Organ preservation after neoadjuvant long-course chemoradiotherapy versus short-course radiotherapy.

BACKGROUND: Potential differences in organ preservation between total neoadjuvant therapy (TNT) regimens integrating long-course chemoradiotherapy (LCCRT) and short-course radiotherapy (SCRT) in rectal cancer remain undefined. PATIENTS AND METHODS: This natural experiment arose from a policy change in response to the COVID-19 pandemic during which our institution switched from uniformly treating patients with LCCRT to mandating that all patients be treated with SCRT. Our study includes 323 locally advanced rectal adenocarcinoma patients treated with LCCRT-based or SCRT-based TNT from January 2018 to January 2021. Patients who achieved clinical complete response were offered organ preservation with watch-and-wait (WW) management. The primary outcome was 2-year organ preservation. Additional outcomes included local regrowth, distant recurrence, disease-free survival (DFS), and overall survival (OS). RESULTS: Patient and tumor characteristics were similar between LCCRT (n = 247) and SCRT (n = 76) cohorts. Median follow-up was 31 months. Similar clinical complete response rates were observed following LCCRT and SCRT (44.5% versus 43.4%). Two-year organ preservation was 40% [95% confidence interval (CI) 34% to 46%] and 31% (95% CI 22% to 44%) among all patients treated with LCCRT and SCRT, respectively. In patients managed with WW, LCCRT resulted in higher 2-year organ preservation (89% LCCRT, 95% CI 83% to 95% versus 70% SCRT, 95% CI 55% to 90%; P = 0.005) and lower 2-year local regrowth (19% LCCRT, 95% CI 11% to 26% versus 36% SCRT, 95% CI 16% to 52%; P = 0.072) compared with SCRT. The 2-year distant recurrence (10% versus 6%), DFS (90% versus 90%), and OS (99% versus 100%) were similar between WW patients treated with LCCRT and SCRT, respectively. CONCLUSIONS: While WW eligibility was similar between cohorts, WW patients treated with LCCRT had higher 2-year organ preservation and lower local regrowth than those treated with SCRT, yet similar DFS and OS. These data support induction LCCRT followed by consolidation chemotherapy as the preferred TNT regimen for patients with locally advanced rectal cancer pursuing organ preservation.

Development and evaluation of the Capability, Opportunity, and Motivation to deliver Physical Activity in School Scale (COM-PASS).

BACKGROUND: Teachers are recognized as 'key agents' for the delivery of physical activity programs and policies in schools. The aim of our study was to develop and evaluate a tool to assess teachers' capability, opportunity, and motivation to deliver school-based physical activity interventions. METHODS: The development and evaluation of the Capability, Opportunity, and Motivation to deliver Physical Activity in School Scale (COM-PASS) involved three phases. In Phase 1, we invited academic experts to participate in a Delphi study to rate, provide recommendations, and achieve consensus on questionnaire items that were based on the Capability, Opportunity, and Motivation Behavior (COM-B) model. Each item was ranked on the degree to which it matched the content of the COM-B model, using a 5-point scale ranging from '1 = Poor match' to '5 = Excellent match'. In Phase 2, we interviewed primary and secondary school teachers using a 'think-aloud' approach to assess their understanding of the items. In Phase 3, teachers (n = 196) completed the COM-PASS to assess structural validity using confirmatory factor analysis (CFA). RESULTS: Thirty-eight academic experts from 14 countries completed three rounds of the Delphi study. In the first round, items had an average rating score of 4.04, in the second round 4.51, and in the third (final) round 4.78. The final tool included 14 items, which related to the six constructs of the COM-B model: physical capability, psychological capability, physical opportunity, social opportunity, reflective motivation, and automatic motivation. In Phase 2, ten teachers shared their interpretation of COM-PASS via a 20-min interview, which resulted in minor changes. In Phase 3, CFA of the 3-factor model (i.e., capability, opportunity, and motivation) revealed an adequate fit to the data (χ2 = 122.6, p < .001, CFI = .945, TLI = .924, RMSEA = .066). The internal consistencies of the three subscale scores were acceptable (i.e., capability: α = .75, opportunity: α = .75, motivation: α = .81). CONCLUSION: COM-PASS is a valid and reliable tool for assessing teachers' capability, opportunity, and motivation to deliver physical activity interventions in schools. Further studies examining additional psychometric properties of the COM-PASS are warranted.

