All-Atom Simulations Uncover Structural and Dynamical Properties of STING Proteins in the Membrane System.

Recent studies have shown that the stimulator of interferon gene (STING) protein plays a central role in the immune system by facilitating the production of type I interferons in cells. The STING signaling pathway is also a prominent activator of cancer-killing T cells that initiate a powerful adaptive immune response. Since biomolecular signaling pathways are complicated and not easily identified through traditional experiments, molecular dynamics (MD) has often been used to study structural and dynamical responses of biological pathways. Here, we carried out MD simulations for full-length chicken and human STING (chSTING and hSTING) proteins. Specifically, we investigated ligand-bound closed (holo) and ligand-unbound open (apo) forms of STING in the membrane system by comparing their conformational and dynamical differences. Our research provides clues for understanding the mechanism of the STING signaling pathway by uncovering detailed insights for the examined systems: the residues from each chain in the binding pocket are strongly correlated to one another in the open STING structure compared with those in the closed STING structure. Ligand-bound closed STING displays ∼174° rotation of the ligand-binding domain (LBD) relative to the open STING structure. The dynamical analysis of residue Cys148 located in the linker region of hSTING does not support the earlier hypothesis that Cys148 can form disulfide bonds between adjacent STING dimers. We also demonstrate that using the full-length proteins is critical, since the MD simulations of the LBD portion alone cannot properly describe the global conformational properties of STING.

Genomic analysis of circulating cell-free DNA infers breast cancer dormancy.

Biomarkers in breast cancer to monitor minimal residual disease have remained elusive. We hypothesized that genomic analysis of circulating free DNA (cfDNA) isolated from plasma may form the basis for a means of detecting and monitoring breast cancer. We profiled 251 genomes using Affymetrix SNP 6.0 arrays to determine copy number variations (CNVs) and loss of heterozygosity (LOH), comparing 138 cfDNA samples with matched primary tumor and normal leukocyte DNA in 65 breast cancer patients and eight healthy female controls. Concordance of SNP genotype calls in paired cfDNA and leukocyte DNA samples distinguished between breast cancer patients and healthy female controls (P < 0.0001) and between preoperative patients and patients on follow-up who had surgery and treatment (P = 0.0016). Principal component analyses of cfDNA SNP/copy number results also separated presurgical breast cancer patients from the healthy controls, suggesting specific CNVs in cfDNA have clinical significance. We identified focal high-level DNA amplification in paired tumor and cfDNA clustered in a number of chromosome arms, some of which harbor genes with oncogenic potential, including USP17L2 (DUB3), BRF1, MTA1, and JAG2. Remarkably, in 50 patients on follow-up, specific CNVs were detected in cfDNA, mirroring the primary tumor, up to 12 yr after diagnosis despite no other evidence of disease. These data demonstrate the potential of SNP/CNV analysis of cfDNA to distinguish between patients with breast cancer and healthy controls during routine follow-up. The genomic profiles of cfDNA infer dormancy/minimal residual disease in the majority of patients on follow-up.

International study on inter-reader variability for circulating tumor cells in breast cancer.

INTRODUCTION: Circulating tumor cells (CTCs) have been studied in breast cancer with the CellSearch® system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement. METHODS: CellSearch® images (N = 272) of either CTCs or white blood cells or artifacts from 109 non-metastatic (M0) and 22 metastatic (M1) breast cancer patients from reported studies were sent to 22 readers from 15 academic laboratories and 8 readers from two Veridex laboratories. Each image was scored as No CTC vs CTC HER2- vs CTC HER2+. The 8 Veridex readers were summarized to a Veridex Consensus (VC) to compare each academic reader using % agreement and kappa (κ) statistics. Agreement was compared according to disease stage and CTC counts using the Wilcoxon signed rank test. RESULTS: For CTC definition (No CTC vs CTC), the median agreement between academic readers and VC was 92% (range 69 to 97%) with a median κ of 0.83 (range 0.37 to 0.93). Lower agreement was observed in images from M0 (median 91%, range 70 to 96%) compared to M1 (median 98%, range 64 to 100%) patients (P < 0.001) and from M0 and <3CTCs (median 87%, range 66 to 95%) compared to M0 and ≥3CTCs samples (median 95%, range 77 to 99%), (P < 0.001). For CTC HER2 expression (HER2- vs HER2+), the median agreement was 87% (range 51 to 95%) with a median κ of 0.74 (range 0.25 to 0.90). CONCLUSIONS: The inter-reader agreement for CTC definition was high. Reduced agreement was observed in M0 patients with low CTC counts. Continuous training and independent image review are required.

Adsorption of terbium (III) on DGA and LN resins: Thermodynamics, isotherms, and kinetics.

