Targeted Intraoperative Radiation Therapy during Breast-Conserving Surgery for Patients with Early Stage Breast Cancer: A Phase II Single Center Prospective Trial.

Purpose: Patients with early stage breast cancer (ESBC) are conventionally treated with breast-conserving surgery (BCS) followed by whole-breast external beam radiation therapy (EBRT). The emergence of targeted intraoperative radiation therapy (TARGIT) with Intrabeam has been used as a therapeutic alternative for patients with risk-adapted ESBC. Here we present our radiation therapy toxicities (RTT), postoperative complications (PC), and short-term outcomes of the prospective phase II trial at the McGill University Health Center. Methods and Materials: Patients aged ≥50 years with biopsy-proven hormone receptor-positive, grade 1 or 2, invasive ductal carcinoma of the breast, cT1N0, were eligible for the study. Enrolled patients underwent BCS followed by immediate TARGIT of 20 Gy in 1 fraction. Upon final pathology, patients with low-risk breast cancer (LRBC) received no further EBRT, and those with high-risk breast cancer (HRBC) received further 15 to 16 fractions of whole breast EBRT. HRBC criteria included pathologic tumor size >2 cm, grade 3, positive lympho-vascular invasion, multifocal disease, close margins (<2 mm), or positive nodal disease. Results: A total of 61 patients with ESBC were enrolled in the study; upon final pathology, 40 (65.6%) had LRBC, and 21 (34.4%) had HRBC. The median follow-up was 3.9 years. The most common HRBC criteria were close margins in 66.6% (n = 14) and lymphovascular invasion in 28.6% (n = 6). No grade 4 RTT were observed in either group. The most common PC were seroma and cellulitis for both groups. The rate of locoregional recurrence was 0% in both groups. The overall survival in LRBC was 97.5% and in HRBC 95.2% with no significant differences. Deaths were nonbreast cancer related. Conclusions: In patients with ESBC undergoing BCS, the use of TARGIT shows low rates of RTT and PC complications. Moreover, our short-term outcomes show no significant difference at 3.9 years median follow-up for locoregional recurrence or overall survival between groups of patients receiving TARGIT alone or TARGIT followed by EBRT. Of all patients, 34.4% required further EBRT, most commonly due to close margins.

In situ Raman spectroscopy and machine learning unveil biomolecular alterations in invasive breast cancer.

Significance: As many as 60% of patients with early stage breast cancer undergo breast-conserving surgery. Of those, 20% to 35% need a second surgery because of incomplete resection of the lesions. A technology allowing in situ detection of cancer could reduce re-excision procedure rates and improve patient survival. Aim: Raman spectroscopy was used to measure the spectral fingerprint of normal breast and cancer tissue ex-vivo. The aim was to build a machine learning model and to identify the biomolecular bands that allow one to detect invasive breast cancer. Approach: The system was used to interrogate specimens from 20 patients undergoing lumpectomy, mastectomy, or breast reduction surgery. This resulted in 238 ex-vivo measurements spatially registered with standard histology classifying tissue as cancer, normal, or fat. A technique based on support vector machines led to the development of predictive models, and their performance was quantified using a receiver-operating-characteristic analysis. Results: Raman spectroscopy combined with machine learning detected normal breast from ductal or lobular invasive cancer with a sensitivity of 93% and a specificity of 95%. This was achieved using a model based on only two spectral bands, including the peaks associated with C-C stretching of proteins around 940 cm - 1 and the symmetric ring breathing at 1004 cm - 1 associated with phenylalanine. Conclusions: Detection of cancer on the margins of surgically resected breast specimen is feasible with Raman spectroscopy.

Breast Implant-Related Adverse Events During Mammography: An Assessment of the Food and Drug Administration Manufacturer and User Facility Device Experience Database.

