Impact of Clinical Trial Design on Recruitment of Racial and Ethnic Minorities.

Knowledge related to how oncology treatment trial design influences enrollment of racial and ethnic minorities is limited. Rigorous identification of clinical trial design parameters that associate favorably with minority accrual provides educational opportunities for individuals interested in designing more representative treatment trials. We identified oncology trials with a minimum of 10 patients at an NCI-Designated Comprehensive Cancer Center from 2010 to 2021. We defined a study endpoint of racial and ethnic minority accrual greater than zero. Multivariable logistic regression was used to determine whether co-variables predicted our study endpoint. P-values of less than 0.05 were considered significant. A total of 352 cancer trials met eligibility criteria. These studies enrolled a total of 7981 patients with a total of 926 racial and ethnic minorities leading to a median enrollment of 10%. Trials open in community sites (yes versus no) were more likely to have a minority patient (OR, 2.21; 95% CI, 1.02-4.96) as well as pilot/phase I studies compared to phase II/III (OR, 3.19; 95% CI, 1.34-8.26). Trials incorporating immunotherapy (yes versus no) were less likely to have a minority patient (OR, 0.47; 95% CI, 0.23-0.94). Trials open in community sites as well as early phase treatment studies were more likely to accrue minority patients. However, studies including immunotherapy were less likely to accrue racial and ethnic minorities. Knowledge gained from our analysis may help individuals design oncology treatment trials that are representative of more diverse populations.

The Impact of Prophylactic Cranial Irradiation and Consolidative Thoracic Radiation Therapy for Extensive Stage Small-Cell Lung Cancer in the Transition to the Chemo-Immunotherapy Era: A Single Institution Series.

INTRODUCTION: Extensive-stage small-cell lung cancer (ES-SCLC) continues to have poor survival due to its aggressive behavior, despite improvements with incorporation of immunotherapy with standard chemotherapy. Controversy exists regarding the role of consolidative thoracic radiation therapy (TRT) and prophylactic cranial irradiation (PCI) in ES-SCLC due to high recurrence rates. We report our institutional result of the benefit of PCI and TRT in ES-SCLC. METHODS: Patients with ES-SCLC without intracranial metastasis at diagnosis (N = 163) were included. All patients completed systemic therapy with or without immunotherapy based on time of standard of care. Cohorts were divided by systemic therapy use and further subdivided by treatment with PCI and TRT. Overall survival (OS) and progression-free survival (PFS) were estimated by the Kaplan-Meier method with log-rank test for comparison. The effects of TRT and PCI were estimated by multivariable (MVA) Cox regression. RESULTS: Seventy-four patients (45.4%) received TRT, and 33.1% (n = 54) received PCI. The median follow-up was 11 months (3-85 months). PCI improved median OS to 15 months from 10 months, P = .02) and median PFS to 8.5 months from 5 months (P = .02) which remained significant on MVA, P = .02 and P = .02, respectively. TRT improved OS on UVA (P = 0.002) but was not significant on MVA. TRT did not improve PFS. CONCLUSION: This study including chemotherapy and chemo-immunotherapy suggests improved outcomes with addition of PCI in patients with ES-SCLC while TRT did not show benefit to either OS or PFS. A future trial is needed to evaluate the role of TRT and PCI in the era of chemo-immunotherapy.

Proton Beam Therapy for Locally Advanced Head and Neck Tumors: An Analysis of Dosimetric and Clinical Outcomes.

