Ecological structure of diversity-dependent diversification in Phanerozoic marine bivalves.

Rigorous analysis of diversity-dependence-the hypothesis that the rate of proliferation of new species is inversely related to standing diversity-requires consideration of the ecology of the organisms in question. Differences between infaunal marine bivalves (living entirely within the sediment) and epifaunal forms (living partially or completely above the sediment-water interface) predict that these major ecological groups should have different diversity dynamics: epifaunal species may compete more intensely for space and be more susceptible to predation and physical disturbance. By comparing detrended standing diversity with rates of diversification, origination, and extinction in this exceptional fossil record, we find that epifaunal bivalves experienced significant, negative diversity-dependence in origination and net diversification, whereas infaunal forms show little appreciable relationship between diversity and evolutionary rates. This macroevolutionary contrast is robust to the time span over which dynamics are analysed, whether mass-extinction rebounds are included in the analysis, the treatment of stratigraphic ranges that are not maximally resolved, and the details of detrending. We also find that diversity-dependence persists over hundreds of millions of years, even though diversity itself rises nearly exponentially, belying the notion that diversity-dependence must imply equilibrial diversity dynamics.

Diversity-Dependent Diversification in the History of Marine Animals.

AbstractBy comparing detrended estimates of diversity (taxonomic richness) and rates of origination, extinction, and net diversification, I show that at the global scale over the course of the Phanerozoic eon, rates of diversification and origination are negatively correlated with diversity. By contrast, extinction rates are only weakly correlated with diversity for the most part. These results hold for both genus- and species-level data and for many alternative analytical protocols. The asymmetry between extinction on the one hand and origination and net diversification on the other hand supports a model whereby extinction is largely driven by abiotic perturbations, with subsequent origination filling the void left by depleted diversity. Diversity dependence is somewhat weaker, but still evident, if the initial Ordovician radiation or rebounds from major mass extinctions are omitted from analysis; thus, diversity dependence is influenced, but not dominated, by these special intervals of Earth history. In the transition from Paleozoic to post-Paleozoic time, diversity dependence of origination weakens while that of extinction strengthens; however, diversity dependence of net diversification barely changes in strength. Despite nuances, individual clades largely yield results consistent with those for the aggregate data on all animals. On the whole, diversity-dependent diversification appears to be a pervasive factor in the macroevolution of marine animal life.

Sex-Based Role Misidentification and Burnout of Resident Physicians: An Observational Study.

OBJECTIVE: This cross-sectional study characterized associations between sex, role misidentification, and burnout among surgical and nonsurgical residents. SUMMARY BACKGROUND DATA: Limited evidence suggests that female resident physicians are more likely to be misidentified as nonphysician team members, with potential negative implications for wellbeing. The prevalence and impact of role misidentification on the trainee experience in surgical as compared to nonsurgical specialties is unknown. METHODS: An anonymous electronic survey was distributed to fourteen different residency programs at 2 academic medical centers in August 2018. The survey included questions about demographics, symptoms of burnout, the frequency of misidentification as another member of the care team, and the effect of misidentification on respondents' well-being. Results: Two-hundred sixty out of 419 (62.1% response rate) resident physicians completed the survey, of whom 184 (77.3%) reported being misidentified as a nonphysician at least weekly. Female sex was associated with a significantly increased odds of being misidentified at least weekly (adjusted OR 23.7, 95% CI 10.9-51.5; P < 0.001), as was training in a surgical program (adjusted OR 3.7, 95% CI 1.7-8.0; P = 0.001). Frequent role misidentification was associated with burnout (OR 2.6, 95% CI 1.2-5.5; P = 0.01). In free-text responses, residents reported that being misidentified invoked a sense of not belonging, caused emotional exhaustion, and interfered with patient communication. CONCLUSIONS: Role misidentification is more prevalent among female residents and surgical residents, compared to male residents and nonsurgical residents, respectively. Physician role misidentification is associated with burnout and has negative implications for resident wellbeing; interventions to reduce role misidentification are needed.

