Elucidation of Physiological, Transcriptomic and Metabolomic Salinity Response Mechanisms in Medicago sativa.

Alfalfa (Medicago sativa L.) is a widely grown perennial leguminous forage crop with a number of positive attributes. However, despite its moderate ability to tolerate saline soils, which are increasing in prevalence worldwide, it suffers considerable yield declines under these growth conditions. While a general framework of the cascade of events involved in plant salinity response has been unraveled in recent years, many gaps remain in our understanding of the precise molecular mechanisms involved in this process, particularly in non-model yet economically important species such as alfalfa. Therefore, as a means of further elucidating salinity response mechanisms in this species, we carried out in-depth physiological assessments of M. sativa cv. Beaver, as well as transcriptomic and untargeted metabolomic evaluations of leaf tissues, following extended exposure to salinity (grown for 3-4 weeks under saline treatment) and control conditions. In addition to the substantial growth and photosynthetic reductions observed under salinity treatment, we identified 1233 significant differentially expressed genes between growth conditions, as well as 60 annotated differentially accumulated metabolites. Taken together, our results suggest that changes to cell membranes and walls, cuticular and/or epicuticular waxes, osmoprotectant levels, antioxidant-related metabolic pathways, and the expression of genes encoding ion transporters, protective proteins, and transcription factors are likely involved in alfalfa's salinity response process. Although some of these alterations may contribute to alfalfa's modest salinity resilience, it is feasible that several may be disadvantageous in this context and could therefore provide valuable targets for the further improvement of tolerance to this stress in the future.

Eliciting Targeted Mutations in Medicago sativa Using CRISPR/Cas9-Mediated Genome Editing: A Potential Tool for the Improvement of Disease Resistance.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) has become a breeding tool of choice for eliciting targeted genetic alterations in crop species as a means of improving a wide range of agronomic traits, including disease resistance, in recent years. With the recent development of CRISPR/Cas9 technology in Medicago sativa (alfalfa), which is an important perennial forage legume grown worldwide, its use for the enhancement of pathogen resistance is almost certainly on the horizon. In this chapter, we present detailed procedures for the generation of a single nonhomologous end-joining-derived indel at a precise genomic locus of alfalfa via CRISPR/Cas9. This method encompasses crucial steps in this process, including guide RNA design, binary CRISPR vector construction, Agrobacterium-mediated transformation of alfalfa explants, and molecular assessments of transformed genotypes for transgene and edit identification.

The arterialised saphenous venous flow-through flap for managing the radial forearm free flap donor site.

BACKGROUND: The radial forearm fasciocutaneous flap (RFFF) is a workhorse flap, however concerns with donor site morbidity include tendon exposure, delayed wound healing, impaired sensitivity, and poor cosmesis, have seen it fall out of favor. We present a method of using an arterialised saphenous flow through flap to reconstruct the RFFF donor site. METHOD: A cohort study of six patients (five male, one female; mean age 59 [range 19-90]) who had their RFFF donor site reconstructed with an arterialised saphenous flow through flap is presented. The use of multiple peripheral efferent venous anastomoses, flap rotation 180 degrees prior to inset, and the ligation of intra-flap connecting veins were three modifications employed. Primary outcomes include complication rates. Secondary outcomes were patient reported outcome measures via the Michigan Hand Outcomes Questionnaire, and patency and flow through the flap. RESULTS: In all six cases, there was flap survival. RFFF dimensions ranging from lengths of 6-15 cm (mean 11.5 cm) and widths of 4-6 cm (mean 5.3 cm), with an average flap area of 58 cm2 (range 24-90). There were no total flap losses, one partial superficial flap loss and one minor donor site delayed healing, over a mean follow-up of 6 months (4-24 months). The average overall patient satisfaction was 91 on Michigan Hand Outcomes Questionnaire. Pain was well tolerated with a low average pain score of 15. CONCLUSION: The modified arterialised saphenous flow through flap is a useful option for reconstructing the soft tissue defect and reconstituting the radial artery after RFFF harvest.

Identification of Differential Drought Response Mechanisms in Medicago sativa subsp. sativa and falcata through Comparative Assessments at the Physiological, Biochemical, and Transcriptional Levels.