ALLIANCE A022104/NRG-GI010: The Janus Rectal Cancer Trial: a randomized phase II/III trial testing the efficacy of triplet versus doublet chemotherapy regarding clinical complete response and disease-free survival in patients with locally advanced rectal cancer.

Background: Recent data have demonstrated that in locally advanced rectal cancer (LARC), a total neoadjuvant therapy (TNT) approach improves compliance with chemotherapy and increases rates of tumor response compared to neoadjuvant chemoradiation (CRT) alone. They further indicate that the optimal sequencing of TNT involves consolidation (rather than induction) chemotherapy to optimize complete response rates. Data, largely from retrospective studies, have also shown that patients with clinical complete response (cCR) after neoadjuvant therapy may be managed safely with the watch and wait approach (WW) instead of preemptive total mesorectal resection (TME). However, the optimal consolidation chemotherapy regimen to achieve cCR has not been established, and a randomized clinical trial has not robustly evaluated cCR as a primary endpoint. Collaborating with a multidisciplinary oncology team and patient groups, we designed this NCI-sponsored study of chemotherapy intensification to address these issues and to drive up cCR rates, to provide opportunity for organ preservation, improve quality of life for patients and improve survival outcomes. Methods: In this NCI-sponsored multi-group randomized, seamless phase II/III trial (1:1), up to 760 patients with LARC, T4N0, any T with node positive disease (any T, N+) or T3N0 requiring abdominoperineal resection or coloanal anastomosis and distal margin within 12 cm of anal verge will be enrolled. Stratification factors include tumor stage (T4 vs T1-3), nodal stage (N+ vs N0) and distance from anal verge (0-4; 4-8; 8-12 cm). Patients will be randomized to receive neoadjuvant long course chemoradiation (LCRT) followed by consolidation doublet (mFOLFOX6 or CAPOX) or triplet chemotherapy (mFOLFIRINOX) for 3-4 months. LCRT in both arms involves 4500 cGy in 25 fractions over 5 weeks + 900 cGy boost in 5 fractions with a fluoropyrimidine (capecitabine preferred). Patients will undergo assessment 8-12 (+/- 4) weeks post-TNT completion. The primary endpoint for the phase II portion will compare cCR between treatment arms. A total number of 296 evaluable patients (148 per arm) will provide statistical power of 90.5% to detect an 17% increase in cCR rate, at a one-sided alpha=0.048. The primary endpoint for the phase III portion will compare disease-free survival (DFS) between treatment arms. A total of 285 DFS events will provide 85% power to detect an effect size of hazard ratio 0.70 at a one-sided alpha of 0.025, requiring enrollment of 760 patients (380 per arm). Secondary objectives include time-to event outcomes (overall survival, organ preservation time and time to distant metastasis) and adverse effects. Biospecimens including archival tumor tissue, plasma and buffy coat in EDTA tubes, and serial rectal MRIs will be collected for exploratory correlative research. This study, activated in late 2022, is open across the NCTN and has a current accrual of 312. Support: U10CA180821, U10CA180882, U24 CA196171; https://acknowledgments.alliancefound.org . Discussion: Building off of data from modern day rectal cancer trials and patient input from national advocacy groups, we have designed the current trial studying chemotherapy intensification via a consolidation chemotherapy approach with the intent to enhance cCR and DFS rates, increase organ preservation rates, and improve quality of life for patients with rectal cancer. Trial Registration: Clinicaltrials.gov ID: NCT05610163 ; Support includes U10CA180868 (NRG) and U10CA180888 (SWOG).

Partial Ribosomal Nontranscribed Spacer Sequences Distinguish Rhagoletis zephyria (Diptera: Tephritidae) From the Apple Maggot, R. pomonella.

