A Nodulation-Proficient Nonrhizobial Inhabitant of Pueraria phaseoloides.

Pueraria phaseoloides is a legume cover crop, found chiefly in the wet zone of Sri Lanka. Nitrogen fixation is performed by nodular inhabitants of this cover crop, comparable to the nodule-dwelling bacteria of most other legume plants. We isolated a bacterium (Sub1) from Pueraria phaseoloides, of coccobacillus cell shape, that showed nodulation, when assessed by hydroponics, showing nodules as early as 3 weeks after reinfection. When a nifH fragment from the genome of this bacterium was amplified using a pair of nifH primers, it yielded an amplicon of 360 bp that, when sequenced, helped us identify the bacterium, as belonging to a species of Pseudacidovorax intermedius, at 99% sequence identity. When we constructed a phylogenetic tree with neighboring sequences, we encountered nifH sequences of Pseudacidovorax, forming a monophyletic cluster, which too contained a single Azospirillum species. The genus Pseudacidovorax is a bacterium that, so far, has not been associated with legume nodules. Sub1 secreted a pair of enzymes to the extracellular medium to degrade cellulose and milk proteins. The Sub1 bacterium showed biofilm formation and secreted into the extracellular medium, indole acetic acid. Sub1 also showed a "bulls eye" swarming pattern for the chemoattractant proline, while showing no significant chemotaxis movement, for naringenin, quercetin, and glutamate. Sub1 too possesses the basic genetic foundation (nifH and nifD) to produce a molybdenum-dependent nitrogenase enzyme. We finally show that this rare nonrhizobial bacterium is able to impact, positively, nodulation and shoot length of Pueraria plants, demonstrating that this beta-proteobacterium can abet the biological vigor of this legume cover crop.

DNA barcoding and real-time PCR detection of Bactrocera xanthodes (Tephritidae: Diptera) complex.

Immature fruit fly stages of the family Tephritidae are commonly intercepted on breadfruit from Pacific countries at the New Zealand border but are unable to be identified to the species level using morphological characters. Subsequent molecular identification showed that they belong to Bactrocera xanthodes, which is part of a species complex that includes Bactrocera paraxanthodes, Bactrocera neoxanthodes and an undescribed species. To establish a more reliable molecular identification system for B. xanthodes, a reference database of DNA barcode sequences for the 5'-fragment of COI gene region was constructed for B. xanthodes from Fiji, Samoa and Tonga. To better understand the species complex, B. neoxanthodes from Vanuatu and B. paraxanthodes from New Caledonia were also barcoded. Using the results of this analysis, real-time TaqMan polymerase chain reaction (PCR) assays for the detection of B. xanthodes complex and for the three individual species of the complex were developed and validated. The assay showed high specificity for the target species, with no cross-reaction observed for closely related organisms. Each of the real-time PCR assays is sensitive, detecting the target sequences at concentrations as low as ten copies µl-1 and can be used as either singleplex or multiplex formats. This real-time PCR assay for B. xanthodes has been successfully applied at the borders in New Zealand, leading to the rapid identification of intercepted Tephritidae eggs and larvae. The developed assays will be useful biosecurity tools for rapid detection of species in the B. xanthodes complex worldwide.

Preliminary data on the presence of an alternate vanadium nitrogenase in a culturable cyanobiont of Azolla pinnata R. Brown: Implications on Chronic Kidney Disease of an unknown etiology (CKDu).

