A proteomic view into infection of greyback canegrubs (<Emphasis Type="Italic">Dermolepida albohirtum</Emphasis>) by <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

Metarhizium anisopliae is a naturally occurring cosmopolitan fungus infecting greyback canegrubs (Dermolepida albohirtum). The main molecular factors involved in the complex interactions occurring between the greyback canegrubs and M. anisopliae (FI-1045) were investigated by comparing the proteomes of healthy canegrubs, canegrubs infected with Metarhizium and fungus only. Differentially expressed proteins from the infected canegrubs were subjected to mass spectrometry to search for pathogenicity related proteins. Immune-related proteins of canegrubs identified in this study include cytoskeletal proteins (actin), cell communication proteins, proteases and peptidases. Fungal proteins identified include metalloproteins, acyl-CoA, cyclin proteins and chorismate mutase. Comparative proteome analysis provided a view into the cellular reactions triggered in the canegrub in response to the fungal infection at the onset of biological control.     

Campylobacter jejuni Glycosylation Island Important in Cell Charge,Legionaminic Acid Biosynthesis,and Colonization of Chickens

Previously, we identified five genes (Cj1321 to Cj1326, of which Cj1325 and Cj1326 are a single gene) in the O-linked flagellin glycosylation island that are highly prevalent in Campylobacter jejuni isolates from chickens. We report mutagenesis, functional, and structural data to confirm that this locus, and Cj1324 in particular, has a significant contributory role in the colonization of chickens by C. jejuni. A motile ΔCj1324 mutant with intact flagella was considerably less hydrophobic and less able to autoagglutinate and form biofilms than the parent strain, 11168H, suggesting that the surface charge of flagella of Cj1324-deficient strains was altered. The physical and functional attributes of the parent were restored upon complementation. Structural analysis of flagellin protein purified from the ΔCj1324 mutant revealed the absence of two legionaminic acid glycan modifications that were present in the parent strain, 11168H. These glycoform modifications were shown to be prevalent in chicken isolates and confirm that differences in the highly variable flagellin glycosylation locus can relate to the strain source. The discovery of molecular mechanisms influencing the persistence of C. jejuni in poultry aids the rational design of approaches to control this problematic pathogen in the food chain.Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide (7). Infection can cause symptoms including abdominal pain and fever with watery to bloody diarrhea (54). Occasionally, postinfectious sequelae follow C. jejuni infection and include reactive arthritis and Guillain-Barré syndrome (8). Recently, C. jejuni has been associated with immunoproliferative small intestine disease, which is a rare type of mucosa-associated lymphoid tissue lymphoma (31). The main source of transmission through the food chain is the consumption and handling of contaminated poultry, but the underlying reasons why chickens are particularly susceptible to colonization by C. jejuni are unknown (15). C. jejuni has also been recovered from nonavian livestock, unpasteurized milk, and contaminated water (7). The socioeconomic burden of this pathogen means that it is imperative that ways of reducing the levels of C. jejuni in the food chain, particularly poultry, are investigated.The glycosylation of flagellin in a number of gram-negative pathogenic bacteria, including Pseudomonas aeruginosa, Helicobacter pylori, and Aeromonas spp., is increasingly recognized as playing significant roles (2, 24, 32, 43, 49). Glycosylation modifications have been shown to influence the cell''s immunogenicity, interaction with eukaryotic cells, and host cell specificity. Aeromonads are waterborne bacteria that can cause disease in fish, reptiles, and amphibians. Mesophilic aeromonads are important human pathogens causing gastrointestinal infections