis and Cdt1 overexpression is in fact often observed in human cancers. The mechanisms by which Cdt1 can damage chromatin without inducing re-replication are unclear at present and this is an interesting research challenge. Elucidation could provide valuable information regarding the essential question of how Cdt1 functions in MCM loading reaction.In mammalian cells, Cdt1 is a very central player in regulation of DNA replication. During the cell cycle, strict control is exerted by geminin, by Cdk phosphorylation and subsequent SCFThe author(s) declare that they have no competing interests.MF wrote the entire manuscript.


Despite a variety of attempts there has been very limited success in the area of environmental control of allergic disease. Our objective was to identify a non-invasive, non-pharmacological method to reduce indoor allergen loads in atopic persons' homes and public environments. We employed a novel Fifty skin test reactive patients were simultaneously skin tested with conventional test materials and the actions of the protein/glycoprotein modifier, aluminum sulfate. Common allergens, dog, cat, dust mite, Alternaria, and cockroach were used in the study.Skin test reactivity was significantly reduced by the modifier aluminum sulfate. Our studies demonstrate that the effects of histamine were not affected by the presence of aluminum sulfate. In fact, skin test reactivity was reduced independent of whether aluminum sulfate was present in the allergen test material or removed prior to testing, indicating that the allergens had in some way been inactivated.in vivo allergic reaction cascade induced by skin testing with common allergens. The exact mechanism is not clear but appears to involve the alteration of IgE-binding epitopes on the allergen. Our results indicate that it may be possible to diminish the allergenicity of an environment by application of the active agent aluminum sulfate, thus producing environmental control without complete removal of the allergen.Aluminum sulfate was found to reduce the  The various clinical manifestations of type 1 hypersensitivities and their resultant comorbid illnesses are well known. A common feature shared by these is the mechanism by which they are induced, driven by cytokines produced by Th2 lymphocytes, resulting in IgE antibody production. Antigen specific IgE antibodies cause multiple preformed mediators to be released from mast cells and blood basophils. These preformed cytokines interact with their receptors on target cells inducing a cascade of reactions with late phase mediator formation, leading to sustained symptoms . Thus, aAlthough various attempts have been made to control environment allergens, most have met only limited success ,6. Chemiin vivo approach to examine the possibility of using aluminum sulfate (AS), to control environmental allergens and to inhibit allergic reactions. This chemical seemed to be a good candidate for this purpose because of its ability to bind to proteins [In this study, we employed a novel proteins , its lonproteins ,19. BecaIndividuals were recruited from a large private allergy practice in Louisville, KY, between 1999 and 2005. The age range was from 10 to 66 yrs. These patients had clinical symptoms of rhinitis, asthma, conjunctivitis, chronic sinusitis or a combination of 2 or more of the manifestations of Type I hypersensitivity reactions. The protocol to be used in the study was explained in detail to the patients and they were given the option of participating in the study, informed consent was granted. If their prescribed routine allergy skin test produced a high level of reactivity to one of the selected test antigens they were included in the study groups. No volunteer was compensated monetarily or otherwise for his or her participation and no funding for the study came from outside sources.2(SO4)3,  was prepared by dissolving proper aliquots in sterile water. Using the same diluent as for the allergens , 8.75% (ASα) and 34.2% (ASβ) solutions of the chemical were prepared. Prior to use, they were filtered using a micropore filter #4, placed in sterile containers, and stored at 4°C until used in skin test.Aluminum sulfate, AlToxicity tests were performed using cultured human endothelial cells  and the trypan blue dye exclusion test to evaluate the safety of using AS in human applications . Cells wInitial screening of patients involved only routine inhalant skin test with the following allergens: animal danders, cockroach, dust mite, mold spores and pollens of grasses, weeds and trees prevalent in the Ohio River Valley. They were obtained from Greer Labs  and used at a standard prick test concentration. Those patients who produced high skin reactions  to cat, dog, Alternaria, dust mites, cockroach or multiples there of were selected to undergo additional skin tests with these same allergens mixed with AS. AS-allergen preparations were created by mixing 0.1 ml ASα (8.75%) to 0.9 ml of the skin test dose allergen or 0.1 ml ASβ (34.2%) to 0.9 ml of the skin test dose allergen. No precipitate