In a recent paper titled "How a taxonomically-ambiguous cyanobiont and vanadate assist in the phytoremediation of cadmium by Azolla pinnata: implications for CKDu" (Atugoda et al., 2018) [1] it was shown by us, that plant health and phytoremediation capacities, of Azolla pinnata R. Brown, were elevated in the presence of vanadate, a vanadium containing ion. This highlighted a possibility, that either the major or minor cyanobionts of Azolla pinnata, could possess a vanadium dependent nitrogenase enzyme, as an alternate nitrogenase, in addition to the molybdenum counterpart. In this data article, we report the isolation of a minor cyanobiont which we name as Fischerella uthpalarensis. We grew Fischerella uthpalarensis, exclusively in N-free media, with only molybdenum (Mo+ V-), with only vanadium (V+ Mo-) and with neither (negative control), to find out the growth patterns in the relevant media. While F. uthpalarensis grew as green colored consistencies, increasing gradually in turbidity, for 4 weeks in culture, both, in the presence of molybdenum (Mo+ V-), as well as vanadium (V+ Mo-), the negative control, showed no, or very little growth. This alludes to the presence of dual nitrogenases in Fischerella uthpalarensis. An attempt was also made by us to unravel the vnf genes, responsible for the V-nitrogenase. However, it was not possible to PCR amplify the vnf genes, from both, the unculturable major (using total DNA from the Azolla-Nostoc azollae symbiosis) and minor (DNA directly from the cultured F. uthpalarensis) cyanobionts. This is the first time, to our knowledge, that an endosymbiotic cyanobacterium inside a plant compartment, has been shown to contain two possible nitrogenase systems.

Could parenchymal enhancement on contrast-enhanced spectral mammography (CESM) represent a new breast cancer risk factor? Correlation with known radiology risk factors.

AIM: To compare background parenchymal enhancement (BPE) on contrast-enhanced (CE) spectral mammography (CESM) with CE magnetic resonance imaging (MRI), and evaluate how these relate to hormonal status, mammographic breast density (MBD) and MRI fibroglandular tissue volume (FGTV). METHODS AND MATERIALS: Between June 2012 to October 2015, participants in a cancer staging study underwent full-field digital mammography (FFDM), CEMRI, and CESM. Two readers independently rated FGTV, MBD, and BPE using the Breast Imaging-Reporting and Data System (BI-RADS) criteria. Inter-reader reliability was estimated using weighted kappa (k) and correlations between BPE, MBD, and FGTV calculated using Spearman's correlation coefficient. Associations with hormonal status were evaluated using multilevel ordinal regression analysis. RESULTS: Of the 96 eligible participants, 66 women (35-77 years) underwent CESM and CEMRI. Reasons for exclusion were declined or withdrawn consent (n=18), inadequate renal function (n=2), claustrophobia (n=2), previous reaction to contrast medium (n=2), mild reaction to contrast medium following CESM (n=2), lack of vascular access (n=1), neoadjuvant chemotherapy (n=1), CESM equipment failure (n=1), and unclear in one case. Inter-reader agreement was substantial (k=0.67) for CESM BPE, slight (k=0.19) for CEMRI BPE, moderate (k=0.57) for MRI FGTV and fair (k=0.35) for MBD. CESM BPE showed significant correlation with MBD (rho=0.36, p<0.0001), FGTV (rho=0.52, p<0.0001), and MRI BPE (rho=0.49, p<0.0001). BPE was significantly reduced in the post-menopausal group for CEMRI and CESM (p<0.05). CESM BPE did not significantly fluctuate during the menstrual cycle. CONCLUSION: CESM BPE is correlated with MBD, FGTV, and CEMRI BPE, has better inter-reader reliability than CEMRI, and is not influenced by the menstrual cycle. Grading the degree of BPE on CESM could be a useful addition to breast cancer risk assessment tools.

Development and validation of a real-time PCR assay for the glassy-winged sharpshooter Homalodisca vitripennis (Hemiptera: Cicadellidae).

The glassy-winged sharpshooter (Homalodisca vitripennis) is an invasive pest organism, which is found throughout Central America and has recently invaded a few countries in the Pacific Islands. As a carrier of the highly virulent plant pathogenic bacterium Xylella fastidiosa, it is of great economic significance to horticulture and is estimated to cost Californian vineyards over US$100 million per year in control and losses. New Zealand is currently free from this pest, but its recent spread through the Pacific has raised concerns of it establishing in New Zealand, potentially as a result of introduction through human travel. We report here a real-time polymerase chain reaction assay for the rapid identification of H. vitripennis. The assay was extensively validated in silico then optimized and tested against a range of Cicadellidae species, both internationally collected and local to New Zealand. This assay was able to correctly identify H. vitripennis samples, and distinguish between H. vitripennis and close relatives, such as the smoke-tree sharpshooter (Homalodisca liturata) and will be of great benefit to New Zealand biosecurity.

