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Author(s): A. J. Mungall (corresponding author) [1, 2]; S. A. Palmer [1]; S. K. Sims [1]; C. A. Edwards [1]; J. L. Ashurst [1]; L. Wilming [1]; M. C. Jones [...]

Agriculture is fundamental for food production, and microbiomes support agriculture through multiple essential ecosystem services. Despite the importance of individual (i.e., niche specific) agricultural microbiomes, microbiome interactions across niches are not well-understood. To observe the linkages between nearby agricultural microbiomes, multiple approaches (16S, 18S, and ITS) were used to inspect a broad coverage of niche microbiomes. Here we examined agricultural microbiome responses to 3 different nitrogen treatments (0, 150, and 300 kg/ha/yr) in soil and tracked linked responses in other neighbouring farm niches (rumen, faecal, white clover leaf, white clover root, rye grass leaf, and rye grass root). Nitrogen treatment had little impact on microbiome structure or composition across niches, but drastically reduced the microbiome network connectivity in soil. Networks of 16S microbiomes were the most sensitive to nitrogen treatment across amplicons, where ITS microbiome networks were the least responsive. Nitrogen enrichment in soil altered soil and the neighbouring microbiome networks, supporting our hypotheses that nitrogen treatment in soil altered microbiomes in soil and in nearby niches. This suggested that agricultural microbiomes across farm niches are ecologically interactive. Therefore, knock-on effects on neighbouring niches should be considered when management is applied to a single agricultural niche., Competing Interests: KR, AB, LJ, ST, NP, and GA were employed by the company AgResearch Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Reilly, Heathcott, Biswas, Johnson, Teasdale, Grelet, Podolyan, Gregorini, Attwood, Palevich and Morales.)

AIMS To determine the frequency of the FAS-ligand gene (FASLG) variant associated with feline autoimmune lymphoproliferative syndrome (FALPS) and the proportion of carriers of the variant in three British shorthair (BSH) breeding catteries in New Zealand. METHODS Buccal swabs were collected from all cats in two BSH breeding catteries from the South Island and one from the North Island of New Zealand. DNA was extracted and was tested for the presence of the FASLG variant using PCR. Cats with the FASLG variant were identified and the frequency of the FASLG variant allele calculated. Pedigree analysis was performed and inbreeding coefficients were calculated for cats with the FASLG variant. RESULTS Of 32 BSH cats successfully tested for the presence of the FASLG variant, one kitten (3%) was homozygous (FALPS-affected), and seven (22%) cats were heterozygous (carriers) for the FASLG variant allele, and 24 (75%) cats were homozygous for the wild type allele. The overall frequency of the FASLG variant allele in these 32 cats was 0.14. Cats carrying the FASLG variant were from all three breeding catteries sampled, including two catteries that had not previously reported cases of FALPS. Pedigree analysis revealed common ancestry of FALPS-affected and carrier cats within six generations, as well as frequent inbreeding, with inbreeding coefficients >0.12 for five cats with the FASLG variant. CONCLUSIONS AND CLINICAL RELEVANCE There was a high frequency of the FASLG variant allele (0.14) in this small sample of BSH cats, with 22% of healthy cats identified as carriers of the FASLG variant. For an inherited disease, lethal at a young age, in a small population in which inbreeding is common, these results are significant. To prevent future cases of disease and stop further spread of the FASLG variant allele within the BSH population in New Zealand, it is recommended that all BSH and BSH-cross cats be tested for the presence of the FASLG variant before mating. Cats identified as carriers of the variant allele should be desexed and not used for breeding. Results support the need for further investigations of the true frequency of the FASLG variant allele and occurrence of FALPS in the wider population of BSH cats in New Zealand.

CASE HISTORY AND CLINICAL FINDINGS A 15-year-old neutered male domestic short-haired cat was presented due to multiple 0.5-2 cm-diameter crusting plaques in the left preauricular region, over the bridge of nose, and in the right periocular region. The plaques did not appear to cause discomfort. HISTOPATHOLOGICAL FINDINGS Biopsy samples of four plaques were examined histologically. Three plaques consisted of well-demarcated foci of mild epidermal hyperplasia overlying markedly hyperplastic sebaceous glands. Approximately 60% of the hyperplastic cells contained a large cytoplasmic vacuole that ranged from being clear to containing prominent grey-blue fibrillar material. The fourth plaque was composed solely of epidermal hyperplasia, consistent with previous descriptions of feline viral plaques. MOLECULAR BIOLOGY Papillomavirus DNA was amplified from all four plaques using PCR. A single DNA sequence was amplified from the plaques with sebaceous differentiation. This sequence was identical to the FdPV-MY sequence previously suggested to be from a putative unclassified papillomavirus type. Felis catus papillomavirus type 2 sequences were amplified from the plaque typical of feline viral plaques. Immunohistochemistry to detect p16 CDKN2A protein (p16) showed marked immunostaining throughout the hyperplastic epidermis and adnexal structures within the plaques with sebaceous differentiation. DIAGNOSIS Multiple feline viral plaques with variable sebaceous differentiation. CLINICAL RELEVANCE Feline viral plaques with sebaceous differentiation have not been previously reported in cats. The presence of unique cell changes within these lesions, the detection of an unclassified papillomavirus type, and the p16 immunostaining within these plaques suggest that they may have been caused by the papillomavirus that contains the FdPV-MY sequence.