was observed upon mixing of the allergen and AS. All skin reactions were read 20 min after the prick test was applied.The diameter of the patients' skin test responses to allergens or allergens mixed with AS were measured using skin test calipers and recorded in terms of size in mm of induration  at the largest diameter. Clinical score: a conventional grading system of + to 4+ was also used as determined by these parameters: (+) = 3 to 5 mm wheal; (++) = 5 to 7 mm wheal; (+++) = 7 to 10 mm wheal; (++++) = 10 mm or greater wheal; erythema (E) and pseudopod (P) were also observed and recorded. P-values were determined using an unpaired two-tailed Student's t-test .Histamine base solution (1.8 mg/ml and 50% glycerol wt/vol), obtained from Allermed Labs , was injected intradermally alone and mixed with AS (at a concentration of 0.9% and 9.0%). Controls included 0.9% NaCl, 0.4% phenol. After 20 min, lesion diameters were measured as before using skin test calipers. In some cases, reactive sites that had previously been injected with only histamine, received a second injection with just AS. As before, responses were read 20 min later.Lyophilized cat dander was purchased from Greer Labs  and suspended in 100 microliters of sterile water to a concentration of 29 mg/ml. Next, 15 microliters was added to either 1 ml sterile water (sample 1) or 1 ml 9% AS (samples 2 & 3). Sample 1 was then dialyzed for 6 h against 1 liter of sterile water, sample 2 was dialyzed for 6 h against 1 liter of 9% AS, and sample 3 was dialyzed for 6 h against 1 liter of sterile water. Final allergen concentration was estimated to be approximately 50,000 BAU. Samples were then used for skin testing in three patients sensitized to cat allergen with inclusion of proper controls, sterile water as sample 4 and 9% AS solution as sample 5. The experiment was repeated using Centricon ultrafiltration devises  as a different means to dialyze the samples. Dialysis was accomplished by addition of 2 ml of either 9% AS or water following complete concentration of the samples, this was repeated four times to ensure complete dialysis of the samples was accomplished.2 at 37°C. Cells were collected and a trypan blue dye exclusion test was performed (see Methods section). The results indicate that 99.9% of the cells were still viable at the end of a 24 h exposure to the chemical, for the dilutions tested. It should also be noted here that no toxicity was observed in vivo using the skin prick test.A preliminary experiment was done to determine if AS exhibited any cellular toxicity. As previously noted, 1:10, 1:20, or 1:30 dilutions  of ASβ were placed in established human endothelial cell cultures and incubated for 24 h with 5% COin vivo can more readily be seen by the photographic evidence presented in Figure In this experiment, a group of 50 patients were used. They were selected for testing with the AS-allergen mixtures based on their level of sensitivity to routine allergy skin tests. For this purpose, only those allergens that had induced a +4 reaction (10 mm or greater) were mixed with AS for further skin testing. The allergens and their concentrations used are indicated in Table Methods section). The results indicate that histamine induced skin reactions cannot be blocked by AS , dialyzed against water to remove the AS, and then used for skin testing in several sensitized patients. Results shown in Figure In order to be sure that AS was altering the allergen, and not modulating the hypersensitivity reaction by some other mechanism, mast cell stabilization for example, a simple experiment was performed. Cat allergen was mixed with AS  will result from AS application to the environment. Active investigation is currently underway to evaluate the effectiveness AS have in inactivating allergens in the environment.Aluminum sulfate does not appear to block histamine-induced responses. Skin reactions were not influenced by mixing AS with histamine or by injecting histamine into sites previously injected with AS Fig. . Its effAS was found to significantly reduce the skin test response in sensitized patients to each of the allergens tested: dust mite, Alternaria, dog, cat, and cockroach. The exact mechanism in which AS produces this effect is not known. However, our results demonstrate that AS does not block the effects of histamine, and produces its effect without being present in the skin test sample. It appears that AS alters the allergen, changing its epitopes, thus reducing the ability of specific IgE to bind and ultimately mast cell degranulation. AS does not stain, is cheap, nontoxic, is stable in solution, and appears to have long acting affects, making it a great candidate for use as an environmental control agent.AS, Aluminum sulfate; ID, intradermal; STR, skin test responseThe author(s) declare that they have no competing interests.CSS performed the patient testing, along with the clinical study design. SAS, TJG, and DEJ performed the laboratory testing and study design. All of the authors contributed to manuscript preparation.