Alfalfa (Medicago sativa L.) is an extensively grown perennial forage legume, and although it is relatively drought tolerant, it consumes high amounts of water and depends upon irrigation in many regions. Given the progressive decline in water available for irrigation, as well as an escalation in climate change-related droughts, there is a critical need to develop alfalfa cultivars with improved drought resilience. M. sativa subsp. falcata is a close relative of the predominantly cultivated M. sativa subsp. sativa, and certain accessions have been demonstrated to exhibit superior performance under drought. As such, we endeavoured to carry out comparative physiological, biochemical, and transcriptomic evaluations of an as of yet unstudied drought-tolerant M. sativa subsp. falcata accession (PI 641381) and a relatively drought-susceptible M. sativa subsp. sativa cultivar (Beaver) to increase our understanding of the molecular mechanisms behind the enhanced ability of falcata to withstand water deficiency. Our findings indicate that unlike the small number of falcata genotypes assessed previously, falcata PI 641381 may exploit smaller, thicker leaves, as well as an increase in the baseline transcriptional levels of genes encoding particular transcription factors, protective proteins, and enzymes involved in the biosynthesis of stress-related compounds. These findings imply that different falcata accessions/genotypes may employ distinct drought response mechanisms, and the study provides a suite of candidate genes to facilitate the breeding of alfalfa with enhanced drought resilience in the future.

The CRISPR/Cas9-Mediated Modulation of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 in Alfalfa Leads to Distinct Phenotypic Outcomes.

Alfalfa (Medicago sativa L.) is the most widely grown perennial leguminous forage and is an essential component of the livestock industry. Previously, the RNAi-mediated down-regulation of alfalfa SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 (MsSPL8) was found to lead to increased branching, regrowth and biomass, as well as enhanced drought tolerance. In this study, we aimed to further characterize the function of MsSPL8 in alfalfa using CRISPR/Cas9-induced mutations in this gene. We successfully generated alfalfa genotypes with small insertions/deletions (indels) at the target site in up to three of four MsSPL8 alleles in the first generation. The efficiency of editing appeared to be tightly linked to the particular gRNA used. The resulting genotypes displayed consistent morphological alterations, even with the presence of up to two wild-type MsSPL8 alleles, including reduced leaf size and early flowering. Other phenotypic effects appeared to be dependent upon mutational dosage, with those plants with the highest number of mutated MsSPL8 alleles also exhibiting significant decreases in internode length, plant height, shoot and root biomass, and root length. Furthermore, MsSPL8 mutants displayed improvements in their ability to withstand water-deficit compared to empty vector control genotypes. Taken together, our findings suggest that allelic mutational dosage can elicit phenotypic gradients in alfalfa, and discrepancies may exist in terms of MsSPL8 function between alfalfa genotypes, growth conditions, or specific alleles. In addition, our results provide the foundation for further research exploring drought tolerance mechanisms in a forage crop.

How should we manage women with fat necrosis following autologous breast reconstruction: An algorithmic approach.

Fat necrosis is a common complication of autologous breast reconstruction; however, diagnostic criteria are yet to be standardized, making comparison of autologous breast reconstructive techniques challenging. A systematic review found six of 556 articles met inclusion criteria. These results were used to generate an algorithm for managing fat necrosis after autologous breast reconstruction.

Rapid increase in contaminant burdens following loss of body condition in canvasbacks (Aythya valisineria) overwintering on the Lake St. Clair region of the Great Lakes.