BACKGROUND: Adverse events arising in patients with breast implants during mammography reported by the Food and Drug Administration include implant rupture, pain, and impaired visualization. However, data supporting these claims were collected in 2004, and since, newer implant generations have been developed with overall rate of implantation increasing by 48%. OBJECTIVES: This article aims to determine the current incidence of implant-related adverse events arising during mammography. METHODS: We analyzed reports regarding silicone and saline breast implants published in the Food and Drug Administration Manufacturer and User Facility Device Experience database between 2008 and November 2018. Search terms included "mammogram," "mammography," "radiograph," "breast cancer screening," "breast cancer test," and "x-ray." RESULTS: Of the 20 539 implant-related adverse events available in the Manufacturer and User Facility Device Experience database, 427 were retrieved using our search strategy and 41 were related to mammography. Thirty-five of identified cases (85.4%) reported implant rupture, of which 19 (54.3%) were confirmed by a healthcare professional, 9 (25.7%) were clinically confirmed by saline implant deflation, and 7 (20.0%) were unverified reports by patients. Sixteen ruptures (45.7%) occurred with silicone implants, whereas 19 ruptures (54.3%) occurred with saline. Other adverse events included pain (29.3%), change in implant appearance (14.6%), and swelling (7.3%). CONCLUSIONS: Although implant rupture, pain, change in implant appearance, and swelling may occur, minimal implant-related adverse events arise during mammography. Given the extremely low reported risk of implant rupture, this should neither prevent patients from adhering to breast cancer screening programs nor deter patients from seeking breast implants. Patients should be aware of these reported risks and discuss screening options with their breast cancer screening team.

Feasibility of Prospectively Comparing Opioid Analgesia With Opioid-Free Analgesia After Outpatient General Surgery: A Pilot Randomized Clinical Trial.

Importance: The overprescription of opioids to surgical patients is recognized as an important factor contributing to the opioid crisis. However, the value of prescribing opioid analgesia (OA) vs opioid-free analgesia (OFA) after postoperative discharge remains uncertain. Objective: To investigate the feasibility of conducting a full-scale randomized clinical trial (RCT) to assess the comparative effectiveness of OA vs OFA after outpatient general surgery. Design, Setting, and Participants: This parallel, 2-group, assessor-blind, pragmatic pilot RCT was conducted from January 29 to September 3, 2020 (last follow-up on October 2, 2020). at 2 university-affiliated hospitals in Montreal, Quebec, Canada. Participants were adult patients (aged ≥18 years) undergoing outpatient abdominal (ie, cholecystectomy, appendectomy, or hernia repair) or breast (ie, partial or total mastectomy) general surgical procedures. Exclusion criteria were contraindications to drugs used in the trial, preoperative opioid use, conditions that could affect assessment of outcomes, and intraoperative or early complications requiring hospitalization. Interventions: Patients were randomized 1:1 to receive OA (around-the-clock nonopioids and opioids for breakthrough pain) or OFA (around-the-clock nonopioids with increasing doses and/or addition of nonopioid medications for breakthrough pain) after postoperative discharge. Main Outcomes and Measures: Main outcomes were a priori RCT feasibility criteria (ie, rates of surgeon agreement, patient eligibility, patient consent, treatment adherence, loss to follow-up, and missing follow-up data). Secondary outcomes included pain intensity and interference, analgesic intake, 30-day unplanned health care use, and adverse events. Between-group comparison of outcomes followed the intention-to-treat principle. Results: A total of 15 surgeons were approached; all (100%; 95% CI, 78%-100%) agreed to have patients recruited and adhered to the study procedures. Rates of patient eligibility and consent were 73% (95% CI, 66%-78%) and 57% (95% CI, 49%-65%), respectively. Seventy-six patients were randomized (39 [51%] to OA and 37 [49%] to OFA) and included in the intention-to-treat analysis (mean [SD] age, 55.5 [14.5] years; 50 [66%] female); 40 (53%) underwent abdominal surgery, and 36 (47%) underwent breast surgery. Seventy-five patients (99%; 95% CI, 93%-100%) adhered to the allocated treatment; 1 patient randomly assigned to OFA received an opioid prescription. Seventeen patients (44%) randomly assigned to OA consumed opioids after discharge. Seventy-three patients (96%; 95% CI, 89%-99%) completed the 30-day follow-up. The rate of missing questionnaires was 37 of 3724 (1%; 95% CI, 0.7%-1.4%). All the a priori RCT feasibility criteria were fulfilled. Conclusions and Relevance: The findings of this pilot RCT support the feasibility of conducting a robust, full-scale RCT to inform evidence-based prescribing of analgesia after outpatient general surgery. Trial Registration: ClinicalTrials.gov Identifier: NCT04254679.