Pacing of Paleozoic macroevolutionary rates by Milankovitch grand cycles.

Periodic fluctuations in past biodiversity, speciation, and extinction have been proposed, with extremely long periods ranging from 26 to 62 million years, although forcing mechanisms remain speculative. In contrast, well-understood periodic Milankovitch climate forcing represents a viable driver for macroevolutionary fluctuations, although little evidence for such fluctuation exists except during the Late Cenozoic. The reality, magnitude, and drivers of periodic fluctuations in macroevolutionary rates are of interest given long-standing debate surrounding the relative roles of intrinsic biotic interactions vs. extrinsic environmental factors as drivers of biodiversity change. Here, we show that, over a time span of 60 million years, between 9 and 16% of the variance in biological turnover (i.e., speciation probability plus species extinction probability) in a major Early Paleozoic zooplankton group, the graptoloids, can be explained by long-period astronomical cycles (Milankovitch "grand cycles") associated with Earth's orbital eccentricity (2.6 million years) and obliquity (1.3 million years). These grand cycles modulate climate variability, alternating times of relative stability in the environment with times of maximum volatility. We infer that these cycles influenced graptolite speciation and extinction through climate-driven changes to oceanic circulation and structure. Our results confirm the existence of Milankovitch grand cycles in the Early Paleozoic Era and show that known processes related to the mechanics of the Solar System were shaping marine macroevolutionary rates comparatively early in the history of complex life. We present an application of hidden Markov models to macroevolutionary time series and protocols for the evaluation of statistical significance in spectral analysis.

Greenhouse-icehouse transition in the Late Ordovician marks a step change in extinction regime in the marine plankton.

Two distinct regimes of extinction dynamic are present in the major marine zooplankton group, the graptolites, during the Ordovician and Silurian periods (486-418 Ma). In conditions of "background" extinction, which dominated in the Ordovician, taxonomic evolutionary rates were relatively low and the probability of extinction was highest among newly evolved species ("background extinction mode"). A sharp change in extinction regime in the Late Ordovician marked the onset of repeated severe spikes in the extinction rate curve; evolutionary turnover increased greatly in the Silurian, and the extinction mode changed to include extinction that was independent of species age ("high-extinction mode"). This change coincides with a change in global climate, from greenhouse to icehouse conditions. During the most extreme episode of extinction, the Late Ordovician Mass Extinction, old species were selectively removed ("mass extinction mode"). Our analysis indicates that selective regimes in the Paleozoic ocean plankton switched rapidly (generally in <0.5 My) from one mode to another in response to environmental change, even when restoration of the full ecosystem was much slower (several million years). The patterns observed are not a simple consequence of geographic range effects or of taxonomic changes from Ordovician to Silurian. Our results suggest that the dominant primary controls on extinction throughout the lifespan of this clade were abiotic (environmental), probably mediated by the microphytoplankton.

Diversity-dependent evolutionary rates in early Palaeozoic zooplankton.

The extent to which biological diversity affects rates of diversification is central to understanding macroevolutionary dynamics, yet no consensus has emerged on the importance of diversity-dependence of evolutionary rates. Here, we analyse the species-level fossil record of early Palaeozoic graptoloids, documented with high temporal resolution, to test directly whether rates of diversification were influenced by levels of standing diversity within this major clade of marine zooplankton. To circumvent the statistical regression-to-the-mean artefact, whereby higher- and lower-than-average values of diversity tend to be followed by negative and positive diversification rates, we construct a non-parametric, empirically scaled, diversity-independent null model by randomizing the observed diversification rates with respect to time. Comparing observed correlations between diversity and diversification rate to those expected from this diversity-independent model, we find evidence for negative diversity-dependence, accounting for up to 12% of the variance in diversification rate, with maximal correlation at a temporal lag of approximately 1 Myr. Diversity-dependence persists throughout the Ordovician and Silurian, despite a major increase in the strength and frequency of extinction and speciation pulses in the Silurian. By contrast to some previous work, we find that diversity-dependence affects rates of speciation and extinction nearly equally on average, although subtle differences emerge when we compare the Ordovician and Silurian.