Overwintering canvasbacks were collected in the Lake St. Clair region of the Great Lakes in the winter of 2008/09 and livers were analyzed for organochlorines, mercury (Hg), and selenium (Se). We found dramatic increases in hepatic concentrations of Hg, Se, sum PCBs, p,p'-DDE, and other organochlorines in canvasbacks in which concentrations in February were greater than concentrations in November when overwintering ducks arrived in the study area. Increases in contaminant burdens were generally greatest between December and January which also coincided with the period when ducks from Lake St. Clair (LSC) moved following freeze-up of the Lake to forage on the St. Clair River (SCR), an area of known historic contamination, and upstream of LSC. Body condition estimated using body metrics and measured using lipid reserves (after controlling for body size) increased in LSC ducks but subsequently decreased in SCR ducks. This rapid loss of body condition through loss of lipid reserves was one factor likely driving the dramatic increase in contaminant burdens and particularly for organochlorines which were inversely related to body condition in SCR ducks. Increased exposure due to foraging in closer proximity to contaminant sources and changes in diet associated with the movement of ducks may have also contributed to temporal trends. Concentrations overall were below those associated with toxicity with the exception of Se for which 30% of ducks exceeded the Se threshold that is considered elevated and one duck exceeded the threshold associated with possible toxicity. Fitness consequences of reduced lipid reserves include reduced survival, delayed migration, reduced breeding propensity, and transfer of contaminant burdens to eggs. Food availability, ice cover, and movements of canvasbacks are additional factors influencing contaminant accumulation and lipid reserves in waterfowl utilizing this important wintering location.

Neoadjuvant Radiotherapy: Changing the Treatment Sequence to Allow Immediate Free Autologous Breast Reconstruction.

BACKGROUND: Locally advanced breast cancer (LABC) is traditionally treated with a multimodal approach of chemotherapy, surgery, and postmastectomy radiotherapy (PMRT). The advantages of immediate breast reconstruction (IBR) are well described and include improved aesthetic outcomes, fewer surgical procedures, shorter treatment period, and a higher quality of life. However, this sequence makes immediate free autologous reconstruction more challenging as PMRT can have deleterious and unpredictable effects on the flap. We have reversed this treatment sequence with neoadjuvant chemotherapy and radiotherapy, followed by mastectomy and immediate free autologous reconstruction. To our knowledge, this is the first series to assess the outcomes of neoadjuvant radiotherapy on immediate free microvascular breast reconstruction. METHODS: A review of patients with LABC who underwent immediate free autologous breast reconstruction post neoadjuvant chemoradiotherapy between 2013 and 2017 was conducted. All reconstructions were performed by a single reconstructive team. The primary end points were flap failure and surgical complications. Secondary end points were pathological response rate and disease recurrence. RESULTS: A total of 40 women with an average age of 48.1 (36-61) and average body mass index of 25.6 (18-37) were included. The most common choice of flap was immediate deep inferior epigastric perforator (DIEP, 31), followed by transverse or diagonal upper gracilis (5), muscle-sparing transversus abdominis (3), and stacked DIEP (1). Our major complication rate was 12.5% and minor complication 15%. There were no cases of local recurrence and only three cases (7.5%) of distant disease progression. CONCLUSION: From our experience, this treatment sequence allows patients to have an immediate gold standard reconstruction without an increase in surgical morbidity. It affords the benefits of IBR without concern in delaying adjuvant therapy and appears to be safe from an oncological perspective.

Effects of halogenated contaminants on reproductive development in wild mink (Neovison vison) from locations in Canada.

The concept of the Anthropocene, that humans are now re-engineering global ecosystems, is in part evidenced by the pervasive pollution by persistent organic pollutants (POPs). Certain POPs are hormone mimics and can disrupt endocrine and hence reproductive processes, shown mainly by laboratory studies with model species. There are, in contrast, fewer confirmations of such disruption from eco-epidemiological studies of wild mammals. Here we used the American mink (Neovison vison) as a sentinel species for such a study. Over the period 1998-2006, 161 mink carcasses were obtained from commercial trappers in the Canadian provinces of British Columbia and Ontario. Mink were aged, sexed, measured, and body condition assessed. Livers were analyzed either individually or pooled for organochlorine (OC) pesticides, polychlorinated biphenyls (PCBs), and subsets for polybrominated diphenyl ethers (PBDEs). We primarily addressed whether contaminants affected male reproductive development by measuring baculum size and assessing the influences of age and body condition. We also considered the influence of spatial variation on relative exposure and size of baculum. Statistical models separated by age class revealed that significant relationships between baculum length or mass and juvenile mink were mostly positive, whereas for adults and first year mink they were mostly negative. A significant negative relationship for adult mink was determined between DDE and both baculum length and mass. For juvenile mink we found significant positive relationships between ∑PCBs, DDE and ∑PBDEs with baculum length. Our results provide some indication of negative effects of halogenated contaminants on male reproductive development in wild mink, and the most likely candidate chemical is the confirmed anti-androgenic compound, DDE, rather than PCBs or other compounds.