Spatially mapping the immune landscape of melanoma using imaging mass cytometry.

Melanoma is an immunogenic cancer with a high response rate to immune checkpoint inhibitors (ICIs). It harbors a high mutation burden compared with other cancers and, as a result, has abundant tumor-infiltrating lymphocytes (TILs) within its microenvironment. However, understanding the complex interplay between the stroma, tumor cells, and distinct TIL subsets remains a substantial challenge in immune oncology. To properly study this interplay, quantifying spatial relationships of multiple cell types within the tumor microenvironment is crucial. To address this, we used cytometry time-of-flight (CyTOF) imaging mass cytometry (IMC) to simultaneously quantify the expression of 35 protein markers, characterizing the microenvironment of 5 benign nevi and 67 melanomas. We profiled more than 220,000 individual cells to identify melanoma, lymphocyte subsets, macrophage/monocyte, and stromal cell populations, allowing for in-depth spatial quantification of the melanoma microenvironment. We found that within pretreatment melanomas, the abundance of proliferating antigen-experienced cytotoxic T cells (CD8+CD45RO+Ki67+) and the proximity of antigen-experienced cytotoxic T cells to melanoma cells were associated with positive response to ICIs. Our study highlights the potential of multiplexed single-cell technology to quantify spatial cell-cell interactions within the tumor microenvironment to understand immune therapy responses.

Growing-season frost is a better predictor of tree growth than mean annual temperature in boreal mixedwood forest plantations.

Increase in frost damage to trees due to earlier spring dehardening could outweigh the expected increase in forest productivity caused by climate warming. We quantified the impact of growing-season frosts on the performance of three spruce species (white, black, and Norway spruce) and various seed sources with different frost tolerance in two plantations, established on both sides of the eastern Canadian boreal-temperate forest ecotone. The objectives of this study were to determine (a) if spruce species and seed sources planted in sites far from their natural provenance would be less adapted to local site conditions, leading to increased frost damage and reduced height growth; (b) at which height above the ground growing-season frosts ceased to damage apical meristems; and (c) if height growth was best predicted by extreme climatic events (growing-season frosts) or by mean annual or summer temperature. At each site and for all spruce species and seed sources, we cross-sectioned spruce trees at different heights above the ground. Tree rings were cross-dated and screened for frost rings, which were then given a severity score based on cellular damage. Frost severity reduced height growth of all spruce species and provenances at both sites. Height growth of the non-native Norway spruce was the most reduced by frost severity and was the smallest species at both sites. Frost caused the highest growth reduction in white spruce at the boreal mixedwood site and had the least effect on black spruce at both sites. For all spruce species, height growth was affected up to 2 m above the ground. Model selection based on corrected Akaike's information criteria (AICc) identified that minimum temperature in May was by far the best climate variable predicting tree growth (AICc weight = 1), highlighting the importance of considering extreme climatic events, which are likely to increase in the future.

Probability of Spring Frosts, Not Growing Degree-Days, Drives Onset of Spruce Bud Burst in Plantations at the Boreal-Temperate Forest Ecotone.

Climate warming-driven early leaf-out is expected to increase forest productivity but concurrently increases leaf exposure to spring frosts, which could reduce forests' net productivity. We hypothesized that due to their damaging effect on buds, spring frosts exert a stronger control on bud phenology than do growing degree-days. We monitored bud flush phenology of three white spruce seed sources (one local seed source from the boreal mixedwood forest and two seed sources from the temperate forest), one black spruce seed source originating from the boreal mixedwood forest and four nonlocal Norway spruce seed sources in 2016 and 2017 in two plantations located on both sides of the temperate-boreal mixedwood forest ecotone in eastern Canada (Quebec). We aimed to determine inter- and intraspecies variations in bud break timing and sensitivity to air temperature and photoperiod. We expected that bud break timing for boreal species and seed sources would be better synchronized with the decrease in frost probability than for nonlocal species and seed sources. We used mixed binomial regressions and AICc model selection to determine the best environmental variables predicting each transition from one stage of bud phenology to the next. At both plantation sites, white spruce bud flush began and ended earlier compared to black and Norway spruce. Buds of all spruce species were sensitive to frost probability for early phenological stages, whereas growing degree-days controlled the remaining stages. Photoperiod sensitivity was higher for white spruce compared to black and Norway spruce and reached its maximum in the temperate forest. At intraspecies level, the two southern white spruce seed sources opened their buds earlier than the local source and were more sensitive to photoperiod, which increased their exposure to spring frosts. Onset of spruce bud flush is driven by spring frosts and photoperiod, but once started, bud phenology responds to temperature. The high photoperiod sensitivity in white spruces could counterbalance climate warming and limit future premature leaf-out, whereas the low photoperiod sensitivity in black spruce should not restrain leaf-out advancement with climate warming. Our results call for adapting the temperature-driven hypotheses of ecophysiological models predicting leaf-out to include spring frost probability.

