Breeding and improving ornamentals, Station Bulletin, no.461

The Bulletin is a publication of the New Hampshire Agricultural Experiment Station, College of Life Sciences and Agriculture, University of New Hampshire, Durham, New Hampshire

Community Science in Support of Ecosystem-Based Management: A Case Study from the Damariscotta River Estuary, Maine, USA

Coastal marine ecosystems are dynamic social-ecological systems (SESs) that support diverse ecosystem services and human activities. The complexity of SESs means that ecosystem-based approaches are increasingly used to support coastal marine ecosystem stewardship. We report how a community science program in Maine, USA offers a model of organizational innovation to expand capacities for shellfish research and management. Since 2019, we have collaborated with local students, shellfish harvesters, and others in data collection, interpretation, and application, contributing to local shellfish management and ecosystem sustainability. We demonstrate how community-based social and ecological research can build adaptive capacities by centering local knowledge; generating long-term and fine-scale datasets; supporting community-led management and civic engagement; and contributing to field methods that people with varied lived experiences and training can implement in a sustainable fashion. Our approach can be adapted to other contexts, particularly in data-limited SESs that are experiencing observable social and ecological change

Declaration of Principles & Aims

Report by special Kentucky Educational Association committee charged to draft a Declaration of Principles or a statement of the aims and purposes of the educational forces of the State of Kentucky. The report was adopted unanimously at the annual meeting of KEA.https://digitalcommons.wku.edu/exhibit_2015/1015/thumbnail.jp

UA3/1/4 Declaration of Principles & Aims

Report by special Kentucky Educational Association committee charged to draft a Declaration of Principles or a statement of the aims and purposes of the educational forces of the State of Kentucky. The report was adopted unanimously at the annual meeting of KEA

Protection From Influenza by Intramuscular Gene Vector Delivery of a Broadly Neutralizing Nanobody Does Not Depend on Antibody Dependent Cellular Cytotoxicity

Cross-subtype neutralizing single domain antibodies against influenza present new opportunities for immunoprophylaxis and pandemic preparedness. Their simple modular structure and single open reading frame format are highly amenable to gene therapy-mediated delivery. We have previously described R1a-B6, an alpaca-derived single domain antibody (nanobody), that is capable of potent cross-subtype neutralization in vitro of H1N1, H5N1, H2N2, and H9N2 influenza viruses, through binding to a highly conserved epitope in the influenza hemagglutinin stem region. To evaluate the potential of R1a-B6 for immunoprophylaxis, we have reformatted it as an Fc fusion for adeno-associated viral (AAV) vector delivery. Our findings demonstrate that a single intramuscular injection in mice of AAV encoding R1a-B6 fused to Fc fragments of different isotypes equipped either, with or without antibody dependent cellular cytotoxicity (ADCC) activity, was able to drive sustained high-level expression (0.5–1.1 mg/mL) in sera with no evidence of reduction for up to 6 months. R1a-B6-Fc fusions of both isotypes gave complete protection against lethal challenge with both pandemic A/California/07/2009 (H1N1)pdm09 and avian influenza A/Vietnam/1194/2004 (H5N1). This data suggests that R1a-B6 is capable of cross-subtype protection and ADCC was not essential for R1a-B6 efficacy. Our findings demonstrate AAV delivery of cross-subtype neutralizing nanobodies may be an effective strategy to prevent influenza infection and provide long-term protection independent of a host induced immune response

Synthesis and Detection of Oxygen-18 Labeled Phosphate

Phosphorus (P) has only one stable isotope and therefore tracking P dynamics in ecosystems and inferring sources of P loading to water bodies have been difficult. Researchers have recently employed the natural abundance of the ratio of 18O/16O of phosphate to elucidate P dynamics. In addition, phosphate highly enriched in oxygen-18 also has potential to be an effective tool for tracking specific sources of P in the environment, but has so far been used sparingly, possibly due to unavailability of oxygen-18 labeled phosphate (OLP) and uncertainty in synthesis and detection. One objective of this research was to develop a simple procedure to synthesize highly enriched OLP. Synthesized OLP is made up of a collection of species that contain between zero and four oxygen-18 atoms and, as a result, the second objective of this research was to develop a method to detect and quantify each OLP species. OLP was synthesized by reacting either PCl5 or POCl3 with water enriched with 97 atom % oxygen-18 in ambient atmosphere under a fume hood. Unlike previous reports, we observed no loss of oxygen-18 enrichment during synthesis. Electrospray ionization mass spectrometertry (ESI-MS) was used to detect and quantify each species present in OLP. OLP synthesized from POCl3 contained 1.2% P18O16O3, 18.2% P18O216O2, 67.7% P18O316O, and 12.9% P18O4, and OLP synthesized from PCl5 contained 0.7% P16O4, 9.3% P18O316O, and 90.0% P18O4. We found that OLP can be synthesized using a simple procedure in ambient atmosphere without the loss of oxygen-18 enrichment and ESI-MS is an effective tool to detect and quantify OLP that sheds light on the dynamics of synthesis in ways that standard detection methods cannot

Discovery of candidate disease genes in ENU-induced mouse mutants by large-scale sequencing, including a splice-site mutation in nucleoredoxin.