OBJECTIVE: Locally advanced tumors of the head and neck region often lie in close proximity to critical organs at risk (OARs). Providing effective treatment coverage to these malignancies while minimizing radiation dose to surrounding OARs is advantageous. Our aim is to compare dosimetric data of OARs from proton beam therapy (PBT) plans to volumetric modulated arc therapy (VMAT) treatment plans, and to evaluate clinical outcomes in patients treated with PBT. METHODS: We identified patients with locally advanced head and neck tumors treated with PBT at our institution from 2016 to 2019. Study endpoints included mean and maximum doses for the OAR structures for each treatment plan, overall survival, time to local-regional or distant progression, and presence of acute and late toxicities. Mean and maximum doses to OAR structures were compared between treatment modalities using a paired Wilcoxon signed-rank test. P-values <0.05 were considered significant. RESULTS: A total of 42 patients were identified. Clinical target volume coverage was >95% for both PBT and VMAT plans. PBT plans showed a significant reduction to the mean doses to all OARs, and max doses to most OARs (P<0.05). The largest reduction mean dose was seen in the contralateral cochlea and parotid glands at 71% and 75%, respectively. Median follow-up was 27 months. Overall survival at 4 years was 44.75%. Freedom from local-regional progression was 73.28% at 2 years. The majority of patients developed Common Terminology Criteria for Adverse Events (CTCAE) grade I dermatitis, mucositis, or both. CONCLUSIONS: PBT resulted in meaningful dose reductions to OARs while maintaining comparable target coverage when compared with VMAT plans. Further refinements to proton therapy may have the potential to further minimize dose to critical structures.

An analysis of a large multi-institutional database reveals important associations between treatment parameters and clinical outcomes for stereotactic body radiotherapy (SBRT) of oligometastatic colorectal cancer.

PURPOSE: In recent years, stereotactic body radiotherapy (SBRT) has emerged as an effective treatment for oligometastatic cancers. Here, we report radiation treatment parameters and clinical outcomes for patients with oligometastatic colorectal cancer (CRC) treated with SBRT using a large multi-institutional database. METHODS: Patients with extra-cranial oligometastatic CRC (≤5 lesions) treated with SBRT at six large academic cancer centers were included. The primary outcome was local recurrence while secondary outcomes included overall survival (OS) progression free survival, oligo-progression, and widespread progression. Survival outcomes were estimated using the Kaplan-Meier method. Univariable and multivariable analyses were performed to determine the relationship between patient and treatment characteristics and clinical outcomes. RESULTS: We identified 235 patients with a total of 381 oligometastatic CRC lesions. The 1- and 5-year local recurrence rate was 13.6% and 44.3% respectively. The median OS was 49 months with a 2-and 5-year OS of 76.1% and 35.9%, respectively. On multivariable analysis, a BED10 of ≥120 Gy, and lung versus liver metastases were associated with a reduction in local recurrence. Larger total PTV size (≥17.5 cc) was associated with worse overall survival, progression free survival, and widespread progression. CONCLUSION: This large multi-institutional analysis found that the use of SBRT for oligometastatic colorectal cancer resulted in favorable overall survival. However, local recurrence is higher than expected for ablative radiation treatment. An increase in BED10 should be considered if feasible and safe.

Stereotactic body radiotherapy (SBRT) for T2N0 (>3 cm) non-small cell lung cancer: Outcomes and failure patterns.

PURPOSE/OBJECTIVES: Outcomes of T2N0 lung cancer patients treated with stereotactic radiotherapy are not well known. METHODS AND MATERIALS: We conducted a single institution retrospective review of patients with T2N0 NSCLC who were treated with SBRT. The local, regional, distant control rates were calculated from available clinical data. Survival outcomes were determined using the Kaplan Meier method. RESULTS: Fifty-six patients met our selection criteria. The two-year local control rate was 84.2%. The two and 5-year disease-free survival (DFS) and OS were 31.9% and 15.3% and 39.9% and 12.1%, respectively. Centroid BED10 > 150Gy was associated with improved DFS, (p = 0.014), and OS on univariable analysis (p=0.0132). CONCLUSIONS: SBRT provides good local control for T2N0 NSCLC, but systemic failure remains problematic.

Novel Treatment for Glioblastoma Delivered by a Radiation Responsive and Radiopaque Hydrogel.