Predicting response to immune checkpoint blockade therapy among mismatch repair-deficient patients using mutational signatures.

Despite the overall efficacy of immune checkpoint blockade (ICB) for mismatch repair deficiency (MMRD) across tumor types, a sizable fraction of patients with MMRD still do not respond to ICB. We performed mutational signature analysis of panel sequencing data (n = 95) from MMRD cases treated with ICB. We discover that T>C-rich single base substitution (SBS) signatures-SBS26 and SBS54 from the COSMIC Mutational Signatures catalog-identify MMRD patients with significantly shorter overall survival. Tumors with a high burden of SBS26 show over-expression and enriched mutations of genes involved in double-strand break repair and other DNA repair pathways. They also display chromosomal instability (CIN), likely related to replication fork instability, leading to copy number losses that trigger immune evasion. SBS54 is associated with transcriptional activity and not with CIN, defining a distinct subtype. Consistently, cancer cell lines with a high burden of SBS26 and SBS54 are sensitive to treatments targeting pathways related to their proposed etiology. Together, our analysis offers an explanation for the heterogeneous responses to ICB among MMRD patients and supports an SBS signature-based predictor as a prognostic biomarker for differential ICB response.

Facts and Hopes in Colorectal Cancer Immunotherapy.

Although a minority of colorectal cancers exhibit mismatch repair deficiency and associated sensitivity to immune checkpoint inhibitors (ICI), the vast majority of colorectal cancers arise in a tolerogenic microenvironment with mismatch repair proficiency, low tumor-intrinsic immunogenicity, and negligible immunotherapy responsiveness. Treatment strategies to augment tumor immunity with combination ICIs and chemotherapy have broadly failed in mismatch repair-proficient tumors. Similarly, although several small single-arm studies have shown that checkpoint blockade plus radiation or select tyrosine kinase inhibition may show improved outcomes compared with historical controls, this finding has not been clearly validated in randomized trials. An evolving next generation of intelligently engineered checkpoint inhibitors, bispecific T-cell engagers, and emerging CAR-T cell therapies may improve immunorecognition of colorectal tumors. Across these modalities, ongoing translational efforts to better define patient populations and biomarkers associated with immune response, as well as combine biologically sound and mutually amplifying therapies, show promise for a new era of immunotherapy in colorectal cancer.

Metastatic well differentiated serotonin-producing pancreatic neuroendocrine tumor with carcinoid heart disease: a case report.

Background: Less than two percent of pancreatic neuroendocrine tumors (NETs) produce serotonin. Serotonin can cause carcinoid syndrome and less commonly carcinoid heart disease (CHD). CHD is associated with increased mortality and requires a more aggressive approach. Here we present a rare case of a serotonin-producing pancreatic NET complicated by CHD at presentation and discuss timing of systemic therapy, liver-directed therapy, and heart failure management. Case Description: A 36-year-old white man presented with diarrhea, lower extremity edema, and exertional dyspnea. He was found to have a well-differentiated serotonin-producing pancreatic NETs grade three with bilobar liver metastasis complicated by carcinoid syndrome and CHD. His symptoms and disease burden improved with somatostatin analog and liver-directed therapy with bland embolization to control carcinoid symptoms and obtain rapid hormonal control to prevent progression of CHD. He concurrently received diuretics to manage his heart failure and was considered for valvular replacement surgery, which was deferred for optimal hormonal control. Conclusions: Our case highlights the importance of multidisciplinary care for patients with pancreatic NETs and early identification and management of CHD. Although uncommon, serotonin-producing pancreatic NETs can present with CHD and require combination of somatostatin analogs, liver-directed therapy, and heart failure management.