However, there are no published data evaluating the impact of genetic manipulations on the allergenicity of GM potatoes. To compare the allergenicity of GM potatoes with that of wild-type potatoes using A total of 1886 patients with various allergic diseases and 38 healthy controls participated in the study. Skin-prick testing and IgE-ELISA were carried out with extracts prepared from wild-type and GM potatoes. An ELISA inhibition test was used to confirm the binding specificity. IgE-binding components in extracts from the two types of potato were identified by SDS-PAGE and IgE-immunoblotting. The effects of digestive enzymes and heat on the allergenicity of the extracts was evaluated by preincubating the potatoes with or without simulated gastric and intestinal fluids in the absence or presence of heat.Positive responses  ≥ 2+) to wild-type or GM potato extracts, as demonstrated by the skin-prick test, were observed in 108 patients (5.7%). Serum-specific IgE was detected in 0–88% of subjects who tested positively. ELISA inhibition tests indicated significant inhibition when extract from each type of potato was added. IgE-immunoblot analysis demonstrated the presence of 14 IgE-binding components within the wild-type potato and 9 within the GM potato. Furthermore, a common 45-kDa binding component that yielded similar IgE-binding patterns was noted in more than 80% of the reactions using sera from patients sensitized to wild-type or GM potato. Exposure to simulated gastric fluid and heat treatment similarly inhibited IgE binding by extracts from wild-type and GM potatoes, whereas minimal changes were obtained following exposure of the extracts to simulated intestinal fluid.Our results strongly suggest that genetic manipulation of potatoes does not increase their allergenic risk. The sensitization rate of adult allergy patients to both types of extract was 5.7%, and a common major allergen (45 kDa) was identified. Food-induced allergic reactions are responsible for a variety of symptoms involving the skin, gastrointestinal tract, and respiratory tract, and proceed through IgE- and non-IgE-mediated mechanisms. The major foods responsible for food-induced allergic reactions are milk, eggs, peanuts, fish, and tree nuts in children, and peanuts, tree nuts, fish, and shellfish in adults . There aAgricultural biotechnology has tremendous implications for both agriculture and the general public. Insect-resistant corn and herbicide-tolerant soybeans are grown on 30–50% of the total acreage planted with these crops in North America . PreviouIn this study, the sensitization rates of adult allergy patients in response to wild-type and GM potatoes were evaluated by skin-prick test and ELISA (enzyme linked immunosorbent assay). SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and IgE-immunoblotting were carried out to identify the major allergens present in the potato extracts. To evaluate the effects of digestive enzymes and heat on the allergenicity of the two types of potato, the extracts were preincubated with or without simulated gastric and intestinal fluids in the presence or absence of heat.Sensitization rates to the two potato extracts were evaluated in 1886 allergy patients and in 38 healthy non-atopic subjects. The participants, who ranged in age from 15 to 65 years, were enrolled in the study by the Department of Allergy and Rheumatology, Ajou University School of Medicine, Suwon, Korea. The GM potato, carrying the neomycin phosphotransferase II (NPTII) and phosphinothricin acetyltransferase (PAT) genes , kept at 4°C overnight, and then centrifuged at 12,000–15,000 rpm for 20 min. The supernatant was dialyzed  against 4 l of PBS at 4°C for 72 h, and the resultant fluid was stored at -20°C until ELISA, ELISA inhibition testing, and immunoblot analyses were carried out. For the skin-prick test, extract was mixed with sterile glycerin at a ratio of 1:1. To evaluate the effects of digestive enzymes, simulated gastric fluid  and simulated intestinal fluid  were preincubated with the extracts in the presence or absence of heat.The presence of specific IgE antibodies to the two types of extract was determined by ELISA, as previously described . MicrotiThe colorimetric reaction was developed with a TMB  substrate solution for 15 min at room temperature. The reaction was stopped by the addition of 100 μl of 2 N sulfuric acid, and absorbance was read at 450 nm using an automated microplate reader . All assays were carried out in duplicate. Positive cutoff values were determined from the mean plus two standard deviations of the absorbance values of the 38 healthy controls.Dermatophagoides pteronyssinus or extract from GM or wild-type potatoes, and then incubated for 12 h in microtiter plates coated with extracts from the two types of potato. The same steps were followed as in the ELISA. As a control, samples were preincubated with equal volumes of PBS (pH 7.5) instead of house dust mite or potato extracts. The percentage of inhibition of serum IgE binding was expressed as: 100 -  × 100.The specificity of IgE binding to the potato extracts was tested and the allergenic potency of the GM and wild-type potato extracts was analyzed by a competitive ELISA inhibition test. Sera from four patients with high levels of specific IgE binding were pooled, preincubated overnight at 4°C with five concentrations (1–100 μg/ml) of either SDS-PAGE and immunoblot analysis were carried out under reducing