Successful treatment of glioblastoma (GBM) is hampered by primary tumor recurrence after surgical resection and poor prognosis, despite adjuvant radiotherapy and chemotherapy. In search of improved outcomes for this disease, quisinostat appeared as a lead compound in drug screening. A delivery system was devised for this drug and to exploit current clinical methodology: an injectable hydrogel, loaded with both the quisinostat drug and radiopaque gold nanoparticles (AuNP) as contrast agent, that can release these payloads as a response to radiation. This hydrogel grants high local drug concentrations, overcoming issues with current standards of care. Significant hydrogel degradation and quisinostat release were observed due to the radiation trigger, providing high in vitro anticancer activity. In vivo, the combination of radiotherapy and the radiation-induced delivery of quisinostat from the hydrogel, successfully inhibited tumor growth in a mice model bearing xenografted human GBM tumors with a total response rate of 67%. Long-term tolerability was observed after intratumoral injection of the quisinostat loaded hydrogel. The AuNP payload enabled precise image-guided radiation delivery and the monitoring of hydrogel degradation using computed tomography (CT). These exciting results highlight this hydrogel as a versatile imageable drug delivery platform that can be activated simultaneously to radiation therapy and potentially offers improved treatment for GBM.

Role of radiation in extensive stage small cell lung cancer: a National Cancer Database registry analysis.

The role of prophylactic cranial irradiation (PCI) and thoracic radiation therapy (TRT) in extensive-stage small cell lung cancer remains controversial. The authors examined the National Cancer Database and identified patients with extensive-stage small cell lung cancer with no brain metastasis. Patients were excluded if they died 30 days from diagnosis, did not receive polychemotherapy, had other palliative radiation or had missing information. A propensity score-matched analysis was also performed. A total of 21,019 patients were identified. The majority of patients did not receive radiation (69%), whereas 10% received PCI and 21% received TRT. The addition of PCI and TRT improved median survival and survival at 1 and 2 years (p ≤ 0.05). The propensity score-matched analysis confirmed the same overall survival benefit with both PCI and TRT. This registry-based analysis of >1500 accredited cancer programs shows that PCI and TRT are not commonly utilized for extensive-stage small cell lung cancer patients who are treated with multiagent chemotherapy. The addition of PCI and TRT significantly improves overall survival in this otherwise poor prognostic group. Further research is needed to confirm the role of PCI and TRT, especially in the era of improved systemic therapy.

Lay abstract The role of radiation therapy in patients with metastatic small cell lung cancer remains controversial. The authors examined the National Cancer Database and identified patients with metastatic small cell lung cancer without brain metastasis. Patients were excluded if they died 30 days from diagnosis, did not receive multiagent chemotherapy, had other palliative radiation or had missing information regarding treatment. A total of 21,019 patients were identified. The majority of patients did not receive radiation (69%), whereas 10% received radiation to the brain and 21% received radiation to their lungs. The addition of brain and lung radiation therapy improved median survival and survival at 1 and 2 years. The addition of prophylactic cranial irradiation and thoracic radiation therapy improves survival in extensive-stage small cell lung cancer. Future research is needed to evaluate the role of radiation in the era of chemoimmunotherapy.

Combined fluorescence-guided surgery and photodynamic therapy for glioblastoma multiforme using cyanine and chlorin nanocluster.