Two commercially available extraction chromatography (EXC) resins containing N,N,N',N'-tetra-n-octyldiglycolamide (DGA Resin, Normal, 50 - 100 µm) and Bis(2-ethylhexyl) phosphate (LN Resin, 100 - 150 µm) were used as adsorbents to study fundamental adsorption properties such as thermodynamic values, equilibrium isotherms, and kinetic uptake models for terbium(III) adsorption. Weight distribution ratios (Dw) for terbium on DGA and LN resins were measured using a [160Tb]Tb3+radiometric tracer in nitric acid as a function of acidity, temperature, initial analyte concentration, and equilibrium time. The Dw values showed increasing binding affinity for DGA resin at high nitric acid concentrations and decreasing binding affinity for LN resins. Thermodynamic studies for DGA and LN resins revealed that the Gibbs free energy (ΔG) increased consistently with temperature. To model equilibrium data, increasingly higher parameter equilibrium isotherm models (Henry (1) < Langmuir, Freundlich (2) < Redlich-Peterson (3) < Fritz-Schluender (4)) were compared on their root mean squared errors (RMSE) and adjusted determination coefficients to determine the most applicable model. In all cases, the empirical four-parameter Fritz-Schluender isotherm demonstrated a superior fit. Similar comparisons for reaction-based kinetic models (Pseudo-first-order < Pseudo-second-order < Pseudo-n-order) revealed that the higher-order PNO model yielded a superior fit of kinetic data for both resins. However, in some cases, adsorption isotherms and kinetic models could also be modeled by a lower-order model with minimal change in error parameters. Weber-Morris plots revealed that two linear sections are observed for each resin, where the first linear segment is attributed to fast (film diffusion) adsorption of terbium, followed by slower intraparticle diffusion of terbium through the pores as the rate-limiting step. Based on the Weber-Morris plot, both film and intraparticle diffusion are involved in controlling the kinetic rate of adsorption for DGA and LN resins.

Do storm overflows influence AMR in the environment and is this relevant to human health? A UK perspective on a global issue.

Antimicrobial resistance (AMR) is a global public health threat, and the environment has been identified as an important reservoir for resistant microorganisms and genes. Storm overflows (SOs) discharge wastewater and stormwater, and are found throughout many wastewater networks. While there are no data currently showing the impact of SOs on the environment with respect to AMR in the UK, there is a small but growing body of evidence globally highlighting the potential role of SOs on environmental AMR. This review aims to provide an overview of the current state of SOs, describe global data investigating the impact of SOs on environmental AMR, and discuss the implications of SOs regarding AMR and human health. In addition, the complexities of studying the effects of SOs are discussed and a set of priority research questions and policy interventions to tackle a potentially emerging threat to public health are presented.

Blunt aortic injuries in the new era: radiologic findings and polytrauma risk assessment dictates management strategy.

PURPOSE: Blunt aortic injuries (BAI) have historically been considered an indication for emergent surgical intervention. Nevertheless, the observation that the outcome of the concomitant traumatic injuries has a major impact on prognosis and the rise of thoracic endovascular aortic repair (TEVAR) as an effective therapy for BAI have significantly changed in recent years the treatment algorithm of this condition. Our objective was to identify findings associated with the aortic injury which would be the best predictor of prognosis, with the objective of guiding the decision-making process for selecting the optimal timing of aortic repair. METHODS: We reviewed blunt aortic injuries from 3 Level I Trauma Centers from July 2008 to December 2016. We analyzed overall and BAI-related 30-day mortality in relation to: hemodynamics, timing of treatment, TEVAR vs open repair, and aortic injury grade as defined by the Society for Vascular Surgery. Based on computed tomographic angiography (CT scan) imaging, we selected the radiologic aortic findings most indicative of high mortality risk, which we defined as "Radiographic Severe Injury" (RSI): (1) total/partial aortic transection, (2) active contrast extravasation, or (3) the association of 2 of more of the following: contained contrast extravasation > 10 mm, periaortic hematoma, and/or mediastinal hematoma with thickness > 10 mm, or significant left pleural effusion. RESULTS: Of a total of 76 consecutive patients, 50 (66%) underwent immediate repair, 24 (31%) delayed aortic repair, and 2 (3%) died prior to repair. 58 patients (76%) had TEVAR, while 16 (24%) had open repair. Overall mortality was 18% and BAI-related mortality was 13%. In BAI-related mortalities, 70% of patients had RSI. Patients with high risk of overall mortality had hypotension and tachycardia (SBP < 100, HR ≥ 100), high ISS, and required vasopressors. Factors only associated with BAI-related mortality included RSI. CONCLUSION: CT scan findings suggestive of RSI are predictive of mortality associated with BAI. Radiologic assessment of the severity of the aortic injury with characterization for the presence of RSI may represent the key factors to determine the optimal timing of treatment of the aortic injury and guide the overall treatment strategy. LEVEL OF EVIDENCE: IV.