Geographic isolation and climatic variability contribute to genetic differentiation in fragmented populations of the long-lived subalpine conifer Pinus cembra L. in the western Alps.

BACKGROUND: Genetic processes shape the modern-day distribution of genetic variation within and between populations and can provide important insights into the underlying mechanisms of evolution. The resulting genetic variation is often unequally partitioned within species' distribution range and especially large differences can manifest at the range limit, where population fragmentation and isolation play a crucial role in species survival. Despite several molecular studies investigating the genetic diversity and differentiation of European Alpine mountain forests, the climatic and demographic constrains which influence the genetic processes are often unknown. Here, we apply non-coding microsatellite markers to evaluate the sporadic peripheral and continuous populations of cembra pine (Pinus cembra L.), a long-lived conifer species that inhabits the subalpine treeline ecotone in the western Alps to investigate how the genetic processes contribute to the modern-day spatial distribution. Moreover, we corroborate our findings with paleoecological records, micro and macro-remains, to infer the species' possible glacial refugia and expansion scenarios. RESULTS: Four genetically distinct groups were identified, with Bayesian and FST based approaches, across the range of the species, situated in the northern, inner and south-western Alps. We found that genetic differentiation is substantially higher in marginal populations than at the center of the range, and marginal stands are characterized by geographic and genetic isolation due to spatial segregation and restricted gene flow. Moreover, multiple matrix regression approaches revealed effects of climatic heterogeneity in species' spatial genetic pattern. Also, population stability tests indicated that all populations had experienced a severe historical bottleneck, no heterozygosity excess was detected, suggesting that more recently population sizes have remained relatively stable. CONCLUSIONS: Our study demonstrated that cembra pine might have survived in multiple glacial refugia and subsequently recolonized the Alps by different routes. Modern-day marginal populations, at the edge of the species' range, could maintain stable sizes over long periods without inbreeding depression and preserve high amounts of genetic variation. Moreover, our analyses indicate that climatic variability has played a major role in shaping differentiation, in addition to past historical events such as migration and demographic changes.

Long-term changes in the impacts of global warming on leaf phenology of four temperate tree species.

Contrary to the generally advanced spring leaf unfolding under global warming, the effects of the climate warming on autumn leaf senescence are highly variable with advanced, delayed, and unchanged patterns being all reported. Using one million records of leaf phenology from four dominant temperate species in Europe, we investigated the temperature sensitivities of spring leaf unfolding and autumn leaf senescence (ST , advanced or delayed days per degree Celsius). The ST of spring phenology in all of the four examined species showed an increase and decrease during 1951-1980 and 1981-2013, respectively. The decrease in the ST during 1981-2013 appears to be caused by reduced accumulation of chilling units. As with spring phenology, the ST of leaf senescence of early successional and exotic species started to decline since 1980. In contrast, for late successional species, the ST of autumn senescence showed an increase for the entire study period from 1951 to 2013. Moreover, the impacts of rising temperature associated with global warming on spring leaf unfolding were stronger than those on autumn leaf senescence. The timing of leaf senescence was positively correlated with the timing of leaf unfolding during 1951-1980. However, as climate warming continued, the differences in the responses between spring and autumn phenology gradually increased, so that the correlation was no more significant during 1981-2013. Our results further suggest that since 2000, due to the decreased temperature sensitivity of leaf unfolding the length of the growing season has not increased any more. These finding needs to be addressed in vegetation models used for assessing the effects of climate change.

Influence of northern limit range on genetic diversity and structure in a widespread North American tree, sugar maple (Acer saccharum Marshall).