conditions according to previously described methods . Extract2CO3 (200 mmol/l). The mixture was then boiled for 5 min and stored at -20°C until further analysis. To evaluate the effects of SIF, 680 μg of the naïve crude extract or the preheated extract were dissolved in 260 μl of prewarmed intestinal control solution  containing 1.0% (w/v) pancreatin . This solution was incubated at 37°C with continuous shaking. Aliquots (26 μl) were withdrawn at 1, 90, and 240 min, mixed with 26 μl of sample buffer (containing 2.5% 2-mercaptoethanol and 1% SDS), and then boiled for 5 min. SDS-PAGE (12%) and IgE-immunoblot analysis were then carried out as described above.Crude extracts were prepared from GM and wild-type potatoes and then heated at 100°C for 5 min. The intrinsic digestibility of the extracts and the digestibility of extracts preheated and incubated in SGF or SIF were examined as previously described . BrieflyDuring the one-year study, 108 (5.7%) of the 1886 patients had an A/H score >2+ in response to the skin-prick test using extracts from GM and wild-type potatoes. All of the subjects who reacted positively to the GM potato also had positive responses to the wild-type potato, as seen in Table D. pteronyssinus. Figure Dermatophagoides pteronyssinus. The same pooled sera from patients sensitized to potatoes were used .The ELISA inhibition test showed significant dose-dependent inhibition for wild-type and GM potato extracts. In addition, the two extracts had similar potencies. By contrast, minimal inhibition was noted using The IgE-binding components within the wild-type and GM potatoes were compared using the sera of eight individuals with high specific IgE levels and sera pooled from the controls. The latter was derived from 10 patients who responded negatively to the two potato extracts on the skin-prick test. Three components  were noted in >50% and 11 components  in 33% of patients sensitized to wild-type potato. One component (45 kDa) was noted in 88% and eight components  in <50% of patients sensitized to GM potato. Thus, the 45-kDa band present in serum was the most frequently bound (>80%) by extracts from wild-type and GM potatoes  by the two types of extract, and ELISA inhibition studies suggested that they had similar potencies. Recent investigations to evaluate the allergic risks of GM corn and soybean demonstrated that allergenicity did not increase after genetic manipulation of wild-type corn  and soybNPTII gene introduced in the potato used in this study encodes an enzyme that confers resistance to aminoglycoside antibiotics and was isolated from the prokaryotic transposon Tn5 [PAT gene was obtained from the aerobic soil bacterium Streptomyces viridochromogenes. If the gene source is bacterial, specific and targeted serum screenings are not necessary because bacterial proteins are rarely allergenic, due to the low exposure levels and lack of allergic sensitization to these proteins [GM crops currently on the market have been thoroughly assessed for safety according to the guidelines developed by the World Health Organization  and the oson Tn5 . The PATproteins . Furtherproteins ,21. Heroproteins  found thA few studies have identified IgE-binding components within potato extracts. Wahl et al.  reportedProteolytic stability is a useful criterion in assessing the allergenic potential of food allergens. The FAO/WHO  decisionWe report that the sensitization rate of patients to wild-type and GM potato extracts was 5.7%, and the prevalence of specific IgE to these extracts in the sera of subjects with a positive skin-prick test was similar. An ELISA inhibition test showed significant dose-dependent inhibition by GM and wild-type potato extracts, and the two extracts had similar potencies. A 45-kDa band was identified as the most frequently bound by both extracts. Our results strongly suggest that genetic manipulation of potatoes using antibiotic resistance and herbicide tolerance genes does not increase their allergenic risk.GM = genetically modifiedELISA = enzyme linked immunosorbent assaySDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresisNPTII = neomycin phosphotransferase IIPAT = phosphinothricin acetyltransferaseSGF = simulated gastric fluidSIF = simulated intestinal fluidSKL: ideas, study design and writingYMY: ideas and writingSHY: laboratory workBOL: preparation of GMO and non -GMO potatoSHK: study design and laboratory workHSP: ideas, study design, laboratory work and writing


As in all western countries, Australia's older population experiences high levels of hearing impairment coupled with relatively low levels of hearing device usage. Poor hearing diminishes the quality of life of affected individuals and their families. This paper discusses how to improve Australian hearing health policies in order to better combat this impairment amongst older Australians.Medline, Meditext and Web of Science to find articles that discussed strategies and innovations to assist the hearing health of older people, and related this material to observations made during the Blue Mountains Hearing Study in NSW between 1997 and 2003.We searched the databases The literature search identified five areas for inclusion in a comprehensive hearing health policy in Australia. These are: early intervention; addressing of hearing aid expense; the use of assisted listening devices; hearing rehabilitation, and; screening and education. Further research in Australia is critical if we are