Molecular Classification of Appendiceal Adenocarcinoma.

PURPOSE: Appendiceal adenocarcinomas (ACs) are rare, histologically diverse malignancies treated as colorectal cancers despite having distinct biology and clinical behavior. To guide clinical decision making, we defined molecular subtypes of AC associated with patient survival, metastatic burden, and chemotherapy response. PATIENTS AND METHODS: A comprehensive molecular analysis was performed in patients with AC to define molecular subtypes. Associations between molecular subtype and overall survival, intraoperative peritoneal cancer index, and first-line chemotherapy response were assessed adjusting for histopathologic and clinical variables using multivariable Cox proportional hazards, linear regression, and logistic regression models. RESULTS: We defined distinct molecular lineages of mucinous appendiceal adenocarcinoma (MAAP) from co-occurring mutations in GNAS, RAS, and TP53. Of 164 MAAP tumors, 24 were RAS-mutant (mut) predominant (RAS-mut/GNAS-wild-type [wt]/TP53-wt) with significantly decreased mutations and chromosomal alterations compared with tumors with GNAS mutations (GNAS-mut predominant) or TP53 mutations (TP53-mut predominant). No patient with RAS-mut predominant subtype metastatic MAAP died of cancer, and overall survival in this subgroup was significantly improved compared with patients with GNAS-mut (P = .05) and TP53-mut (P = .004) predominant subtypes. TP53-mut predominant subtypes were highly aneuploid; increased tumor aneuploidy was independently (P = .001) associated with poor prognosis. The findings retained significance in patients with any metastatic AC. RAS-mut predominant metastases exhibited reduced peritoneal tumor bulk (P = .04) and stromal invasion (P < .001) compared with GNAS-mut or TP53-mut predominant tumors, respectively. Patients with RAS-mut predominant MAAP responded more to first-line chemotherapy (50%) compared with patients with GNAS-mut predominant tumors (6%, P = .03). CONCLUSION: AC molecular patterns identify distinct molecular subtypes: a clinically indolent RAS-mut/GNAS-wt/TP53-wt subtype; a chemotherapy-resistant GNAS-mut predominant subtype; and an aggressive, highly aneuploid TP53-mut predominant subtype. Each subtype exhibits conserved clinical behavior irrespective of histopathology.

The Impact of Germline Alterations in Appendiceal Adenocarcinoma.

PURPOSE: More than 10% of assessed patients with appendiceal adenocarcinoma have a pathogenic (P) or likely pathogenic (LP) germline variant, including genes implicated in heritable gastrointestinal cancer syndromes, such as Lynch syndrome. We defined the clinical and molecular impact of heritable alterations in appendiceal adenocarcinoma to evaluate the need for dedicated appendiceal screening and prevention strategies in patients with LP/P germline variants. EXPERIMENTAL DESIGN: We performed an integrated germline and somatic molecular analysis for patients with confirmed appendiceal adenocarcinoma. Patients underwent paired tumor-normal sequencing for up to 90 hereditary cancer risk genes and 505 genes for somatic mutation profiling. We defined the cooccurrence of LP/P germline variants and second-hit pathogenic somatic alterations. The associations between germline variants and patient clinicopathologic features were also evaluated. RESULTS: Twenty-five of 237 patients (10.5%) carried pathogenic or likely pathogenic germline variants in cancer susceptibility genes. Clinicopathologic characteristics and appendiceal adenocarcinoma-specific survival were similar in patients with or without germline variants. Most (92%, N = 23/25) patients with germline variants demonstrated no second-hit somatic alterations, including loss of heterozygosity. Two patients with a germline APC I1307K low-penetrance founder variant exhibited secondary somatic pathogenic alterations in APC. However, only one patient tumor exhibited APC-mediated WNT signaling dysregulation: a plausible consequence of multiple somatic APC mutations with no germline variant contribution. Four patients had germline variants in PMS2 or MSH2 associated with Lynch syndrome, yet their cancers were microsatellite-stable. CONCLUSIONS: Germline variants are likely incidental without a contributory driver role in appendiceal adenocarcinoma. Appendiceal adenocarcinoma screening in patients with germline variants is not clearly merited.