Due to climate change, the ranges of many North American tree species are expected to shift northward. Sugar maple (Acer saccharum Marshall) reaches its northern continuous distributional limit in northeastern North America at the transition between boreal mixed-wood and temperate deciduous forests. We hypothesized that marginal fragmented northern populations from the boreal mixed wood would have a distinct pattern of genetic structure and diversity. We analyzed variation at 18 microsatellite loci from 23 populations distributed along three latitudinal transects (west, central, and east) that encompass the continuous-discontinuous species range. Each transect was divided into two zones, continuous (temperate deciduous) and discontinuous (boreal mixed wood), based on sugar maple stand abundance. Respective positive and negative relationships were found between the distance of each population to the northern limit (D_north), and allelic richness (AR) and population differentiation (FST). These relations were tested for each transect separately; the pattern (discontinuous-continuous) remained significant only for the western transect. structure analysis revealed the presence of four clusters. The most northern populations of each transect were assigned to a distinct group. Asymmetrical gene flow occurred from the southern into the four northernmost populations. Southern populations in Québec may have originated from two different postglacial migration routes. No evidence was found to validate the hypothesis that northern populations were remnants of a larger population that had migrated further north of the species range after the retreat of the ice sheet. The northernmost sugar maple populations possibly originated from long-distance dispersal.

Fine-scale assessment of genetic diversity of trembling aspen in northwestern North America.

BACKGROUND: In North America, the last ice age is the most recent event with severe consequences on boreal species' ranges. Phylogeographic patterns of range expansion in trembling aspen (Populus tremuloides) suggested that Beringia is likely to be a refugium and the "ice-free corridor" in Alberta may represent a region where small populations persisted during the last glacial maximum (LGM). The purpose of this study was to ascertain whether the origins of trembling aspen in western North America are reflected in the patterns of neutral genetic diversity and population structure. A total of 28 sites were sampled covering the northwestern part of aspen's distribution, from Saskatchewan to Alaska. Twelve microsatellite markers were used to describe patterns of genetic diversity. The genetic structure of trembling aspen populations was assessed by using multivariate analyses, Mantel correlograms, neighbor-joining trees and Bayesian analysis. RESULTS: Microsatellite markers revealed little to no neutral genetic structure of P. tremuloides populations in northwestern North America. Low differentiation among populations and small isolation by distance (IBD) were observed. The most probable number of clusters detected by STRUCTURE was K = 3 (∆K = 5.9). The individuals in the populations of the 3 clusters share a common gene pool and showed a high level of admixture. No evidence was found that either Beringia or the "ice-free corridor" were refugia. Highest allelic richness (AR) and lowest heterozygosity (Ho) were observed in Alberta foothills of the Rocky Mountains. CONCLUSIONS: Contrary to our hypothesis, our results showed that microsatellite markers revealed little to no genetic structure in P. tremuloides populations. Consequently, no divergent populations were observed near supposed refugia. The lack of detectable refugia in Beringia and in the "ice-free corridor" was due to high levels of gene flow between trembling apsen populations. More favorable environmental conditions for sexual reproduction and successful trembling aspen seedling establishment may have contributed to increase allelic richness through recombination in populations from the Albertan foothills of the Rocky Mountains.

Genetic consequences of selection cutting on sugar maple (Acer saccharum Marshall).

Selection cutting is a treatment that emulates tree-by-tree replacement for forests with uneven-age structures. It creates small openings in large areas and often generates a more homogenous forest structure (fewer large leaving trees and defective trees) that differs from old-growth forest. In this study, we evaluated whether this type of harvesting has an impact on genetic diversity of sugar maple (Acer saccharum Marshall). Genetic diversity among seedlings, saplings, and mature trees was compared between selection cut and old-growth forest stands in Québec, Canada. We found higher observed heterozygosity and a lower inbreeding coefficient in mature trees than in younger regeneration cohorts of both forest types. We detected a recent bottleneck in all stands undergoing selection cutting. Other genetic indices of diversity (allelic richness, observed and expected heterozygosity, and rare alleles) were similar between forest types. We concluded that the effect of selection cutting on the genetic diversity of sugar maple was recent and no evidence of genetic erosion was detectable in Québec stands after one harvest. However, the cumulative effect of recurring applications of selection cutting in bottlenecked stands could lead to fixation of deleterious alleles, and this highlights the need for adopting better forest management practices.