A new species in the indigenous New Zealand soft scale insect genus Kalasiris Henderson & Hodgson (Hemiptera: Sternorrhyncha: Coccomorpha: Coccidae) on Gahnia setifolia (Cyperaceae).

The genus Kalasiris Henderson & Hodgson (Hemiptera: Sternorrhyncha: Coccomorpha: Coccidae) is currently only known from New Zealand. The adult female and pupa of a new species, K. martini Hodgson & Richmond are described and illustrated below and the possible taxonomic relationships of the genus to other New Zealand genera are discussed.

A comprehensive bioinformatics analysis of the Nudix superfamily in Arabidopsis thaliana.

Nudix enzymes are a superfamily with a conserved common reaction mechanism that provides the capacity for the hydrolysis of a broad spectrum of metabolites. We used hidden Markov models based on Nudix sequences from the PFAM and PROSITE databases to identify Nudix hydrolases encoded by the Arabidopsis genome. 25 Nudix hydrolases were identified and classified into 11 individual families by pairwise sequence alignments. Intron phases were strikingly conserved in each family. Phylogenetic analysis showed that all multimember families formed monophyletic clusters. Conserved familial sequence motifs were identified with the MEME motif analysis algorithm. One motif (motif 4) was found in three diverse families. All proteins containing motif 4 demonstrated a degree of preference for substrates containing an ADP moiety. We conclude that HMM model-based genome scanning and MEME motif analysis, respectively, can significantly improve the identification and assignment of function of new members of this mechanistically-diverse protein superfamily.

Mannan mucin-1 peptide immunization: influence of cyclophosphamide and the route of injection.

The mucin MUC1 is greatly increased in breast cancer and is a potential target for immunotherapy. In mice, MUCI conjugated to oxidized mannan (MUC1-mannan fusion protein [M-FP]) targets the mannose receptor and induces a high frequency of cytotoxic T lymphocytes and anti-tumor responses. On this basis, three phase I trials were performed in patients with adenocarcinoma to evaluate the toxicity and the immunologic responses to mannan MUCI. Forty-one patients with metastatic or locally advanced carcinoma of the breast (trial 1), colon (trial 2), and various adenocarcinomas (trial 3) received increasing doses of M-FP (1 to 300 microg). The immunizations were given at weekly intervals (weeks 1 to 3) and repeated in weeks 7 to 9. Cyclophosphamide (to increase cellular immunity) was given on weeks 1 and 4. M-FP was given intramuscularly in trial 1 and intraperitoneally in trial 2. No toxic effects occurred, and delayed-type hypersensitivity responses were present only as a microscopic lymphocytic infiltration. Overall, approximately 60% of the patients had high-titer MUC1 immunoglobulin G1 antibody responses, with the intraperitoneal route yielding approximately 10-fold higher responses. Cellular responses (proliferation, cytotoxic T cells, or CD8 T cells secreting tumor necrosis factor-alpha alphand interferon-gamma in response to MUC1 stimulation in vitro) were found in 28% of the patients, which was similar to that seen without cyclophosphamide. In most patients, disease progressed, but in five it remained stable. In addition, there were no objective responses. M-FP is not toxic and induces immune responses that were amplified by the intraperitoneal route of immunization. Cyclophosphamide was of no benefit.

Acute pulmonary edema in Brucella myocarditis and interstitial pneumonitis.

A case of Brucella pneumonitis and myocarditis complicated by acute pulmonary edema is presented. The clinical, laboratory and roentgenographic findings are discussed.