and, in severe cases, wound disease and septicemia in healthy and immunocompromised patients (63). Flagella of the mesophilic aeromonad Aeromonas caviae have been shown to be glycosylated (43) with a derivative of pseudaminic acid (50). In the plant pathogen Pseudomonas syringae pv. glycinea, the mutation of three genes located in a flagellin glycosylation island results in alterations to host specificity (61). Mutants of P. syringae pv. glycinea fail to cause symptoms in the normal host, soybean plants, but can grow on nonhost tobacco leaves, causing symptom-like changes on leaves. Takeuchi et al. proposed that the posttranslational modification of flagellin may be an adaptation of the bacterium to avoid recognition by host defenses (61). In P. aeruginosa strain PAK, a flagellin glycosylation island comprising 14 genes was discovered and shown to cause glycosylation exclusively for P. aeruginosa isolates expressing a-type flagellin (2). Further studies have shown that there appears to be variation in the glycosylation islands of strains containing the a-type flagellin (4). A glycosylation island comprising four genes in the type b flagellin strain P. aeruginosa PAO has been found. When a mutant unable to glycosylate flagellin was tested in a murine model of burn wound infection, it exhibited a reduction in virulence compared to that of the wild type (3). Thus, it appears that in P. aeruginosa different glycoforms on the flagellin are required for the colonization of different hosts or environments and that these glycoforms may provide the bacterium with a specific survival advantage.We recently examined 111 strains of C. jejuni, including human, chicken, bovine, ovine, and environmental isolates, using comparative phylogenomics (whole-genome comparisons of microbes using DNA microarrays combined with Bayesian-based phylogenies) (10). Isolates fell into two distinct clades, which based on the origins of the isolates were defined as livestock-associated and non-livestock-associated clades. Over 40 genes were identified as being significantly prevalent in either of the clades. Among these was a set of six genes, Cj1321, Cj1322, Cj1323, Cj1324, Cj1325, and Cj1326 (as identified in the initial annotation by Parkhill et al. for the original sequenced C. jejuni strain, NCTC11168 [42]), that lie within a region of the genome encoding the flagellin O-linked glycosylation system. Thus, although genes Cj1321 to Cj1326 are located within a region of the genome which has variability, they are conserved among some C. jejuni strains that are often associated with livestock. Microarray data have shown that the six genes are all transcribed in the same orientation, but it is unknown if they are an operon (N. Dorrell and B. W. Wren, unpublished data). NCTC11168 has since been reannotated, and as a result, Cj1325 and Cj1326 are now considered to be one gene, hereinafter referred to as Cj1325/6 (22). Previous BLAST analyses have shown that the Cj1321 protein has amino acid similarity to many bacterial acetyl transferases, both Cj1322 and Cj1323 proteins are similar to hydroxyacyl dehydrogenases, and the product of Cj1324 is similar to WbpG, a protein involved in lipopolysaccharide synthesis in many bacteria.In C. jejuni NCTC11168 (the original sequenced strain, found in the livestock clade), the O-linked flagellar glycosylation system is thought to consist of a cluster of approximately 50 genes (Cj1293 to Cj1342) adjacent to flaA and flaB which encode the structural flagellin proteins (42). The full glycan structure(s) in NCTC11168 (and most other strains associated with livestock) is unknown, but given the considerably larger size of the O-linked glycosylation loci in the livestock-associated strains than in the non-livestock-associated strains, it is likely that the livestock-associated strains may have additional modifications to the pseudaminic acid basic structure, as well as other unique glycan moieties, compared to those of the non-livestock-associated strains. The flagellin O-linked glycosylation locus in C. jejuni 81-176 (a