Physiological integration of connected balsam poplar ramets.

Clonal integration between ramets can be an ecological advantage of clonal plant species in environments where resources are patchily distributed. We investigated physiological integration among Populus balsamifera L. ramets under drought stress in order to demonstrate water sharing between connected ramets. Pairs of connected ramets were grown in separate pots in the greenhouse where half of ramets had the parental root connection severed and half were left intact. Drought stress was applied to one ramet, and growth, specific leaf area (SLA), net photosynthesis, stomatal conductance, leaf water potential and carbon isotopic composition (δ(13)C) were measured after an 8-week growing period. Droughted ramets connected to watered ramets were able to maintain high gas exchange activity and water potential, similar to watered ramets. Leaf water potential and SLA results showed that the root connection was more beneficial for proximal compared with distal ramets. The parental root connection also allowed droughted ramets to discriminate more against (13)C compared with severed ramets. In conclusion, this study shows compelling evidence of physiological integration of connected P. balsamifera ramets through water sharing.

The clonal root system of balsam poplar in upland sites of Quebec and Alberta.

Balsam poplar seeds are short-lived and require moist seedbeds soon after they are released to germinate. In addition to sexual reproduction, balsam poplar stands can regenerate clonally by root suckering. The origin of stands will in turn affect their genetic structure and root system architecture, which are poorly understood for upland forest stands. Three stands were hydraulically excavated in Quebec (moist) and Alberta (dry) to determine the origin of trees and to characterize root systems with respect to presence of parental roots and root grafts connections. Clones were identified using single-nucleotide polymorphism (SNPs), and all stems, roots and root grafts were aged using dendrochronology techniques. All 82 excavated trees were of sucker origin, and four of the six stands contained a single clone. Parental root connections were found between 22% and 25% of excavated trees, and 53% and 48% of trees were linked with a root graft between the same or different clones, in Alberta and Quebec, respectively. Mean distance between trees connected by parental root was significantly lower than the distance between unconnected trees (0.47 ± 0.25 m vs. 3.14 ± 0.15 m and 1.55 ± 0.27 m vs. 4.25 ± 0.13 m) in Alberta and in Quebec, respectively. The excavations also revealed many dead stumps with live roots, maintained through root connections with live trees. This research highlights that balsam poplar growing in upland stands is a clonal species that can maintain relatively high genotypic diversity, with frequent root connections between trees at maturity. Maintaining an extensive root system through root connections increases the chances of a clone surviving when the above ground tree is dead and may also enhance the resilience of balsam poplar stands after disturbance.

Bluejoint Is an Effective Bio-Barrier Species on Mine Covers.

Covers with capillary barrier effects (CCBE) are used to prevent acid mine drainage from mine wastes in the short term. However, the long-term efficiency of CCBE can be affected by trees because their roots may reduce the ability of covers to limit oxygen migration and also physically damage the CCBE. Two plant species that are native to boreal Canada, bluejoint () and sheep laurel (, were selected as bio-barrier species (BBS) to test if they reduce the growth and root system architecture of trees established on mine covers (balsam poplar [], willow [ spp], and black spruce []). The experiment was established in 2008 on a mine tailings impoundment located in northwestern Quebec, Canada. Trees were measured for height, diameter, and biomass. Coarse roots were excavated from the plots and digitized in three dimensions. Compared with the control (no BBS), bluejoint strongly decreased tree height and diameter increment, biomass, maximum root depth and radial extension, total root length and volume, and number of second- and third-order tree roots. Height and diameter increment, biomass, maximum root depth and volume, and number of second-order roots of balsam poplar increased with sheep laurel compared with control conditions, whereas willow showed no response to this treatment. Most characteristics of black spruce (except root-to-shoot ratio and number of second-order roots) improved in the presence of sheep laurel compared with the control. Thus, bluejoint was a more efficient BBS than sheep laurel. Bio-barriers comprised of bluejoint can be used as a countermeasure for controlling tree invasion of CCBE.

Infiltrating syringomatous adenoma of the nipple: Sonographic and mammographic features with pathologic correlation.