The apple maggot, Rhagoletis pomonella (Walsh), was introduced into the apple-growing regions of the Pacific Northwest in the U.S.A. during the past 60-100 yr. Apple maggot (larvae, puparia, and adults) is difficult to distinguish from its morphologically similar sister species, Rhagoletis zephyria Snow, which is native and abundant in the Pacific Northwest. While morphological identifications are common practice, a simple, inexpensive assay based on genetic differences would be very useful when morphological traits are unclear. Here we report nucleotide substitution and insertion-deletion mutations in the nontranscribed spacer (NTS) of the ribosomal RNA gene cistron of R. pomonella and R. zephyria that appear to be diagnostic for these two fly species. Insertion-deletion variation is substantial and results in a 49 base-pair difference in PCR amplicon size between R. zephyria and R. pomonella that can be scored using agarose gel electrophoresis. PCR amplification and DNA sequencing of 766 bp of the NTS region from 38 R. pomonella individuals and 35 R. zephyria individuals from across their geographic ranges led to the expected PCR fragments of approx. 840 bp and 790 bp, respectively, as did amplification and sequencing of a smaller set of 26 R. pomonella and 16 R. zephyria flies from a sympatric site in Washington State. Conversely, 633 bp mitochondrial COI barcode sequences from this set of flies were polyphyletic with respect to R. pomonella and R. zephyria. Thus, differences in NTS PCR products on agarose gels potentially provide a simple way to distinguish between R. pomonella and R. zephyria.

Geographic distribution of Ovavesicula popilliae in the United States and sensitivity of visual diagnosis compared with qPCR detection.

The Japanese beetle (Popillia japonica) is one of the most destructive invasive pests in North America, causing significant economic impact to many fruit crops, turfgrass and the nursery industry. A microsporidian pathogen of Japanese beetle, Ovavesicula popilliae, discovered in 1985, proliferates in the Malpighian tubules of larvae and adults, disrupting waste-removal, mineral filtering, and fluid balance in heavily infected individuals. Most infected larvae do not survive from fall to spring, and egg production by infected females is reduced by 50%. Ovavesicula popilliae is promising as a classical biological control agent for Japanese beetle, but outside of surveys completed in Connecticut and Michigan little is known about its geographic distribution in North America. The objective of this research is to obtain a better understanding of the distribution of O. popilliae in North America. Japanese beetles were collected at 59 locations in a total of 19 different states in the USA for pathogen analysis. Overall, the proportion of Japanese beetle adults infected by O. popilliae was much greater in Michigan, Ohio, Tennessee and four states in the northeastern USA compared with sites located west of the Mississippi River (18.6 ± 13.3% and 0.6 ± 1.2%, respectively). Nucleotide sequences of the gene encoding a small subunit of nuclear ribosomal RNA (ssrDNA), obtained from GenBank for O. popilliae was used to develop a highly specific qPCR test for O. popilliae DNA. A subsample of 110 individual Japanese beetles were visually diagnosed first, then analyzed via qPCR. Visual diagnosis and qPCR detection agreed for 80.9% of the beetles tested. The qPCR assay is more sensitive than visual diagnosis (56 visually positive, 73 qPCR positive), is highly specific for O. popilliae, and will be useful for detecting the pathogen in large batches of beetles, or in beetle frass.

Identification of immune and non-immune cells in regenerating axolotl limbs by single-cell sequencing.

Immune cells are known to be critical for successful limb regeneration in the axolotl (Ambystoma mexicanum), but many details regarding their identity, behavior, and function are yet to be resolved. We isolated peripheral leukocytes from the blood of adult axolotls and then created two samples for single-cell sequencing: 1) peripheral leukocytes (N = 7889) and 2) peripheral leukocytes with presumptive macrophages from the intraperitoneal cavity (N = 4998). Using k-means clustering, we identified 6 cell populations from each sample that presented gene expression patterns indicative of erythrocyte, thrombocyte, neutrophil, B-cell, T-cell, and myeloid cell populations. A seventh, presumptive macrophage cell population was identified uniquely from sample 2. We then isolated cells from amputated axolotl limbs at 1 and 6 days post-amputation (DPA) and performed single cell sequencing (N = 8272 and 9906 cells respectively) to identify immune and non-immune cell populations. Using k-means clustering, we identified 8 cell populations overall, with the majority of cells expressing erythrocyte-specific genes. Even though erythrocytes predominated, we used an unbiased approach to identify infiltrating neutrophil, macrophage, and lymphocyte populations at both time points. Additionally, populations expressing genes for epidermal cells, fibroblast-like cells, and endothelial cells were also identified. Consistent with results from previous experimental studies, neutrophils were more abundant at 1 DPA than 6 DPA, while macrophages and non-immune cells exhibited inverse abundance patterns. Of note, we identified a small population of fibroblast-like cells at 1 DPA that was represented by considerably more cells at 6 DPA. We hypothesize that these are early progenitor cells that give rise to the blastema. The enriched gene sets from our work will aid future single-cell investigations of immune cell diversity and function during axolotl limb regeneration.