INTRODUCTION: Glioblastoma multiforme (GBM) is the most common primary intracranial malignancy; survival can be improved by maximizing the extent-of-resection. METHODS: A near-infrared fluorophore (Indocyanine-Green, ICG) was combined with a photosensitizer (Chlorin-e6, Ce6) on the surface of superparamagnetic-iron-oxide-nanoparticles (SPIONs), all FDA-approved for clinical use, yielding a nanocluster (ICS) using a microemulsion. The physical-chemical properties of the ICS were systematically evaluated. Efficacy of photodynamic therapy (PDT) was evaluated in vitro with GL261 cells and in vivo in a subtotal resection trial using a syngeneic flank tumor model. NIR imaging properties of ICS were evaluated in both a flank and an intracranial GBM model. RESULTS: ICS demonstrated high ICG and Ce6 encapsulation efficiency, high payload capacity, and chemical stability in physiologic conditions. In vitro cell studies demonstrated significant PDT-induced cytotoxicity using ICS. Preclinical animal studies demonstrated that the nanoclusters can be detected through NIR imaging in both flank and intracranial GBM tumors (ex: 745 nm, em: 800 nm; mean signal-to-background 8.5 ± 0.6). In the flank residual tumor PDT trial, subjects treated with PDT demonstrated significantly enhanced local control of recurrent neoplasm starting on postoperative day 8 (23.1 mm3 vs 150.5 mm3, p = 0.045), and the treatment effect amplified to final mean volumes of 220.4 mm3 vs 806.1 mm3 on day 23 (p = 0.0055). CONCLUSION: A multimodal theragnostic agent comprised solely of FDA-approved components was developed to couple optical imaging and PDT. The findings demonstrated evidence for the potential theragnostic benefit of ICS in surgical oncology that is conducive to clinical integration.

Evaluation of Diagnostic Accuracy Following the Coadministration of Delta-Aminolevulinic Acid and Second Window Indocyanine Green in Rodent and Human Glioblastomas.

PURPOSE: Fluorescence-guided-surgery offers intraoperative visualization of neoplastic tissue. Delta-aminolevulinic acid (5-ALA), which targets enzymatic abnormality in neoplastic cells, is the only approved agent for fluorescence-guided neurosurgery. More recently, we described Second Window Indocyanine Green (SWIG) which targets neoplastic tissue through enhanced vascular permeability. We hypothesized that SWIG would demonstrate similar clinical utility in identification of high-grade gliomas compared with 5-ALA. PROCEDURES: Female C57/BL6 and nude/athymic mice underwent intracranial implantation of 300,000 GL261 and U87 cells, respectively. Tumor-bearing mice were euthanized after administration of 5-ALA (200 mg/kg intraperitoneal) and SWIG (5 mg/kg intravenous). Brain sections were imaged for protoporphyrin-IX and ICG fluorescence. Fluorescence and H&E images were registered using semi-automatic scripts for analysis. Human subjects with HGG were administered SWIG (2.5 mg/kg intravenous) and 5-ALA (20 mg/kg oral). Intraoperatively, tumors were imaged for ICG and protoporphyrin-IX fluorescence. RESULTS: In non-necrotic tumors, 5-ALA and SWIG demonstrated 90.2 % and 89.2 % tumor accuracy (p value = 0.52) in U87 tumors and 88.1 % and 87.7 % accuracy (p value = 0.83) in GL261 tumors. The most distinct difference between 5-ALA and SWIG distribution was seen in areas of tumor-associated necrosis, which often showed weak/no protoporphyrin-IX fluorescence, but strong SWIG fluorescence. In twenty biopsy specimens from four subjects with HGG, SWIG demonstrated 100 % accuracy, while 5-ALA demonstrated 75-85 % accuracy; there was 90 % concordance between SWIG and 5-ALA fluorescence. CONCLUSION: Our results provide the first direct comparison of the diagnostic utility of SWIG vs 5-ALA in both rodent and human HGG. Given the broader clinical utility of SWIG compared with 5-ALA, our data supports the use of SWIG in tumor surgery to improve the extent of safe resections. CLINICAL TRIAL: NCT02710240 (US National Library of Medicine Registry; https://www.clinicaltrials.gov/ct2/show/NCT02710240?id=NCT02710240&draw=2&rank=1 ).

A Patient-Derived Glioblastoma Organoid Model and Biobank Recapitulates Inter- and Intra-tumoral Heterogeneity.