We report the case of a 39-year-old woman complaining of painless unilateral nipple enlargement for 3 weeks. She had no family history of breast cancer. Clinical examination revealed left nipple enlargement without pain, erythema, or skin changes m no associated palpable breast or axillary masses. Ultrasound showed several bright foci in the left nipple suggestive of microcalcifications. Neither solid nor cystic masses were detected. The mammogram performed subsequently confirmed the presence of multiple pleomorphic microcalcifications within the nipple. Wedge biopsy showed a syringomatous adenoma. Wide local excision of the nipple was performed. The postoperative course was uneventful.

Development and multiplexed amplification of SSR markers for Thuja occidentalis (Cupressaceae) using shotgun pyrosequencing.

PREMISE OF THE STUDY: Sixteen novel, polymorphic, multiplexed microsatellite loci were developed for eastern white cedar (Thuja occidentalis) using simple sequence repeat (SSR)-enriched shotgun pyrosequencing. • METHODS AND RESULTS: Sixteen loci were tested on a panel of 24 individuals from different populations. The number of observed alleles ranged from four to 22. Four sets of multiplex PCR for the 16 loci were then carried out on 60 individuals of two populations from islands of FERLD Duparquet Forest, Canada. Mean number of alleles, observed heterozygosity, and expected heterozygosity were respectively 5.75, 0.594, and 0.574 for Island 58, and 5.50, 0.704, and 0.624 for Island 134. • CONCLUSIONS: Four sets of multiplex microsatellite loci can be used for future genetic studies, which includes investigating genetic diversity and structure, and fragmentation and regeneration studies.

Genetic consequences of fragmentation in "arbor vitae," eastern white cedar (Thuja occidentalis L.), toward the northern limit of its distribution range.

We tested the hypothesis that marginal fragmented populations of eastern white cedar (EWC) are genetically isolated due to reduced pollen and gene flow. In accordance with the central-marginal model, we predicted a decrease in population genetic diversity and an increase in differentiation along the latitudinal gradient from the boreal mixed-wood to northern coniferous forest. A total of 24 eastern white cedar populations were sampled along the north-south latitudinal gradient for microsatellite genotyping analysis. Positive F(is) values and heterozygote deficiency were observed in populations from the marginal (F(is) = 0.244; P(HW) = 0.0042) and discontinuous zones (F(is) = 0.166; P(HW) = 0.0042). However, populations from the continuous zone were in HW equilibrium (F(is) = -0.007; P(HW) = 0.3625). There were no significant latitudinal effects on gene diversity (H(s)), allelic richness (AR), or population differentiation (F(st)). Bayesian and NJT (neighbor-joining tree) analyses demonstrated the presence of a population structure that was partly consistent with the geographic origins of the populations. The impact of population fragmentation on the genetic structure of EWC is to create a positive inbreeding coefficient, which was two to three times higher on average than that of a population from the continuous zone. This result indicated a higher occurrence of selfing within fragmented EWC populations coupled with a higher degree of gene exchange among near-neighbor relatives, thereby leading to significant inbreeding. Increased population isolation was apparently not correlated with a detectable effect on genetic diversity. Overall, the fragmented populations of EWC appear well-buffered against effects of inbreeding on genetic erosion.

Herbivore-simulated induction of defenses in clonal networks of trembling aspen (Populus tremuloides).

Trembling aspen (Populus tremuloides Michx.) as a clonal tree species possesses a complex root system through which trees of the same or different clones are connected. Root connections have been studied with respect to resource sharing, but the nature, quantities or extent of what is shared between trees is relatively unknown. In this study, we posed the hypothesis that systemic defense induction signals could also spread through these root networks and trigger defenses in neighboring ramets before arrival of pests. Temporal expression pattern of Kunitz trypsin inhibitor (KTI) and dihydroflavonol reductase (DFR) genes, two markers of poplar defense, was followed by quantitative real-time polymerase chain reaction. The expression was quantified in systemic leaves of wounded and healthy plants that shared the same parental root and in untreated controls grown in separate pots. Untreated interconnected plants did not show induced resistance upon herbivore-simulated attack. Although wound-treated ramets induced defense genes, untreated interconnected plants produced an expression pattern similar to non-connected controls. Root connections do not automatically lead to induction of defensive traits that are expressed in plants directly under damage thought to simulate herbivory. Rather, it seems that other communication means such as airborne volatiles can serve as signal transmission pathways among neighboring plants.