Risk factors associated with progression to referable retinopathy: a type 2 diabetes mellitus cohort study in the Republic of Ireland.

AIM: To determine factors associated with progression to referable diabetic retinopathy in people with type 2 diabetes in the Republic of Ireland. RESEARCH DESIGN AND METHODS: The study was conducted in a dynamic cohort of 2770 people with type 2 diabetes, recruited between April 2005 and July 2013. Systemic factors (systolic and diastolic blood pressure, HbA1c , lipid levels, BMI) and baseline diabetic retinopathy grading results were evaluated at 4-monthly and yearly intervals, respectively. Associations between risk factors (most recently recorded value, and rate of change in value between pairs of consecutive systemic evaluations) and development of referable diabetic retinopathy were estimated using Cox proportional hazards models. RESULTS: There was a fourfold increased risk of progression to referral when retinopathy was present at baseline vs no retinopathy at baseline (hazard ratio 4.02, 95% CI 2.80-5.78; P<0.001). Higher current values of HbA1c (hazard ratio 1.22, 95% CI 1.11-1.34; P<0.001), systolic blood pressure (hazard ratio 1.29, 95% CI 1.15-1.45; P<0.001) and triglycerides (hazard ratio 1.10, 95% CI 1.03-1.18; P=0.004) were associated with increased risk of referral. Higher current BMI (hazard ratio 0.83, 95% CI 0.73-0.95; P=0.007) and diastolic blood pressure (hazard ratio 0.91, 95% CI 0.85-0.97; P=0.006) were associated with reduced risk of referral. CONCLUSIONS: Presence of retinopathy at baseline was strongly associated with increased risk of referral. Modest associations between systemic factors and risk of progression to referable retinopathy were detected.

Postoperative chemotherapy improves survival in patients with resected high-risk Stage II colorectal cancer: results of a systematic review and meta-analysis.

AIM: The aim was to assess the benefit of adjuvant chemotherapy in high-risk Stage II colorectal cancer. METHOD: A systematic literature review and meta-analysis was performed comparing survival in patients with resected Stage II colorectal cancer and high-risk features having postoperative chemotherapy vs no chemotherapy. RESULTS: Of 1031 articles screened, 29 were included, reporting on 183 749 participants. Adjuvant chemotherapy significantly improved overall survival [hazard ratio (HR) 0.61, P < 0.0001], disease-specific survival (HR = 0.73, P = 0.05) and disease-free survival (HR = 0.59, P < 0.0001) compared to no chemotherapy. Adjuvant chemotherapy significantly increased 5-year overall survival (OR = 0.53, P = 0.0008) and 5-year disease-free survival (OR = 0.50, P = 0.001). Overall survival and disease-free survival remained significantly prolonged during subgroup analysis of studies published from 2015 onwards (HR = 0.60, P < 0.0001; HR = 0.65, P = 0.0001; respectively), in patients with two or more high-risk features (HR = 0.59, P = 0.0001; HR = 0.70, P = 0.03; respectively) and in colon cancer (HR = 0.61, P < 0.0001; HR = 0.51, P = 0.0001; respectively). Overall survival, disease-specific survival and disease-free survival during subgroup analysis of individual high-risk features were T4 tumour (HR = 0.58, P < 0.0001; HR = 0.50, P = 0.003; HR = 0.75, P = 0.05), < 12 lymph nodes harvested (HR = 0.67, P = 0.0002; HR = 0.80, P = 0.17; HR = 0.72, P = 0.02), poor differentiation (HR = 0.84, P = 0.35; HR = 0.85, P = 0.23; HR = 0.61, P = 0.41), lymphovascular or perineural invasion (HR = 0.55, P = 0.05; HR = 0.59, P = 0.11; HR = 0.76, P = 0.05) and emergency surgery (HR = 0.60, P = 0.02; HR = 0.68, P = 0.19). CONCLUSION: Adjuvant chemotherapy in high-risk Stage II colorectal cancer results in a modest survival improvement and should be considered on an individual patient basis. Due to potential heterogeneity and selection bias of the included studies, and lack of separate rectal cancer data, further large randomized trials with predefined inclusion criteria and standardized chemotherapy regimens are required.