Organochlorine contaminants in wild mink from the lower Great Lakes basin, Canada, 1998-2006.

Organochlorine contaminants were measured in livers of wild mink (Neovison vison) trapped in the lower Great Lakes basin from 1998 to 2006. To assess exposure and potential risk in mink feeding on Great Lakes biota, concentrations of contaminants were compared in mink trapped within 7.8 km of the shoreline as well as at inland sites (i.e., 8-40 km). Overall, significant spatial variation in mean hepatic concentrations of sum PCBs and seven other organochlorines was found in mink from 13 Great Lakes sites, many of which are within the Great Lakes Areas of Concern. Mean sum PCB concentrations, on a lipid weight basis, ranged from 2 µg/g in mink from inland Lake Ontario sites to 44 µg/g in mink from western Lake Erie. Concentrations of other organochlorines in mink were generally low. Mink from western Lake Erie had the highest mean cumulative organochlorine burdens dominated largely by PCBs. A significant age effect was found with 1-year-old mink having significantly higher PCB burdens than mink less than 1 year in age. With respect to published PCB threshold effect concentrations, some mink exceeded those associated with effects on reproduction and survival as well as the presence of jaw lesions. This was most consistently found in western Lake Erie where the health of populations of wild mink may be adversely affected and where no mink 2 years of age or older were collected.

Electronic microarray assays for avian influenza and Newcastle disease virus.

Microarrays are suitable for multiplexed detection and typing of pathogens. Avian influenza virus (AIV) is currently classified into 16 H (hemagglutinin) and 9 N (neuraminidase) subtypes, whereas Newcastle disease virus (NDV) strains differ in virulence and are broadly classified into high and low pathogenicity types. In this study, three assays for detection and typing of poultry viruses were developed on an automated microarray platform: a multiplex assay for simultaneous detection of AIV and detection and pathotyping of NDV, and two separate assays for differentiating all AIV H and N subtypes. The AIV-NDV multiplex assay detected all strains in a 63 virus panel, and accurately typed all high pathogenicity NDV strains tested. A limit of detection of 10(1)-10(3) TCID(50)/mL and 200-400 EID(50)/mL was obtained for NDV and AIV, respectively. The AIV typing assays accurately typed all 41 AIV strains and a limit of detection of 4-200 EID(50)/mL was obtained. Assay validation showed that the microarray assays were generally comparable to real-time RT-PCR. However, the AIV typing microarray assays detected more positive clinical samples than the AIV matrix real-time RT-PCR, and also provided information regarding the subtype. The AIV-NDV multiplex and AIV H typing microarray assays detected mixed infections and could be useful for detection and typing of AIV and NDV.

Mercury and other heavy metals in free-ranging mink of the lower Great Lakes basin, Canada, 1998-2006.

Mercury concentrations were examined in livers of free-ranging mink (Neovison vison) trapped in the lower Great Lakes basin from 1998 to 2006. Significant geographic variation in total mercury levels was found in mink from 13 Great Lakes locations, many of which are within Great Lakes Areas of Concern (AOCs). Total mercury levels in mink from these locations were generally low to moderate, with means (±SE) ranging from 0.99 (±0.36) µg/g dry weight in mink from inland Lake Erie to 7.31 (±1.52) µg/g in mink from Walpole Island in the St. Clair River AOC. Overall, mercury exposure was highest in mink trapped in locations associated with large riverine wetlands. Total mercury concentrations were negatively related to the percentage of mercury present as methylmercury and positively related to selenium concentrations, consistent with increasing demethylation of methylmercury with increasing total mercury concentrations. One-year old mink had significantly higher levels of mercury and selenium than mink under 1 year but similar to mink 2 years of age and older. Mercury levels were below those associated with mercury toxicity. Concentrations of cadmium, lead and arsenic were low and largely associated with environmental background levels. Significant age and sex effects were found for cadmium. Lead levels were significantly lower in mink infected by the lung nematode parasite compared to uninfected animals. Further studies of biomagnification of methylmercury in wetland environments, where top predators such as mink may be an increased risk of exposure, are warranted.