frequently studied human strain found in the non-livestock-associated clade) is far simpler than that in C. jejuni NCTC11168, comprising just 26 genes (21). Two modifications predominantly decorate FlaA and FlaB of strain 81-176, the nine-carbon sugar pseudaminic acid (5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-α-l-manno-nonulosonic acid [Pse5Ac7Ac]) and an acetamidino form of pseudaminic acid, 5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-l-glycero-α-l-manno-nonulosonic acid (Pse5Ac7Am). Derivatives of Pse5Ac7Am also decorate the flagellin of 81-176 in minor quantities (34, 37, 62). Genetic analysis of 81-176 showed that pse genes are involved in the biosynthesis of pseudaminic acid and its derivatives (21, 37, 62). More recently, the full biosynthetic pathway for pseudaminic acid was determined; in a six-step reaction, UDP-N-acetylglucosamine (UDP-GlcNAc) is converted to pseudaminic acid through the actions of PseB/Cj1293, PseC/Cj1294, PseH/Cj1313, PseG/Cj1312, PseI/Cj1317, and PseF/Cj1311 proteins (The Cj designations refer to predicted coding sequences in C. jejuni NCTC11168) (11, 19, 21, 35, 52, 62).The most detailed analysis of the flagellin O-linked glycosylation locus has been undertaken with C. jejuni strain 81-176 and the related species Campylobacter coli (strain VC167) (34). Structural studies of the flagellum modifications of C. coli VC167 revealed that in addition to Pse5Ac7Ac, acetamidino and N-methylacetimidoyl derivatives of legionaminic acid [5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto-nonulosonic acid (Leg5Am7Ac) and 5-E/Z-N-(N-methlyacetimidoyl)-7-acetamidino-3,5,7,9-tetradeoxy-d-galacto-nonulosonic acid (Leg5AmNMe7Ac), respectively] decorate the C. coli flagellin, the first demonstration of a legionaminic acid derivative modification of bacterial flagellin (36). Biosynthesis of these legionaminic acid derivatives involves a distinct pathway encoded by the posttranslational modification (ptm) genes (34, 36). Although the precise pathway for the production of legionaminic acid has yet to be determined, tentative functions have been assigned which have identified PtmA to PtmH to be required for biosynthesis (PtmA, PtmB, PtmC, PtmD, PtmE, PtmF, PtmG, and PtmH are equivalent to the Cj1332, Cj1331, Cj1327, Cj1328, Cj1329, Cj1330, Cj1324, and Cj1325/6 proteins, respectively) (36). This ptm pathway is absent in C. jejuni strain 81-176. PtmG and PtmH from C. coli VC167 show 86 and 76% amino acid sequence similarity, respectively, to two hypothetical proteins, the Cj1324 and Cj1325/6 proteins of C. jejuni NCTC11168. The enzyme(s) involved in the attachment of glycan(s) to the flagellin protein of Campylobacter strains and the consensus sequence for the O-linked glycosylation process have yet to be identified.In C. jejuni strain 81-176, glycosylation of flagellin has been shown to be necessary for the assembly of flagella and subsequent motility (19). There is extensive polymorphism in the C. jejuni O-linked glycosylation cluster, suggesting that selective pressure may cause the bacterium to alter surface antigens in attempts to evade the host immune defenses (59). Evidence supporting this possibility is demonstrated by comparing the glycan moieties of the flagella of C. jejuni 81-176 and C. coli VC167, as these strains produce unique modifications on their flagella which affect serospecificity (34).Given the diversity of the O-linked glycosylation system in C. jejuni and the prevalence of the locus of Cj1321 to Cj1325/6 in chicken isolates, we hypothesized that these genes may be important for the abilities of some C. jejuni strains to colonize poultry and that colonization may be mediated through structural and surface charge changes in the glycan that modifies the flagellin. In this study, we demonstrate that Cj1324 is involved in the biosynthesis of two novel legionaminic acid modifications found on the flagellin of strain 11168H. The presence of these modifications affects autoagglutination, cell charge, and the efficiency with which C. jejuni 11168H colonizes chickens.     