Changes in the multidisciplinary management of rectal cancer from 2009 to 2015 and associated improvements in short-term outcomes.

AIM: Significant recent changes in management of locally advanced rectal cancer (LARC) include preoperative staging, use of extended neoadjuvant therapies and minimally invasive surgery (MIS). This study was aimed at characterizing these changes and associated short-term outcomes. METHOD: We retrospectively analysed treatment and outcome data from patients with T3/4 or N+ LARC ≤ 15 cm from the anal verge who were evaluated at a comprehensive cancer centre in 2009-2015. RESULTS: In total, 798 patients were identified and grouped into five cohorts based on treatment year: 2009-2010, 2011, 2012, 2013 and 2014-2015. Temporal changes included increased reliance on MRI staging, from 57% in 2009-2010 to 98% in 2014-2015 (P < 0.001); increased use of total neoadjuvant therapy, from 17% to 76% (P < 0.001); and increased use of MIS, from 33% to 70% (P < 0.001). Concurrently, median hospital stay decreased (from 7 to 5 days; P < 0.001), as did the rates of Grade III-V complications (from 13% to 7%; P < 0.05), surgical site infections (from 24% to 8%; P < 0.001), anastomotic leak (from 11% to 3%; P < 0.05) and positive circumferential resection margin (from 9% to 4%; P < 0.05). TNM downstaging increased from 62% to 74% (P = 0.002). CONCLUSION: Shifts toward MRI-based staging, total neoadjuvant therapy and MIS occurred between 2009 and 2015. Over the same period, treatment responses improved, and lengths of stay and the incidence of complications decreased.

Effectiveness of a multidisciplinary patient care bundle for reducing surgical-site infections.

BACKGROUND: Surgical-site infection (SSI) is associated with significant healthcare costs. To reduce the high rate of SSI among patients undergoing colorectal surgery at a cancer centre, a comprehensive care bundle was implemented and its efficacy tested. METHODS: A pragmatic study involving three phases (baseline, implementation and sustainability) was conducted on patients treated consecutively between 2013 and 2016. The intervention included 13 components related to: bowel preparation; oral and intravenous antibiotic selection and administration; skin preparation, disinfection and hygiene; maintenance of normothermia during surgery; and use of clean instruments for closure. SSI risk was evaluated by means of a preoperative calculator, and effectiveness was assessed using interrupted time-series regression. RESULTS: In a population with a mean BMI of 30 kg/m2 , diabetes mellitus in 17·5 per cent, and smoking history in 49·3 per cent, SSI rates declined from 11·0 to 4·1 per cent following implementation of the intervention bundle (P = 0·001). The greatest reductions in SSI rates occurred in patients at intermediate or high risk of SSI: from 10·3 to 4·7 per cent (P = 0·006) and from 19 to 2 per cent (P < 0·001) respectively. Wound care modifications were very different in the implementation phase (43·2 versus 24·9 per cent baseline), including use of an overlying surface vacuum dressing (17·2 from 1·4 per cent baseline) or leaving wounds partially open (13·2 from 6·7 per cent baseline). As a result, the biggest difference was in wound-related rather than organ-space SSI. The median length of hospital stay decreased from 7 (i.q.r. 5-10) to 6 (5-9) days (P = 0·002). The greatest reduction in hospital stay was seen in patients at high risk of SSI: from 8 to 6 days (P < 0·001). SSI rates remained low (4·5 per cent) in the sustainability phase. CONCLUSION: Meaningful reductions in SSI can be achieved by implementing a multidisciplinary care bundle at a hospital-wide level.