The NMR-directed investigation of the New Zealand marine sponge Hamigera tarangaensis has afforded ten new compounds of the hamigeran family, and a new 13-epi-verrucosane congener. Notably, hamigeran F (6) possesses an unusual carbon–carbon bond between C-12 and C-13, creating an unprecedented skeleton within this class. In particular, the structural features of 6, hamigeran H (10) and hamigeran J (12) imply a diterpenoid origin, which has allowed the putative biogenesis of three hamigeran carbon skeletons to be proposed based on geranyl geranyl pyrophosphate. All new hamigerans exhibited micromolar activity towards the HL-60 promyelocytic leukaemic cell line, and hamigeran G also selectively displayed antifungal activity in the budding yeast Saccharomyces cerevisiae. Homozygous deletion profiling (HOP) analysis suggests Golgi apparatus function as a potential target of this unusual class of sponge-derived terpenoids.

Neothyonidioside is a triterpene glycoside (TG) isolated from the sea cucumber, Australostichopus mollis, that is potently cytotoxic to S. cerevisiae, but does not permeabilize cellular membranes. We mutagenized S. cerevisiae and isolated a neothionidioside-resistant (neo(R)) strain. Using synthetic genetic array mapping and sequencing, we identified NCP1 as the resistance locus. Quantitative HPLC revealed that neo(R)/ncp1 mutants have reduced ergosterol content. Ergosterol added to growth media reversed toxicity, demonstrating that neothionidioside binds directly to ergosterol, similar to the polyene natamycin. Ergosterol synthesis inhibitors ketoconazole and atorvastatin conferred resistance to neothionidioside in a dose-dependent manner showing that a threshold ergosterol concentration is required for toxicity. A genome-wide screen of deletion mutants against neothionidioside revealed hypersensitivity of many of the component genes in the ESCRT complexes relating to multivesicular body formation. Confocal microscopy of cells stained with a vital dye showed blockage at this step. Thus, we propose neothionidioside may affect membrane curvature and fusion capability in the endosome-vacuole pathway.

Cyclooxygenase-2 (COX-2), the prostaglandin (PG)-synthesizing enzyme overexpressed in colorectal cancer (CRC), has pleiotropic, cancer-promoting effects. COX-2 inhibitors (CIBs) interfere with many cancer-associated processes and show promising antineoplastic activity, however, a common mechanism of CIB action has not yet been established. We therefore investigated by microarray the global response towards the CIB APHS at a dose significantly inhibiting the growth of three COX-2-positive CRC but not of two COX-2-negative cell lines. None of the genes significantly (p = 0.005) affected by APHS were common to all three cell lines and 83% of the altered pathways were cell line-specific. Quantitative polymerase chain reaction (QPCR) on selected pathways confirmed cell line-specific expression alterations induced by APHS. A low stringency data analysis approach using BRB array tools coupled with QPCR, however, identified small expression changes shared by all COX-2-positive cell lines in genes related to the WNT pathway, the key driver of colonic carcinogenesis. Our data indicates a substantial cell line-specificity of APHS-induced expression alterations in CRC cells and helps to explain the divergent effects reported for CIBs. Further, the shared inhibition of the WNT pathway by APHS suggests one potential common mechanism behind the antineoplastic effects of COX-2 inhibition.

Anaplastic histology and metastasis are each associated with higher relapse and mortality rates in Wilms tumor patients. However, not all anaplastic tumors relapse and some nonanaplastic tumors relapse unexpectedly. To identify more accurate early prognostic indicators, we analyzed expression of 4,900 cancer-related genes in 26 primary Wilms tumors. This analysis revealed that expression of a set of four genes predicts future relapse of primary Wilms tumors with high accuracy, independent of anaplasia. Random permutation testing of this prognostic gene expression signature yielded P = 0.003. Real-time reverse transcription-PCR analysis of the four genes in an independent primary tumor set resulted in correct prediction of future relapse with an accuracy of 92%. One of the four genes in the prognostic signature, CCAAT/enhancer binding protein beta (C/EBPB), is expressed at higher levels in both primary relapsing tumors and metastatic tumors than in primary nonrelapsing tumors. Short interfering RNA-mediated down-regulation of C/EBPB expression in WiT49, a cell line derived from a metastatic Wilms tumor, resulted in spontaneous apoptosis. These findings suggest that C/EBPB is a critical survival factor for Wilms tumor cells and that its expression contributes to the prognosis of Wilms tumor patients.

The development of radiation hybrid (RH) mapping (Cox et al., 1990) and the availability of large numbers of STS markers, together with extensive bacterial clone resources provided a means to accelerate the process of mapping a human chromosome and preparing bacterial clone contigs ready to sequence. Our aim is to construct physical clone maps covering those regions of chromosome 6 that are not currently extensively mapped, and use these to determine the DNA sequence of the whole chromosome. We report here a strategy which initially involves establishing a high density framework map using RH mapping. The framework markers are then used for the identification of bacterial genomic clones covering the chromosome. The bacterial clones are analysed by restriction enzyme fingerprinting and STS-content analysis to identify sequence-ready contigs. Contig gap closure will also be performed by clone walking.