A comparison of three-dimensional ultrasound, two-dimensional ultrasound and dissections for determination of lesion volume in tendons.

The purpose of this work was to evaluate the accuracy and precision of a freehand three-dimensional (3-D) ultrasonography system in the determination of lesion volume in tendons. The accuracy and precision of a 3-D ultrasonography system was assessed by performing repeated measurements on a phantom of known volume. Volume measurements of tendon lesions performed with 3-D ultrasonography were compared with measurements based on a series of two-dimensional (2-D) ultrasound (US) scans and to direct measurements from dissections. A novel method for the creation of tendon lesions in vitro was developed. 3-D US showed excellent precision and accuracy in measurements of the phantom (mean measured volume = 3.76 mL, calculated volume = 3.77 mL, coefficient of variation (CoV) = 0.54%) and good repeatability in the determination of tendon lesions (repeatability coefficient = 0.00047). All three methods examined were repeatable (repeatability coefficient for 2-D US = 0.00032, repeatability coefficient for dissections = 0.00076). However, each of the methods produced different results and no constant relationship could be found between any of the measurement methods. Both 3-D and 2-D US proved to be repeatable techniques for the measurement of the volume of a tendon lesion. Even if they produced different results, each of them can be repeatedly used individually. It was not possible to define which one provided the most accurate value as a result of difficulties encountered in lesion identification on histology, and therefore the lack of a gold standard.

The efficacy of lapatinib in metastatic breast cancer with HER2 non-amplified primary tumors and EGFR positive circulating tumor cells: a proof-of-concept study.

BACKGROUND: Analysis of circulating tumor cells (CTCs) provides real-time measures of cancer sub-populations with potential for CTC-directed therapeutics. We examined whether lapatinib which binds both HER2 and EGFR could induce depletion of the EGFR-positive pool of CTCs, which may in turn lead to clinical benefits. PATIENTS AND METHODS: Patients with metastatic breast cancer and HER2 non-amplified primary tumors with EGFR-positive CTCs were recruited and lapatinib 1500 mg daily was administered, in a standard two step phase 2 trial. RESULTS: There were no responses leading to termination at the first analysis with 16 patients recruited out of 43 screened. In 6 out of 14 (43%) individuals eligible for the efficacy analysis, a decrease in CTCs was observed with most of these having a greater decrease in their EGFR-positive CTC pool. CONCLUSIONS: This is one of the first studies of CTC-directed therapeutics and suggests that lapatinib monotherapy is not having any demonstrable clinical effects by reducing the EGFR-positive pool of CTCs in HER2 non-amplified primary tumors. Our attempt to expand the pool of patients eligible for a targeted therapy was unsuccessful; the role of clonal populations in cancer biology and therapeutic strategies to control them will require extensive evaluation in years to come. TRIAL REGISTRATION: Clinical trials.gov NCT00820924.

Circulating tumor cells and plasma DNA analysis in patients with indeterminate early or metastatic breast cancer.

BACKGROUND: Circulating tumor cells (CTCs) and tumor-specific alterations in cell-free plasma DNA can both be used as markers of prognosis in breast cancer. To date, there have been no studies that have compared these as markers for subclinical metastases in the follow-up of early breast cancer. In this study, we measured CTCs and plasma DNA in a published group of patients with multiple pulmonary nodules and indeterminate metastatic disease. PATIENTS & METHODS: A single blood sample for CTC and plasma DNA measurement was taken approximately 1.5 years after surgery from 19 women with histologically confirmed primary breast cancer and small pulmonary nodules. The CellSearch system was used to enrich and enumerate CTCs from peripheral blood. DNA was isolated from plasma and was analyzed by quantitative real-time PCR for DNA concentration, integrity and evidence of HER2 amplification. RESULTS: Of the 19 individuals with 'indeterminate' early or metastatic breast cancer, 17 demonstrated no evidence of CTCs, one had one CTC and one had three CTCs. The mean plasma DNA concentration was low and within the range detected in healthy female controls, as were the values for DNA integrity. HER2 amplification was detected in the plasma DNA in four of the eight patients with HER2 immunohistochemistry 3+ tumors, but there was no overlap with the two CTC-positive patients. None of the patients have relapsed thus far (median follow-up: 3.5 years). CONCLUSION: Both CTC and plasma DNA analyses together suggested that these patients had little evidence of metastatic disease. Future studies will be designed to assess the utility of these biomarkers in the follow-up of a larger number of women with breast cancer.