Lymph node yield in right colectomy for cancer: a comparison of open, laparoscopic and robotic approaches.

AIM: Studies have demonstrated a relationship between lymph node (LN) yield and survival after colectomy for cancer. The impact of surgical technique on LN yield has not been well explored. METHOD: This is a retrospective study of right colectomy (RC) for cancer at a single institution from 2012 to 2014. Exclusion criteria were previous colectomy and emergent and palliative operations. All data were collected by chart review. Primary outcomes were LN yield and the LN to length of surgical specimen (LN-LSS) ratio. Multivariable mixed models were created with surgeon and pathologist as random effects. Sensitivity analyses were performed to exclude Stage IV cancers and to analyse groups on an 'as-treated' basis. RESULTS: We identified 181 open (O-RC), 163 laparoscopic (L-RC) and 119 robotic (R-RC) right colectomies. O-RC was more commonly performed in women with metastatic disease. The mean LN yield was 28, 29 and 34 in O-RC, L-RC and R-RC, respectively; the respective mean LN-LSS ratios were 0.83, 0.91 and 1.0. The R-RC approach produced a higher LN yield than the other approaches (P < 0.01), and a higher LN-LSS ratio than O-RC (P < 0.01). These findings were unchanged in sensitivity analyses. CONCLUSION: Robotic right colectomy improves LN yield and the LN-LSS ratio, which may reflect better mesocolic excision. The effect of these findings on survival requires further investigation.

Methodological considerations and impact of school-based interventions on objectively measured physical activity in adolescents: a systematic review and meta-analysis.

OBJECTIVE: The aims of this systematic review and meta-analysis are (i) to determine the impact of school-based interventions on objectively measured physical activity among adolescents and (ii) to examine accelerometer methods and decision rule reporting in previous interventions. METHODS: A systematic search was performed to identify randomized controlled trials targeting adolescents (age: ≥10 years), conducted in the school setting, and reporting objectively measured physical activity. Random effects meta-analyses were conducted to determine the pooled effects of previous interventions on total and moderate-to-vigorous physical activity. Potential moderators of intervention effects were also explored. RESULTS: Thirteen articles met the inclusion criteria, and twelve were included in the meta-analysis. The pooled effects were small and non-significant for both total physical activity (standardized mean difference = 0.02 [95% confidence interval = -0.13 to 0.18]) and moderate-to-vigorous physical activity (standardized mean difference = 0.24 [95% confidence interval = -0.08 to 0.56]). Sample age and accelerometer compliance were significant moderators for total physical activity, with a younger sample and higher compliance associated with larger effects. CONCLUSION: Previous school-based physical activity interventions targeting adolescents have been largely unsuccessful, particularly for older adolescents. There is a need for more high-quality research using objective monitoring in this population. Future interventions should comply with best-practice recommendations regarding physical activity monitoring protocols.

Myeloid translocation genes differentially regulate colorectal cancer programs.

Myeloid translocation genes (MTGs), originally identified as chromosomal translocations in acute myelogenous leukemia, are transcriptional corepressors that regulate hematopoietic stem cell programs. Analysis of The Cancer Genome Atlas (TCGA) database revealed that MTGs were mutated in epithelial malignancy and suggested that loss of function might promote tumorigenesis. Genetic deletion of MTGR1 and MTG16 in the mouse has revealed unexpected and unique roles within the intestinal epithelium. Mtgr1-/- mice have progressive depletion of all intestinal secretory cells, and Mtg16-/- mice have a decrease in goblet cells. Furthermore, both Mtgr1-/- and Mtg16-/- mice have increased intestinal epithelial cell proliferation. We thus hypothesized that loss of MTGR1 or MTG16 would modify Apc1638/+-dependent intestinal tumorigenesis. Mtgr1-/- mice, but not Mtg16-/- mice, had a 10-fold increase in tumor multiplicity. This was associated with more advanced dysplasia, including progression to invasive adenocarcinoma, and augmented intratumoral proliferation. Analysis of chromatin immunoprecipitation sequencing data sets for MTGR1 and MTG16 targets indicated that MTGR1 can regulate Wnt and Notch signaling. In support of this, immunohistochemistry and gene expression analysis revealed that both Wnt and Notch signaling pathways were hyperactive in Mtgr1-/- tumors. Furthermore, in human colorectal cancer (CRC) samples MTGR1 was downregulated at both the transcript and protein level. Overall our data indicates that MTGR1 has a context-dependent effect on intestinal tumorigenesis.