Clinical Outcomes Associated With Pembrolizumab Monotherapy Among Adults With Diffuse Malignant Peritoneal Mesothelioma.

Importance: Diffuse malignant peritoneal mesothelioma (DMPM) represents a rare and clinically distinct entity among malignant mesotheliomas. Pembrolizumab has activity in diffuse pleural mesothelioma but limited data are available for DMPM; thus, DMPM-specific outcome data are needed. Objective: To evaluate outcomes after the initiation of pembrolizumab monotherapy in the treatment of adults with DMPM. Design, Setting, and Participants: This retrospective cohort study was conducted in 2 tertiary care academic cancer centers (University of Pennsylvania Hospital Abramson Cancer Center and Memorial Sloan Kettering Cancer Center). All patients with DMPM treated between January 1, 2015, and September 1, 2019, were retrospectively identified and followed until January 1, 2021. Statistical analysis was performed between September 2021 and February 2022. Exposures: Pembrolizumab (200 mg or 2 mg/kg every 21 days). Main Outcomes and Measures: Median progression-free survival (PFS) and median overall survival (OS) were assessed using Kaplan-Meier estimates. The best overall response was determined using RECIST (Response Evaluation Criteria in Solid Tumors) criteria, version 1.1. The association of disease characteristics with partial response was evaluated using the Fisher exact test. Results: This study included 24 patients with DMPM who received pembrolizumab monotherapy. Patients had a median age of 62 years (IQR, 52.4-70.6 years); 14 (58.3%) were women, 18 (75.0%) had epithelioid histology, and most (19 [79.2%]) were White. A total of 23 patients (95.8%) received systemic chemotherapy prior to pembrolizumab, and the median number of lines of prior therapy was 2 (range, 0-6 lines). Of the 17 patients who underwent programmed death ligand 1 (PD-L1) testing, 6 (35.3%) had positive tumor PD-L1 expression (range, 1.0%-80.0%). Of the 19 evaluable patients, 4 (21.0%) had a partial response (overall response rate, 21.1% [95% CI, 6.1%-46.6%]), 10 (52.6%) had stable disease, and 5 (26.3%) had progressive disease (5 of 24 patients [20.8%] were lost to follow-up). There was no association between a partial response and the presence of a BAP1 alteration, PD-L1 positivity, or nonepithelioid histology. With a median follow-up of 29.2 (95% CI, 19.3 to not available [NA]) months, the median PFS was 4.9 (95% CI, 2.8-13.3) months and the median OS was 20.9 (95% CI, 10.0 to NA) months from pembrolizumab initiation. Three patients (12.5%) experienced PFS of more than 2 years. Among patients with nonepithelioid vs epithelioid histology, there was a numeric advantage in median PFS (11.5 [95% CI, 2.8 to NA] vs 4.0 [95% CI, 2.8-8.8] months) and median OS (31.8 [95% CI, 8.3 to NA] vs 17.5 [95% CI, 10.0 to NA] months); however, this did not reach statistical significance. Conclusions and Relevance: The results of this retrospective dual-center cohort study of patients with DMPM suggest that pembrolizumab had clinical activity regardless of PD-L1 status or histology, although patients with nonepithelioid histology may have experienced additional clinical benefit. The partial response rate of 21.0% and median OS of 20.9 months in this cohort with 75.0% epithelioid histology warrants further investigation to identify those most likely to respond to immunotherapy.

Clinical Importance of Clonal Hematopoiesis in Metastatic Gastrointestinal Tract Cancers.