to develop a strong approach to the increasing prevalence of age-related hearing loss.Australia needs to act now to address hearing impairment as it is a major cause of disability in those aged 55 and over. Federal and State governments should collaborate to construct a comprehensive hearing health policy that tackles poor levels of hearing health through early intervention, addressing hearing aid expense, encouraging the use of assisted listening devices, rehabilitation, screening and education. A good start would be to declare age related hearing impairment as a National Health Priority Area. Policy-making around hearing health in Australia needs not only commitment but creativity. High rates of hearing impairment among older people, coupled with low rates of hearing aid use, demonstrate that the current policy approach is not working. This is alarming in a society whose population is ageing, as hearing loss is strongly associated with age: in Australia it affects 50% of the population aged 55 and over . There apolicy research. Given the Federal Government's backing of 'Ageing Well, Ageing Productively' as a research priority "to develop better social, medical and population health strategies to improve the mental and physical capacities of ageing people", more attention needs to be paid to the identification and treatment of hearing impairment in older people [The Federal Government has to some extent recognised hearing loss as a growing problem for Australia as the population ages, and has increased funding to hearing services provision . But polr people .Australia needs a stronger commitment to better hearing among older people through an effective and cohesive, national policy on hearing health. This policy needs the backing of a strong research program to provide a firm empirical base – for both its development and its evaluation.This paper will now examine areas that are important in the development of a well-rounded hearing health policy and identify strategies that might lead to the creation of a comprehensive, effective approach to age-related hearing loss in Australia.Medline, Meditext and Web of Science for articles about age-related hearing loss, hearing policy and hearing aids published during the past ten years. 555 articles were identified. We perused the abstracts of these articles and obtained the full text of relevant articles. From there, we used a 'snowballing' technique and looked for relevant articles from the reference lists of the papers not captured in the database search. Using an online search, we identified Australian state and federal government policy documents and guidelines and policy papers from the major hearing lobby groups in Australia, such as the Deafness Forum and Better Hearing.We conducted a literature search to identify what should be included in a hearing health policy for older people for the next ten years for Australia. We searched the databases Five areas emerged as important for the construction of a hearing health policy for older Australians. These areas were: early intervention; addressing of hearing aid expense; the use of assisted listening devices; the construction of a wide-ranging rehabilitation program, and; screening and education.We also sought to identify areas which, while addressed in literature from overseas, provided no specific research data with respect to the Australian population.That hearing loss is associated with poor quality of life among older people has been firmly established -11. EvenBy contrast, with hearing loss, the successful use of hearing aids has been shown to be associated with a significant improvement in both quality and length of life among the hearing impaired ,7,15,16.These findings are supported by other smaller studies, which have also demonstrated similar improvements ,17. In aDespite evidence for a link between increased life quality and hearing aid use by older people with a hearing impairment, both the initial uptake and successful use of hearing devices amongst this group remain low -22. A stThe data from Australian studies into hearing also demonstrate patchy hearing aid use and are largely comparable to those found in studies overseas ,9,12. ThWe will now discuss factors that have been identified in the literature on age-related hearing loss as critical concerns for the development of a comprehensive policy approach to this problem.Earlier intervention for hearing loss through the provision of hearing rehabilitation and/or hearing aids would benefit older people and their families , especiaFirst, auditory deprivation, an inability to adequately recognize speech, may develop as a result of reduced auditory stimulation of the central auditory system ,18,25. ISecond, delayed hearing rehabilitation contributes to the lack of hearing aid retention and use in older people. If a hearing impaired person starts using a hearing aid at an earlier stage, they gain greater benefit from the device ,26. ThisReasons why older individuals may not seek hearing aids at an earlier age include expense, a lack of education about the benefits of both hearing aids and assisted hearing devices and an underestimation  of the level of their hearing impairment . LimitedProhibitive costs are associated with the poor use of hearing devices amongst the elderly Australian population , especiaChing et al. (2003) suggested that the cost is hard to justify for many older people, especially pensioners . The cosWhen