Patterns of care for cervical cancer in Auckland, New Zealand, 2003-2007

Introduction: The purpose of this review is to document current patterns of care for the International Federation of Gynecology and Obstetrics (FIGO) stage IB1 to IVA cervical cancer in a New Zealand cancer centre. Methods: This is a retrospective review of women with newly diagnosed FIGO Stage IB1–IVA cervical cancer in the Auckland/Northland regions between 2003 and 2007. Results: Two hundred seven patients were identified. Fifty‐three percent were stage IB, 24% stage II, 19% stage III and 3% stage IVA. Factors associated with stage ≥IIB were age >50, lack of participation in cervical screening and public first specialist assessment. Ninety percent (90/100) of stage IB1 patients and 73% (8/11) of stage IB2 patients were treated with primary surgery. Thirty‐eight percent of surgically treated stage IB1 and 100% of surgically treated stage IB2 tumours had indications for adjuvant radiotherapy. Radiotherapy utilisation rates were: stage IB 49% (IB1 44%, IB2 91%); stage II 93%; stage III 90%; and stage IVA 71%. Brachytherapy utilisation rate (BTU) for stages IIB to IVA was 64% overall and 75% in definitively treated patients. Seventy‐five percent of patients treated with definitive radiotherapy received concurrent cisplatin chemotherapy. Conclusion: Both radiotherapy and brachytherapy utilization rates were below optimal and are being addressed. No formal surgical or chemotherapy utilisation estimates exist for comparison; however, the use of concurrent cisplatin chemotherapy was similar to other groups. A high rate of adjuvant (chemo)radiotherapy was noted in surgically treated Stage IB patients, suggesting a need for an increased consideration of primary chemoradiotherapy in these patients to avoid the unnecessary toxicity of trimodality therapy. Future outcome analysis is planned.     

Rôle de l’irradiation locorégionale adjuvante en l’absence d’envahissement ganglionnaire après chimiothérapie néoadjuvante, mastectomie totale et lymphadénectomie axillaire pour un cancer du sein. Expérience de l’hôpital René-Huguenin–institut Curie

Purpose

Neoadjuvant chemotherapy generally induces significant changes in the pathological extent of disease and challenges the standard indications of adjuvant postmastectomy radiation therapy. We retrospectively evaluated the impact of postmastectomy radiation therapy in breast cancer patients with negative lymph nodes (pN0) after neoadjuvant chemotherapy.

Patients and materials

Among 1054 breast cancer patients treated with neoadjuvant chemotherapy in our institution between 1990 and 2004, 134 patients had pN0 status after neoadjuvant chemotherapy and mastectomy. Demographic data, tumor characteristics, metastatic sites, and treatments were prospectively recorded. The impact of postmastectomy radiation therapy on locoregional recurrence-free survival and overall survival was evaluated by multivariate analysis including known prognostic factors.

Results

Among 134 eligible patients, 78 patients (58.2%) received postmastectomy radiation therapy, and 56 patients (41.8%) did not. With a median follow-up time of 91.4 months, the 10-year locoregional recurrence-free survival and overall survival rates were 96.2% and 77.2% with postmastectomy radiation therapy and 86.8% and 87.7% without radiation therapy, respectively (no significant difference). In multivariate analysis, there was a trend towards poorer overall survival among patients who did not have a pathologically complete primary tumour response after neoadjuvant chemotherapy (hazard ratio [HR], 6.65; 95% CI, 0.82–54.12; P = 0.076). Postmastectomy radiation therapy had no effect on either locoregional recurrence-free survival (HR, 0.37; 95% CI, 0.09–1.61; P = 0.18) or overall survival (HR, 2.06; 95% CI, 0.71–6; P = 0.18). There was a trend towards poorer overall survival among patients who did not have pathologically complete in-breast tumour response after neoadjuvant chemotherapy (HR, 6.65; 95% CI, 0.82–54.12; P = 0.076).

Conclusions

This retrospective study showed no increase in the risk of distant metastasis, locoregional recurrence or death when postmastectomy radiation therapy was omitted in breast cancer patients with pN0 status after neoadjuvant chemotherapy and mastectomy. Whether the omission of postmastectomy radiation therapy is acceptable for these patients should be addressed prospectively.     