An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU) mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn), inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing

Photochemical internalisation of chemotherapy potentiates killing of multidrug-resistant breast and bladder cancer cells

Multidrug resistance (MDR) is the major confounding factor in adjuvant solid tumour chemotherapy. Increasing intracellular amounts of chemotherapeutics to circumvent MDR may be achieved by a novel delivery method, photochemical internalisation (PCI). PCI consists of the co-administration of drug and photosensitiser; upon light activation the latter induces intracellular release of organelle-bound drug. We investigated whether co-administration of hypericin (photosensitiser) with mitoxantrone (MTZ, chemotherapeutic) plus illumination potentiates cytotoxicity in MDR cancer cells. We mapped the extent of intracellular co-localisation of drug/photosensitiser. We determined whether PCI altered drug-excreting efflux pump P-glycoprotein (Pgp) expression or function in MDR cells. Bladder and breast cancer cells and their Pgp-overexpressing MDR subclones (MGHU1, MGHU1/R, MCF-7, MCF-7/R) were given hypericin/MTZ combinations, with/without blue-light illumination. Pilot experiments determined appropriate sublethal doses for each. Viability was determined by the 3-[4,5-dimethylthiazolyl]-2,5-diphenyltetrazolium bromide assay. Intracellular localisation was mapped by confocal microscopy. Pgp expression was detected by immunofluorescence and Pgp function investigated by Rhodamine123 efflux on confocal microscopy. MTZ alone (0.1–0.2 μg ml−1) killed up to 89% of drug-sensitive cells; MDR cells exhibited less cytotoxicity (6–28%). Hypericin (0.1–0.2 μM) effects were similar for all cells; light illumination caused none or minimal toxicity. In combination, MTZ /hypericin plus illumination, potentiated MDR cell killing, vs hypericin or MTZ alone. (MGHU1/R: 38.65 and 36.63% increase, P<0.05; MCF-7/R: 80.2 and 46.1% increase, P<0.001). Illumination of combined MTZ/hypericin increased killing by 28.15% (P<0.05 MGHU1/R) compared to dark controls. Intracytoplasmic vesicular co-localisation of MTZ/hypericin was evident before illumination and at serial times post-illumination. MTZ was always found in sensitive cell nuclei, but not in dark resistant cell nuclei. In illuminated resistant cells there was some mobilisation of MTZ into the nucleus. Pgp expression remained unchanged, regardless of drug exposure. Pgp efflux was blocked by the Pgp inhibitor verapamil (positive control) but not impeded by hypericin. The increased killing of MDR cancer cells demonstrated is consistent with PCI. PCI is a promising technique for enhancing treatment efficacy

STRIDES - STudying Risk to Improve DisparitiES in Cervical Cancer in Mississippi – Design and Baseline Results of a Statewide Cohort Study

Cervical cancer rates in Mississippi are disproportionately high, particularly among Black individuals; yet, research in this population is lacking. We designed a statewide, racially diverse cohort of individuals undergoing cervical screening in Mississippi. Here, we report the baseline findings from this study. We included individuals aged 21 years and older undergoing cervical screening with cytology or cytology-human papillomavirus (HPV) co-testing at the Mississippi State Health Department (MSDH) and the University of Mississippi Medical Center (UMMC) (December 2017-May 2020). We collected discarded cytology specimens for future biomarker testing. Demographics and clinical results were abstracted from electronic medical records and evaluated using descriptive statistics and chi-square tests. A total of 24,796 individuals were included, with a median age of 34.8 years. The distribution of race in our cohort was 60.2% Black, 26.4% White, 7.5% other, and 5.9% missing. Approximately 15% had abnormal cytology and, among those who underwent co-testing at MSDH (n=6,377), HPV positivity was 17.4% and did not vary significantly by race. Among HPV positives, Black individuals were significantly less likely to be HPV16/18 positive and more likely to be positive for other high-risk 12 HPV types compared to White individuals (20.5% vs. 27.9%, and 79.5% and 72.1%, respectively, p=0.011). Our statewide cohort represents one of the largest racially diverse studies of cervical screening in the U.S. We show a high burden of abnormal cytology and HPV positivity, with significant racial differences in HPV genotype prevalence. Future studies will evaluate cervical precancer risk, HPV genotyping, and novel biomarkers in this population

Hydrophilic interaction liquid chromatography (HILIC) in proteomics

In proteomics, nanoflow multidimensional chromatography is now the gold standard for the separation of complex mixtures of peptides as generated by in-solution digestion of whole-cell lysates. Ideally, the different stationary phases used in multidimensional chromatography should provide orthogonal separation characteristics. For this reason, the combination of strong cation exchange chromatography (SCX) and reversed-phase (RP) chromatography is the most widely used combination for the separation of peptides. Here, we review the potential of hydrophilic interaction liquid chromatography (HILIC) as a separation tool in the multidimensional separation of peptides in proteomics applications. Recent work has revealed that HILIC may provide an excellent alternative to SCX, possessing several advantages in the area of separation power and targeted analysis of protein post-translational modifications