Histories of host shifts and cospeciation among free-living parasitoids of Rhagoletis flies.

Host shifts by specialist insects can lead to reproductive isolation between insect populations that use different hosts, promoting diversification. When both a phytophagous insect and its ancestrally associated parasitoid shift to the same novel host plant, they may cospeciate. However, because adult parasitoids are free living, they can also colonize novel host insects and diversify independent of their ancestral host insect. Although shifts of parasitoids to new insect hosts have been documented in ecological time, the long-term importance of such shifts to parasitoid diversity has not been evaluated. We used a genus of flies with a history of speciation via host shifting (Rhagoletis [Diptera: Tephritidae]) and three associated hymenopteran parasitoid genera (Diachasma, Coptera and Utetes) to examine cophylogenetic relationships between parasitoids and their host insects. We inferred phylogenies of Rhagoletis, Diachasma, Coptera and Utetes and used distance-based cophylogenetic methods (ParaFit and PACo) to assess congruence between fly and parasitoid trees. We used an event-based method with a free-living parasitoid cost model to reconstruct cophylogenetic histories of each parasitoid genus and Rhagoletis. We found that the current species diversity and host-parasitoid associations between the Rhagoletis flies and parasitoids are the primary result of ancient cospeciation events. Parasitoid shifts to ancestrally unrelated hosts primarily occur near the branch tips, suggesting that host shifts contribute to recent parasitoid species diversity but that these lineages may not persist over longer time periods. Our analyses also stress the importance of biologically informed cost models when investigating the coevolutionary histories of hosts and free-living parasitoids.

Anthropogenic impacts drive niche and conservation metrics of a cryptic rattlesnake on the Colorado Plateau of western North America.

Ecosystems transition quickly in the Anthropocene, whereas biodiversity adapts more slowly. Here we simulated a shifting woodland ecosystem on the Colorado Plateau of western North America by using as its proxy over space and time the fundamental niche of the Arizona black rattlesnake (Crotalus cerberus). We found an expansive (= end-of-Pleistocene) range that contracted sharply (= present), but is blocked topographically by Grand Canyon/Colorado River as it shifts predictably northwestward under moderate climate change (= 2080). Vulnerability to contemporary wildfire was quantified from available records, with forested area reduced more than 27% over 13 years. Both 'ecosystem metrics' underscore how climate and wildfire are rapidly converting the Plateau ecosystem into novel habitat. To gauge potential effects on C. cerberus, we derived a series of relevant 'conservation metrics' (i.e. genetic variability, dispersal capacity, effective population size) by sequencing 118 individuals across 846 bp of mitochondrial (mt)DNA-ATPase8/6. We identified five significantly different clades (net sequence divergence = 2.2%) isolated by drainage/topography, with low dispersal (F ST = 0.82) and small sizes (2N ef = 5.2). Our compiled metrics (i.e. small-populations, topographic-isolation, low-dispersal versus conserved-niche, vulnerable-ecosystem, dispersal barriers) underscore the susceptibility of this woodland specialist to a climate and wildfire tandem. We offer adaptive management scenarios that may counterbalance these metrics and avoid the extirpation of this and other highly specialized, relictual woodland clades.

A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality.

BACKGROUND: Target selection for oncology is a crucial step in the successful development of therapeutics. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 editing of specific loci offers an alternative method to RNA interference and small molecule inhibitors for determining whether a cell line is dependent on a specific gene product for proliferation or survival. In our initial studies using CRISPR-Cas9 to verify the dependence on EZH2 activity for proliferation of a SMARCB1/SNF5/INI1 mutant malignant rhabdoid tumor (MRT) cell line, we noted that the initial reduction in proliferation was lost over time. We hypothesized that in the few cells that retain proliferative capacity, at least one allele of EZH2 remains functional. To verify this, we developed an assay to analyze 10s-100s of clonal cell populations for target gene disruption using restriction digest and fluorescent fragment length analyses. RESULTS: Our results clearly show that in cell lines in which EZH2 is essential for proliferation, at least one potentially functional allele of EZH2 is retained in the clones that survive. CONCLUSION: This assay clearly indicates whether or not a specific gene is essential for survival and/or proliferation in a given cell line. Such data can aid the development of more robust therapeutics by increasing confidence in target selection.