Multiplex RT-PCR detection and microarray typing of vesicular disease viruses.

A vesicular disease multiplex reverse transcription (RT)-PCR with an accompanying microarray assay was developed for simultaneous detection and typing of foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV), and for the detection of swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV). The multiplex RT-PCR successfully detected viral RNA from a collection of 49 strains of vesicular viruses, including multiple strains from all seven serotypes of FMDV and both serotypes of VSV. The multiplex RT-PCR was also able to produce amplified products from the RNA genome of all four viruses simultaneously in mixed samples. An indirect (post-PCR labelling) amplicon labelling method and a direct (concurrent labelling with PCR) amplicon labelling method were compared for the purpose of microarray detection and typing. Accurate detection and typing was achieved with all strains tested in the microarray assay which utilized 163 virus- and serotype-specific probes. It was observed that microarray increased detection for some samples compared to using multiplex RT-PCR alone. This was most likely due to signal amplification resulting from fluorescent labelling. The limit of detection of the microarray assay was as low as 4.6TCID(50)/mL for FMDV. No amplification products or microarray reactivity was observed with non-target livestock pathogens tested or with samples collected from healthy cattle, sheep and pigs. All FMDV and VSV serotypes were detected as early as 2 days post-inoculation from oral swabs obtained from cattle infected experimentally.

Lead in the tissues of terrestrial raptors in southern Ontario, Canada, 1995-2001.

Terrestrial raptors which feed on upland hunted game species may increase their risk to lead exposure and lead poisoning by ingesting lead shot found in the tissues of prey. Lead exposure in 225 individuals of nineteen species of terrestrial raptors, collected as carcasses in southern Ontario from 1995-2001, was examined through the analysis of bone, liver and kidney tissues. In this study, one red-tailed hawk contained liver lead concentrations and exhibited signs consistent with lead poisoning. Liver and kidney concentrations of one turkey vulture were also significantly higher than that associated with subclinical effects. This same bird, plus another turkey vulture and a northern harrier, had elevated bone lead concentrations (>10 microg/g dw) associated with possible toxicity. Turkey vultures had the highest mean concentrations of lead in bone and kidney compared to other raptor species. While it appeared only a single bird suffered acute lead poisoning, lead levels exceeded threshold concentrations associated with subclinical or acute toxicity in 4 of 225, almost 2%, of terrestrial raptors assessed. Given the association between lead exposure in raptors and hunting of game species reported in other studies, the continued use of lead shot for upland hunting in Ontario likely remains as one of the primary sources of lead and a continued risk to these birds of prey.

A microsphere immunoassay for detection of antibodies to avian influenza virus.

A microsphere immunoassay (MIA) was developed for the detection of serum antibodies to avian influenza virus. A recombinant influenza A nucleoprotein expressed in baculovirus was conjugated to microspheres and incubated with antibodies. High median fluorescent intensities (MFIs) were obtained with a monoclonal antibody and positive chicken sera. Chickens were inoculated with 10 strains of avian influenza virus representing different subtypes, including high and low pathogenic H5 and H7 subtypes. Three hundred and fifty-four samples from experimentally infected chickens and controls were tested with a competitive ELISA (cELISA) and the MIA. MFIs were converted to positive/negative (PN) ratios. The results of both tests, as percentage inhibition and PN ratio, showed a high correlation (R2 = 0.77). From the comparison data, a ratio of > or =4.5 was selected as the cut-off value for positivity in the MIA. Using this cut-off value, the sensitivity and specificity of the MIA relative to the cELISA when all discordant experimental samples were retested was 99.3 and 93.1%, respectively. The relative specificity increased to 94.7% when additional negative sera (n = 68) were tested. The MIA may be useful for surveillance testing and as a screening test for flocks infected with low pathogenic avian influenza virus and could be expanded for simultaneous detection of antibodies against other avian infectious disease agents.