Importance: Clonal hematopoiesis (CH) has been associated with development of atherosclerosis and leukemia and worse survival among patients with cancer; however, the association with cancer therapy efficacy, in particular immune checkpoint blockade (ICB), and toxicity has not yet been established. Given the widespread use of ICB and the critical role hematopoietic stem cell-derived lymphocytes play in mediating antitumor responses, CH may be associated with therapeutic efficacy and hematologic toxicity. Objective: To determine the association between CH and outcomes, hematologic toxicity, and therapeutic efficacy in patients with metastatic gastrointestinal tract cancers being treated with systemic therapy, both in the first-line metastatic treatment setting and in ICB. Design, Setting, and Participants: This retrospective cohort study included 633 patients with stage IV colorectal (CRC) and esophagogastric (EGC) cancer who were treated with first-line chemotherapy and/or ICB at Memorial Sloan Kettering Cancer Center. Patients underwent matched tumor and peripheral blood DNA sequencing using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets next-generation sequencing assay between January 1, 2006, and December 31, 2020. Exposures: Clonal hematopoiesis-related genetic alterations were identified by next-generation sequencing of patients' tumor and normal blood buffy coat samples, with a subset of these CH alterations annotated as likely putative drivers (CH-PD) based upon previously established criteria. Main Outcomes and Measures: Patients with CH and CH-PD in peripheral blood samples were identified, and these findings were correlated with survival outcomes (progression-free survival [PFS] and overall survival [OS]) during first-line chemotherapy and ICB, as well as baseline white blood cell levels and the need for granulocyte colony-stimulating factor (G-CSF) support. Results: Among the 633 patients included in the study (390 men [61.6%]; median age, 58 [IQR, 48-66] years), the median age was 52 (IQR, 45-63) years in the CRC group and 61 (IQR, 53-69) years in the EGC group. In the CRC group, 161 of 301 patients (53.5%) were men, compared with 229 of 332 patients (69.0%) in the EGC group. Overall, 62 patients (9.8%) were Asian, 45 (7.1%) were Black or African American, 482 (76.1%) were White, and 44 (7.0%) were of unknown race or ethnicity. Presence of CH was identified in 115 patients with EGC (34.6%) and 83 with CRC (27.6%), with approximately half of these patients harboring CH-PD (CRC group, 44 of 83 [53.0%]; EGC group, 55 of 115 [47.8%]). Patients with EGC and CH-PD exhibited a significantly worse median OS of 16.0 (95% CI, 11.6-22.3) months compared with 21.6 (95% CI, 19.6-24.3) months for those without CH-PD (P = .01). For patients with CRC and EGC, CH and CH-PD were not associated with PFS differences in patients undergoing ICB or first-line chemotherapy. Neither CH nor CH-PD were correlated with baseline leukocyte levels or increased need for G-CSF support. Conclusions and Relevance: These findings suggest CH and CH-PD are not directly associated with the treatment course of patients with metastatic gastrointestinal tract cancer receiving cancer-directed therapy.

Neoplasia risk in patients with Lynch syndrome treated with immune checkpoint blockade.

Metastatic and localized mismatch repair-deficient (dMMR) tumors are exquisitely sensitive to immune checkpoint blockade (ICB). The ability of ICB to prevent dMMR malignant or pre-malignant neoplasia development in patients with Lynch syndrome (LS) is unknown. Of 172 cancer-affected patients with LS who had received ≥1 ICB cycles, 21 (12%) developed subsequent malignancies after ICB exposure, 91% (29/32) of which were dMMR, with median time to development of 21 months (interquartile range, 6-38). Twenty-four of 61 (39%) ICB-treated patients who subsequently underwent surveillance colonoscopy had premalignant polyps. Within matched pre-ICB and post-ICB follow-up periods, the overall rate of tumor development was unchanged; however, on subgroup analysis, a decreased incidence of post-ICB visceral tumors was observed. These data suggest that ICB treatment of LS-associated tumors does not eliminate risk of new neoplasia development, and LS-specific surveillance strategies should continue. These data have implications for immunopreventative strategies and provide insight into the immunobiology of dMMR tumors.