Rapid, specific, simple, in-field detection of Xanthomonas campestris pathovar musacearum by loop-mediated isothermal amplification.

AIMS: To develop and evaluate a loop-mediated isothermal amplification (LAMP) assay for Xanthomonas campestris pathovar musacearum (Xcm), the causal agent of banana Xanthomonas wilt, a major disease of banana in Africa. METHODS AND RESULTS: LAMP primers were designed to the general secretion pathway protein D gene and tested against 17 isolates of Xcm encompassing the known genetic and geographic diversity of the bacterium and all isolates were detected. Seventeen other Xanthomonas isolates, including closely related Xanthomonas vasicola, other bacterial pathogens/endophytes of Musa and two healthy Musa varieties gave negative results with the LAMP assay. The assay showed good sensitivity, detecting as little as 51 fg of Xcm DNA, a greater level of sensitivity than that of an Xcm PCR assay. Amplification with the LAMP assay was very rapid, typically within 9 min from bacterial cultures. Symptomatic field samples of Musa from Uganda were tested and all produced amplification in less than 13 min. CONCLUSIONS: The LAMP assay provides rapid, sensitive detection of the pathogen that is ideally suited for deployment in laboratories with basic facilities and in-field situations. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first LAMP assay for Xcm which provides a significant improvement compared to existing diagnostics.

The early management of the burned patient in the Naval service.

Burns represent a perpetual threat in military combat and are a pervasive threat in the land, maritime and air arenas. Therefore, it is imperative that military clinicians are well versed in managing burns in order to ensure optimal survival and recovery. This article aims to explore the epidemiology, pathophysiology, assessment and early management of the burned patient with a focus on the austere maritime environment and current military guidance.

Risk stratification by the Appendicitis Inflammatory Response score to guide decision-making in patients with suspected appendicitis.

BACKGROUND: Current management of suspected appendicitis is hampered by the overadmission of patients with non-specific abdominal pain and a significant negative exploration rate. The potential benefits of risk stratification by the Appendicitis Inflammatory Response (AIR) score to guide clinical decision-making were assessed. METHODS: During this 50-week prospective observational study at one institution, the AIR score was calculated for all patients admitted with suspected appendicitis. Appendicitis was diagnosed by histological examination, and patients were classified as having non-appendicitis pain if histological findings were negative or surgery was not performed. The diagnostic performance of the AIR score and the potential for risk stratification to reduce admissions, optimize imaging and prevent unnecessary explorations were quantified. RESULTS: A total of 464 patients were included, of whom 210 (63·3 per cent) with non-appendicitis pain were correctly classified as low risk. However, 13 low-risk patients had appendicitis. Low-risk patients accounted for 48·1 per cent of admissions (223 of 464), 57 per cent of negative explorations (48 of 84) and 50·7 per cent of imaging requests (149 of 294). An AIR score of 5 or more (intermediate and high risk) had high sensitivity for all severities of appendicitis (90 per cent) and also for advanced appendicitis (98 per cent). An AIR score of 9 or more (high risk) was very specific (97 per cent) for appendicitis, and the majority of patients with appendicitis in the high-risk group (21 of 30, 70 per cent) had perforation or gangrene. Ultrasound imaging could not exclude appendicitis in low-risk patients (negative likelihood ratio (LR) 1·0) but could rule-in the diagnosis in intermediate-risk patients (positive LR 10·2). CT could exclude appendicitis in low-risk patients (negative LR 0·0) and rule-in appendicitis in the intermediate group (positive LR 10·9). CONCLUSION: Risk stratification of patients with suspected appendicitis by the AIR score could guide decision-making to reduce admissions, optimize utility of diagnostic imaging and prevent negative explorations.