Body iron stores and breast cancer risk in female atomic bomb survivors

Iron can be a potent pro-oxidant and, on this basis, elevated body iron may increase the risk of cancer. Although epidemiological evidence is mixed, there is overall support for this possibility. In addition, because of this same oxidative capacity, body iron levels may alter radiation sensitivity. In the present study, a nested case-control study of breast cancer was conducted in Japanese atomic bomb survivors. Stored serum samples from the Adult Health Study cohort were assayed for ferritin levels and joint statistical analyses were conducted of ferritin and radiation dose on the risk of breast cancer. Serum ferritin is the best feasible indicator of body iron levels in otherwise healthy people. A total of 107 cases and 212 controls were available for analysis. The relative risk (RR) of breast cancer for a 1 log unit increase in ferritin was 1.4 (95% confidence interval 1.1-1.8). This translates to an RR of 1.64 comparing high and low values of the interquartile range among controls (58 and 13.2 ng/mL, respectively). The results support the hypothesis that elevated body iron stores increase the risk of breast cancer. However, the study was inconclusive regarding the question of whether body iron alters radiation-induced breast cancer risk.     

Presence of Helicobacter pylori in supragingival dental plaque of individuals with periodontal disease and upper gastric diseases

Background

Helicobacter pylori is a Gram-negative microorganism which is able to colonize the gastric mucosa and is associated with peptic ulcer, gastric carcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. Several studies have detected this bacterium in the oral cavity, suggesting it as a potential reservoir. The aim of this study was to investigate the presence of H. pylori in the oral cavity of individuals with periodontal disease and gastric diseases.

Methods

115 individuals, with mean age 49.6 (±5.8) years, were divided in 4 groups: (A) with gastric diseases and periodontal disease; (B) with gastric diseases and no periodontal disease; (C) without gastric diseases and without periodontal disease, (D) without gastric diseases and with periodontal disease. Supra and subgingival plaque samples were collected from posterior teeth of the individuals with sterile paper points, and prepared for Polymerase Chain Reaction analysis. Fisher's exact test was used for detecting statistical differences between groups (p < 0.05).

Results

H. pylori was detected in supragingival plaque of 9/36 (25%) of group A, 1/31 (0.3%) of group B, 0 (0%) of group C and 3/36 (8.3%) of group D. No subgingival samples were positive for H. pylori. There was a statistically higher prevalence of H. pylori in groups A and D when compared to B and C (p < 0.05).

Conclusion

H. pylori was detected in the supragingival plaque, but not in the subgingival plaque, of individuals with periodontal disease and upper gastric diseases. There was an association between the supragingival colonization of H. pylori and oral hygiene parameters such as the presence of plaque and gingival bleeding.     

Adherence to Pharmacological Thromboprophylaxis Orders in Hospitalized Patients

Objective

We compared adherence to unfractionated heparin (UFH) 2 or 3 times daily prophylaxis orders versus low-molecular-weight heparin (LMWH) once daily orders. Our goals were to determine which strategy demonstrated the best adherence in terms of timing and frequency of dose administration, and to determine reasons for ordered heparin not being administered.

Methods

We queried our electronic medication administration record where nurses document reasons for delayed administration or omitted doses. We identified 250 consecutive patients who were prescribed prophylaxis with UFH 2 or 3 times daily or LMWH once daily. We followed patients for their hospitalization to determine adherence to physicians' prophylaxis orders.

Results

Adherence, defined as the ratio of prophylaxis doses given to doses ordered, was greater with LMWH (94.9%) than UFH 3 times daily (87.8%) or UFH twice daily (86.8%) regimens (P <.001). Patients receiving LMWH more often received all of their scheduled prophylaxis doses (77%) versus UFH 3 times daily (54%) or UFH twice daily (45%) (P <.001). There were no differences between regimens regarding reasons for omitted doses. The most common reason for late or omitted doses was patient refusal, which explained 44% of the UFH and 39% of the LMWH orders that were not administered.

Conclusions

LMWH once a day had better adherence than UFH 2 or 3 times daily. For both LMWH and UFH, patient refusal was the most common reason for not administering prophylaxis as prescribed. These findings require consideration when evaluating pharmacological prophylaxis strategies. Educational programs, explaining the rationale, may motivate patients to improve adherence during hospitalization.