Killing tumor-associated bacteria with a liposomal antibiotic generates neoantigens that induce anti-tumor immune responses.

Increasing evidence implicates the tumor microbiota as a factor that can influence cancer progression. In patients with colorectal cancer (CRC), we found that pre-resection antibiotics targeting anaerobic bacteria substantially improved disease-free survival by 25.5%. For mouse studies, we designed an antibiotic silver-tinidazole complex encapsulated in liposomes (LipoAgTNZ) to eliminate tumor-associated bacteria in the primary tumor and liver metastases without causing gut microbiome dysbiosis. Mouse CRC models colonized by tumor-promoting bacteria (Fusobacterium nucleatum spp.) or probiotics (Escherichia coli Nissle spp.) responded to LipoAgTNZ therapy, which enabled more than 70% long-term survival in two F. nucleatum-infected CRC models. The antibiotic treatment generated microbial neoantigens that elicited anti-tumor CD8+ T cells. Heterologous and homologous bacterial epitopes contributed to the immunogenicity, priming T cells to recognize both infected and uninfected tumors. Our strategy targets tumor-associated bacteria to elicit anti-tumoral immunity, paving the way for microbiome-immunotherapy interventions.

A syringe-focused ultrasound device for simultaneous injection of DNA and gene transfer.

BACKGROUND: Although known for its success in ameliorating muscle disorders, ultrasound (US)-mediated gene transfer in skeletal muscle has recently shown potential for treating a wide range of therapeutic applications. The disadvantages of the technique, however, including its relatively low transfer efficiency and toxic effects resulting in tissue damage, have impeded clinical progress. We describe a novel syringe-focused US device of our design that combines simultaneous DNA injection and US irradiation to resolve these issues, achieving improved DNA uptake for high expression and lowering the incidence of tissue injury. METHODS: The novel, syringe-focused US device was used to introduce purified plasmid DNA encoding human neprilysin (hNEP) into mouse hindlimb skeletal muscle concurrent with US protocol (1.7 MHz, 1 W/cm(2), 1-min exposure time). hNEP expression was measured in local skeletal muscle, blood serum and brain by immunoassay at different post-injection time points. The incidence of muscle fiber damage was subsequently assessed by Evans Blue dye (EBD) uptake using fluorescence microscopy. RESULTS: The syringe-US device at the first time enabled simultaneous combination of DNA transfer and US stimulation gives rise to two months of gene expression of the encoded polypeptide as it is measured, at the same time as withstanding minor tissue damage. In addition, DNA injection without US treatment failed to induce hNEP expression. CONCLUSIONS: This new technique provides a simple, safe and efficient nonviral gene delivery approach in skeletal muscle and shows promising applications for gene therapy of human disease, such as Alzheimer's disease.

Evolutionary dynamics of taxonomic structure.

The distribution of species among genera and higher taxa has largely untapped potential to reveal among-clade variation in rates of origination and extinction. The probability distribution of the number of species within a genus is modelled with a stochastic, time-homogeneous birth-death model having two parameters: the rate of species extinction, µ, and the rate of genus origination, γ, each scaled as a multiple of the rate of within-genus speciation, λ. The distribution is more sensitive to γ than to µ, although µ affects the size of the largest genera. The species : genus ratio depends strongly on both γ and µ, and so is not a good diagnostic of evolutionary dynamics. The proportion of monotypic genera, however, depends mainly on γ, and so may provide an index of the genus origination rate. Application to living marine molluscs of New Zealand shows that bivalves have a higher relative rate of genus origination than gastropods. This is supported by the analysis of palaeontological data. This concordance suggests that analysis of living taxonomic distributions may allow inference of macroevolutionary dynamics even without a fossil record.