hearing aids alone cannot address hearing impairment, assisted listening devices can be used either on their own or in conjunction with hearing aids ,28,36,38Several authors have stated that proposing hearing aids as the preferred solution for hearing impairment amongst the elderly population is inadequate and ignores the value of other strategies ,36,37. TFor people who have problems with visual or manual dexterity or who need hearing assistance in certain situations in which hearing aids function poorly (such as nursing homes), assisted listening devices can be more appropriate ,36,38. CGovernment policies need to enable the more widespread use of assisted listening devices and offer rehabilitation services and greater subsidies for these devices ,16.Hearing impairment requires service as well as technology. Both Australian and international studies demonstrate the benefit of counselling and hearing rehabilitation programs to supplement the provision of hearing aids ,41,42,27Hearing rehabilitation programs should operate both pre- and post-hearing aid fitting and address other non-hearing aid focused approaches ,42. TheyRehabilitation programs should also support families and friends or nursing home staff ,38. HearThe benefits of a strong hearing rehabilitation program are numerous. If counselling is offered when a hearing impairment is first diagnosed, it may help with hearing aid retention. This is because poor use of hearing aids is due partly to a lack of understanding of the consequences that hearing loss has for an individual's social, emotional and physical wellbeing . Prior tCounselling is more successful if it includes a support person who understands both the reality of the hearing loss and is able to assist with the treatment path being followed . AccordiThe combination of hearing aid provision and audiologic rehabilitation has been shown to give a better return than the provision of hearing aids alone . HoweverScreening and case-finding programs allow early and effective intervention. They can be used to identify hearing loss at a younger age and may encourage people who otherwise lack the confidence and self-motivation to have their hearing tested . CurrentScreening is currently conducted through various government and community-based schemes, such as those offered by Australian Hearing and Better Hearing which travel to various locations around the country and offer free hearing testing . HoweverEducation about the frequency and impact of hearing impairment amongst older people could be expanded and made one element of a hearing health policy for Australia ,52. EducStudies from the United States and the Netherlands have discussed the relation of cost of audiological rehabilitation to the benefits of intervention for hearing impairment ,53,54. TConsidering the large numbers of Australians affected by age-related hearing impairment, it is surprising that more has not already been done by both federal and state governments to develop policies. The population who suffer from age-related hearing loss is generally a politically weak one who, possibly because of the nature of their hearing loss may find difficulty in effectively lobbying for policy change.This literature search has identified several important areas that need to be addressed in the construction of a comprehensive hearing health policy for Australia. These areas are early intervention, hearing aid expense, the use of assisted listening devices, rehabilitation, screening and education. Given the burden imposed by untreated or under treated hearing loss amongst Australians over 55, a strong and coordinated policy response to the issues should be developed for Australia.Clearly, given the enormity of the problem and the insufficient nature of current treatment options, hearing loss needs to become a priority for Australian Federal and State governments . The AusMore research is needed into most aspects of the treatment of age-related hearing impairment in Australia. Current research undertaken by Australian Hearing is important and relevant, but misses large areas identified in this literature search. Gaps in our knowledge of this problem in Australia include the impact of cost of hearing aids and devices on those unable to access free government hearing services, the utility of current hearing screening and audiological rehabilitation programs.Prevention of Deafness and Hearing Impairment Program, with the goal of halving preventable hearing impairment by the year 2010 [It is 20 years since the World Health Organisation launched its ear 2010 . This prear 2010 . CurrentThe author(s) declare that they have no competing interests.JS, PM, JJW and SRL conceived and designed this study. JS conducted the literature search and analysis. JS constructed the draft of the paper. PM, JJW and SRL made significant contributions to the revision of the draft. All authors read and approved the final manuscript.


Study of expression of ras-related oncogenes in human premalignant polyps and malignant tumours of the colorectum, as well as in normal colorectal mucosa, shows a significant elevation in the premalignant and malignant tissues as compared to their respective colorectal mucosa. These results suggest that activation of the ras oncogene family occurs in the development of colorectal tumours and that elevated expression at a premalignant stage may well be critical in the process of carcinogenesis but not in itself sufficient.