Glioblastomas exhibit vast inter- and intra-tumoral heterogeneity, complicating the development of effective therapeutic strategies. Current in vitro models are limited in preserving the cellular and mutational diversity of parental tumors and require a prolonged generation time. Here, we report methods for generating and biobanking patient-derived glioblastoma organoids (GBOs) that recapitulate the histological features, cellular diversity, gene expression, and mutational profiles of their corresponding parental tumors. GBOs can be generated quickly with high reliability and exhibit rapid, aggressive infiltration when transplanted into adult rodent brains. We further demonstrate the utility of GBOs to test personalized therapies by correlating GBO mutational profiles with responses to specific drugs and by modeling chimeric antigen receptor T cell immunotherapy. Our studies show that GBOs maintain many key features of glioblastomas and can be rapidly deployed to investigate patient-specific treatment strategies. Additionally, our live biobank establishes a rich resource for basic and translational glioblastoma research.

Rapid and ultrasensitive digital PCR (dPCR) profiling of EGFRvIII in tumor cells and tissues.

BACKGROUND: Amplification of the epidermal growth factor receptor (EGFR) gene is commonly found in glioblastoma (GBM). About 57% GBM overexpresses EGFR and are associated with tumor progression, poor prognosis, and shorter life expectancy. Molecular profiling of solid tumors usually takes several weeks and may be biased by intrinsic tumor heterogeneity. METHODS: The unique sequence created by the fusion of exon 1 and exon 8 in EGFRvIII was used to guide the design of primers and a Minor Groove Binder (MGB) probe. Extracted total RNA was reverse transcribed and pre-amplified by PCR, followed by detection of the EGFRvIII mutation by dPCR. RESULTS: The lowest limit of quantification of our EGFRvIII assay was 0.003%. The EGFRvIII variant was identified in patient-derived glioma neurosphere cell lines, xenograft mouse model, and patient-derived tumor specimens. The overall workflow can be accomplished within 24 hours. In certain samples, EGFRvIII was detected when next-generation sequencing was unable to identify the variant. This finding highlights the ability of the dPCR assay to identify EGFRvIII mutations in heterogeneous solid tumors such as GBM in a rapid fashion by profiling samples from spatially distinct areas of tumors from the same patient. CONCLUSIONS: In this study, we developed a highly sensitive digital PCR (dPCR) platform and leveraged our assay to detect the variant III alteration of EGFR (EGFRvIII) and amplified EGFR in patient-derived glioma neurosphere cell lines, orthotopic xenograft GBM mouse models, and patient-derived tumor specimens in less than 24 hours from minute quantities of starting material.

An Integrated Stress Response Agent that Modulates DR5-Dependent TRAIL Synergy Reduces Patient-Derived Glioma Stem Cell Viability.

The high incidence of glioblastoma recurrence necessitates additional therapeutic strategies. Heterogeneous populations of cells, including glioma stem cells (GSC) have been implicated in disease recurrence. GSCs are able to survive irradiation and temozolomide (TMZ) treatment due to upregulation of DNA damage pathways. One potential strategy to target treatment-resistant tumor populations may be via the integrated stress response (ISR). Modulation of the ISR pathway also allows for sensitization of treatment-resistant cells to TRAIL. We generated a novel cell-based death receptor assay to identify potent inducers of ISR-dependent DR5 expression. We used this assay to screen compounds from three commercially available libraries, and identified 1-benzyl-3-cetyl-2-methylimidazolium iodide (NH125) as a potent inducer of DR5 expression. NH125 engages the EIF2α-ATF4-CHOP axis culminating in DR5 expression at low micromolar doses. Expression of CHOP plays a critical role in NH125-mediated TRAIL synergy. Treatment of GSC with NH125 produces a marked reduction in viability when compared with other cell lines. NH125-treated GSC also synergize with lower doses of TRAIL when compared with all other cell lines tested. Transcriptional analysis of NH125-treated GSC uncovers a unique profile that involves activation of ISR and GADD45 pathways. Treatment of GSC xenografts with encapsulated PEG-PCL-NH125 leads to a sustained decrease in tumor volume. IMPLICATIONS: Taken together, these data suggest that engaging the ISR pathway represents a promising strategy to target treatment refractory GSC that have been implicated in glioblastoma recurrence.

The second window ICG technique demonstrates a broad plateau period for near infrared fluorescence tumor contrast in glioblastoma.

INTRODUCTION: Fluorescence-guided surgery has emerged as a powerful tool to detect, localize and resect tumors in the operative setting. Our laboratory has pioneered a novel way to administer an FDA-approved near-infrared (NIR) contrast agent to help surgeons with this task. This technique, coined Second Window ICG, exploits the natural permeability of tumor vasculature and its poor clearance to deliver high doses of indocyanine green (ICG) to tumors. This technique differs substantially from established ICG video angiography techniques that visualize ICG within minutes of injection. We hypothesized that Second Window ICG can provide NIR optical contrast with good signal characteristics in intracranial brain tumors over a longer period of time than previously appreciated with ICG video angiography alone. We tested this hypothesis in an intracranial mouse glioblastoma model, and corroborated this in a human clinical trial. METHODS: Intracranial tumors were established in 20 mice using the U251-Luc-GFP cell line. Successful grafts were confirmed with bioluminescence. Intravenous tail vein injections of 5.0 mg/kg (high dose) or 2.5 mg/kg (low dose) ICG were performed. The Perkin Elmer IVIS Spectrum (closed field) was used to visualize NIR fluorescence signal at seven delayed time points following ICG injection. NIR signals were quantified using LivingImage software. Based on the success of our results, human subjects were recruited to a clinical trial and intravenously injected with high dose 5.0 mg/kg. Imaging was performed with the VisionSense Iridium (open field) during surgery one day after ICG injection. RESULTS: In the murine model, the NIR signal-to-background ratio (SBR) in gliomas peaks at one hour after infusion, then plateaus and remains strong and stable for at least 48 hours. Higher dose 5.0 mg/kg improves NIR signal as compared to lower dose at 2.5 mg/kg (SBR = 3.5 vs. 2.8; P = 0.0624). Although early (≤ 6 hrs) visualization of the Second Window ICG accumulation in gliomas is stronger than late (≥24 hrs) visualization (SBR = 3.94 vs. 2.32; p<0.05) there appears to be a long plateau period of stable ICG NIR signal accumulation within tumors in the murine model. We call this long plateau period the "Second Window" of ICG. In glioblastoma patients, the delayed visualization of intratumoral NIR signal was strong (SBR 7.50 ± 0.74), without any significant difference within the 19 to 30 hour visualization window (R2 = 0.019). CONCLUSION: The Second Window ICG technique allows neurosurgeons to deliver NIR optical contrast agent to human glioblastoma patients, thus providing real-time tumor identification in the operating room. This nonspecific tumor accumulation of ICG within the tumor provides strong signal to background contrast, and is not significantly time dependent between 6 hours to 48 hours, providing a broad plateau for stable visualization. This finding suggests that optimal imaging of the "Second Window of ICG" may be within this plateau period, thus providing signal uniformity across subjects.

Distribution of Force in the Medial Collateral Ligament Complex During Simulated Clinical Tests of Knee Stability.

BACKGROUND: Pivot-shift injury commonly results in combined anterior cruciate ligament (ACL)/medial collateral ligament (MCL) injury, yet the contribution of the components of the MCL complex to restraining multiplanar rotatory loads forming critical subcomponents of the pivot shift is not well understood. PURPOSE: To quantify the role of the MCL complex in restraining multiplanar rotatory loads. STUDY DESIGN: Controlled laboratory study. METHODS: A robotic manipulator was used to apply combined valgus and internal rotation torques in a simplified model of the pivot-shift examination in 12 cadaveric knees (49 ± 11 years). Tibiofemoral kinematics were recorded with the ACL intact. Loads borne by the superficial MCL (sMCL), posterior oblique ligament (POL), deep MCL (dMCL), and ACL were determined via the principle of superposition. RESULTS: The POL bore about 50% of the load carried by the ACL in response to the combined torques at 5° and 15° of flexion. The POL bore load during the internal rotation component of the combined torques, while the sMCL carried load during the valgus and internal rotation phases of the simulated pivot. Load in the dMCL was always <10% of the ACL in response to combined valgus and internal rotation torques. CONCLUSION: The POL provides complementary load bearing to the ACL near extension in response to combined torques, which capture key components of the pivot-shift examination. The sMCL resists the valgus component of the maneuver alone, a loading pattern unique from those of the POL and ACL. The dMCL is not loaded during clinical tests of rotational knee stability in the ACL-competent knee. CLINICAL RELEVANCE: Both the sMCL and POL work together with the ACL to resist combined moments, which form key components of the pivot-shift examination.

Novel measure of articular instability based on contact stress confirms that the anterior cruciate ligament is a critical stabilizer of the lateral compartment.

Knee instability following anterior cruciate ligament (ACL) rupture is common, compromising function, and causing cartilage and meniscal damage. In this study, instability at the level of the articular surfaces was characterized with a new measure: articular instability. Articular instability was defined as the change in location of the center of contact stress per unit of applied load. The effect of ACL-deficiency on articular instability was quantified in response to combined abduction and internal rotation moments simulating the clinical pivot shift, which recreates the sensation of instability. Eleven cadaver knees were loaded using a robotic manipulator and tibiofemoral contact stress was measured using a stress transducer. Sectioning the ACL led to pronounced articular instability on the lateral compartment in 4 of 11 knees. In these 4 knees articular instability increased posteriorly up to 403% and increased laterally up to 754%. Factors driving inter-specimen variations in articular instability might include articular morphology, ligamentous laxity, and the applied loads. This novel description of contact mechanics confirms that the ACL prevents sudden changes in the relative position of the lateral articular surfaces. It is applicable to any loading conditions and provides a unique measure to quantify the effects of ACL injury and reconstruction.

Stability analysis of phylogenetic trees.

MOTIVATION: Phylogenetics, or reconstructing the evolutionary relationships of organisms, is critical for understanding evolution. A large number of heuristic algorithms for phylogenetics have been developed, some of which enable estimates of trees with tens of thousands of taxa. Such trees may not be robust, as small changes in the input data can cause major differences in the optimal topology. Tools that can assess the quality and stability of phylogenetic tree estimates and identify the most reliable parts of the tree are needed. RESULTS: We define measures that assess the stability of trees, subtrees and individual taxa with respect to changes in the input sequences. Our measures consider changes at the finest granularity in the input data (i.e. individual nucleotides). We demonstrate the effectiveness of our measures on large published datasets. Our measures are computationally feasible for phylogenetic datasets consisting of tens of thousands of taxa. AVAILABILITY: This software is available at http://bioinformatics.cise.ufl.edu/phylostab CONTACT: sheikh@cise.ufl.edu

In utero and lactational exposure to vinclozolin and genistein induces genomic changes in the rat mammary gland.

Exposure to low doses of environmental estrogens such as bisphenol A and genistein (G) alters mammary gland development. The effects of environmental anti-androgens, such as the fungicide vinclozolin (V), on mammary gland morphogenesis are unknown. We previously reported that perinatal exposure to G, V, and the GV combination causes histological changes in the mammary gland during the peripubertal period, suggesting alterations to the peripubertal hormone response. We now investigate whether perinatal exposure to these compounds alters the gene expression profiles of the developing glands to identify the dysregulated signaling pathways and the underlying mechanisms. G, V, or GV (1 mg/kg body weight per day) was added to diet of Wistar rats, from conception to weaning; female offspring mammary glands were collected at postnatal days (PNDs) 35 and 50. Genes displaying differential expression and belonging to different functional categories were validated by quantitative PCR and immunocytochemistry. At PND35, G had little effect; the slight changes noted were in genes related to morphogenesis. The changes following exposure to V concerned the functional categories associated with development (Cldn1, Krt17, and Sprr1a), carbohydrate metabolism, and steroidogenesis. The GV mixture upregulated genes (Krt17, Pvalb, and Tnni2) involved in muscle development, indicating effects on myoepithelial cells during mammary gland morphogenesis. Importantly, at PND50, cycling females exposed to GV showed an increase in the expression of genes (Csn2, Wap, and Elf5) related to differentiation, consistent with the previously reported abnormal lobuloalveolar development previously described. Thus, perinatal exposure to GV alters the mammary gland hormone response differently at PND35 (puberty) and in animals with established cycles.

A graph-theoretic approach for classification and structure prediction of transmembrane ß-barrel proteins.

BACKGROUND: Transmembrane ß-barrel proteins are a special class of transmembrane proteins which play several key roles in human body and diseases. Due to experimental difficulties, the number of transmembrane ß-barrel proteins with known structures is very small. Over the years, a number of learning-based methods have been introduced for recognition and structure prediction of transmembrane ß-barrel proteins. Most of these methods emphasize on homology search rather than any biological or chemical basis. RESULTS: We present a novel graph-theoretic model for classification and structure prediction of transmembrane ß-barrel proteins. This model folds proteins based on energy minimization rather than a homology search, avoiding any assumption on availability of training dataset. The ab initio model presented in this paper is the first method to allow for permutations in the structure of transmembrane proteins and provides more structural information than any known algorithm. The model is also able to recognize ß-barrels by assessing the pseudo free energy. We assess the structure prediction on 41 proteins gathered from existing databases on experimentally validated transmembrane ß-barrel proteins. We show that our approach is quite accurate with over 90% F-score on strands and over 74% F-score on residues. The results are comparable to other algorithms suggesting that our pseudo-energy model is close to the actual physical model. We test our classification approach and show that it is able to reject α-helical bundles with 100% accuracy and ß-barrel lipocalins with 97% accuracy. CONCLUSIONS: We show that it is possible to design models for classification and structure prediction for transmembrane ß-barrel proteins which do not depend essentially on training sets but on combinatorial properties of the structures to be proved. These models are fairly accurate, robust and can be run very efficiently on PC-like computers. Such models are useful for the genome screening.

Using the fast fourier transform to accelerate the computational search for RNA conformational switches.

Using complex roots of unity and the Fast Fourier Transform, we design a new thermodynamics-based algorithm, FFTbor, that computes the Boltzmann probability that secondary structures differ by [Formula: see text] base pairs from an arbitrary initial structure of a given RNA sequence. The algorithm, which runs in quartic time O(n(4)) and quadratic space O(n(2)), is used to determine the correlation between kinetic folding speed and the ruggedness of the energy landscape, and to predict the location of riboswitch expression platform candidates. A web server is available at http://bioinformatics.bc.edu/clotelab/FFTbor/.

Abnormal peripubertal development of the rat mammary gland following exposure in utero and during lactation to a mixture of genistein and the food contaminant vinclozolin.

The impact of early exposure to endocrine disruptor mixtures on mammary gland development is poorly known. Here, we identify the effects of a conception to weaning exposure of rats to the phytoestrogen genistein (G) and/or the antiandrogen vinclozolin (V) at 1mg/kg-d, alone or in association. Using several approaches, we found that G- and GV-exposed rats displayed significantly greater epithelial branching and proliferation, wider terminal end buds than controls at PND35, as well as ductal hyperplasia and periductal fibrosis. Focal branching defects were present in V-exposed rats. An increased ER and AR expression was observed in G- and GV- as compared to V-exposed rats at PND35. Surprisingly, a significant number of GV- and to a lesser extent, V-exposed animals displayed abnormal hyperplasic alveolar structures at PND50. Thus, gestational and lactational exposure to low doses of genistein plus vinclozolin may seriously affect peripubertal development of the rat mammary gland.