Epidermal growth factor receptor status in early stage breast cancer is associated with cellular proliferation but not cross-talk.

The epidermal growth factor receptor (EGFR) is a therapeutic target in a number of settings in solid malignancies, but its role in breast cancer has remained unclear and controversial. In 810 primary breast cancers derived from patients suitable for cytotoxic chemotherapy, EGFR was prospectively measured and interactions with tumour and clinical correlates were tested to observe whether postulated cross-talk mechanisms are likely to modulate breast cancer metastasis and proliferation. A minority (79 tumours, 9.8%) were EGFR positive; in a multivariate analysis the likelihood of being EGFR positive was significantly increased for patients with grade 3 disease, compared with grade 1 (OR 15.6; 95% CI 2 to 122, p=0.0001), and for oestrogen receptor-negative status compared with positive (OR 24.1; 95% CI 12.7 to 46.00, p=0.0001). EGFR expression may play a role in breast cancer proliferation, but appears unlikely to modify tumour pathology via postulated mechanisms of oestrogen receptor/EGFR-mediated cross-talk.

Measurements of EGFR expression on circulating tumor cells are reproducible over time in metastatic breast cancer patients.

AIMS: Studies of EGFR expression in breast cancer have shown inconsistent results due in part to a large range of methods used. Anti-EGFR therapy trials have often not used patient selection because of this. We describe the use of the CellSearch system (Veridex LLC, NJ, USA) to enumerate and measure EGFR expression on the surface of circulating tumor cells (CTCs), derived from the peripheral blood of individuals with metastatic breast cancer over time. MATERIALS & METHODS: The CellSearch system was used to quantify CTCs and EGFR measurement was performed on all samples. The specificity of EGFR phenotyping was further examined by spiking with cell lines with increased and low (or absent) levels of EGFR expression using the CellSearch system to enrich and phenotype the CTCs. RESULTS: Serial samples were obtained from 33 individuals with metastatic breast cancer. CTCs derived from these individuals had consistent levels of EGFR expression at different time points, and none of the patients 'switched' from a positive to negative EGFR phenotype or vice versa. The specificity of EGFR phenotyping by the CellSearch system was verified by staining of EGFR only being present in a high EGFR expressing EGFR cell line (MDA-MB-468), as confirmed by Western blotting. CONCLUSIONS: Measurement of EGFR on the surface of CTCs, derived from individuals with metastatic breast cancer patients is possible using the CellSearch system and showed consistent positivity over time. The use of this system will now be validated in a prospective study aiming to identify patients for anti-EGFR therapy based on the expression profile of CTCs.

Inertial properties of hominoid limb segments.

Quantitative, accurate data regarding the inertial properties of body segments are of paramount importance when developing musculo-skeletal locomotor models of living animals and, by inference, their ancestors. The limited number of available primate cadavers, and the destructive nature of the post-mortem, result in such data being very rare for primates. This study builds on the work of Crompton et al. (Am. J. Phys. Anthropol. 1996, 99, 547-570) and reports inertial properties of the body segments of gorillas, chimpanzees, orangutans and gibbons. Segment mass, centre of mass and the radius of gyration of five ape cadavers were measured using a complex-pendulum technique and compared with the results derived from external measurements of segment lengths and diameters on the same animals. With additional data from external measurements of eight more hominoid cadavers, and published data, intergeneric differences between the inertial properties and the distribution of mass between limb segments are analysed and related to the locomotor habits of the species. We found that segment inertial properties show extensive overlap between ape genera as a result of large interindividual variation. Segment mass distribution also overlaps between apes and humans, with the exception of the shank segment. However, owing to a different distribution of mass between the limb segments, the centre of mass of both the arms and the legs is located more distally in apes than in humans, and the natural pendular period of ape forelimbs is larger than that of the hindlimbs. This suggests that, in contrast to the limbs of cursorial mammals and cercopithecoid primates, hominoid limbs are not optimized for efficiency in quadrupedal walking, but rather reflect a compromise between various locomotor modes. Common chimpanzees may have secondarily evolved a more efficient quadrupedal gait.

Functional analysis of the foot and ankle myology of gibbons and bonobos.

This study investigates the foot and ankle myology of gibbons and bonobos, and compares it with the human foot. Gibbons and bonobos are both highly arboreal species, yet they have a different locomotor behaviour. Gibbon locomotion is almost exclusively arboreal and is characterized by speed and mobility, whereas bonobo locomotion entails some terrestrial knuckle-walking and both mobility and stability are important. We examine if these differences in locomotion are reflected in their foot myology. Therefore, we have executed detailed dissections of the lower hind limb of two bonobo and three gibbon cadavers. We took several measurements on the isolated muscles (mass, length, physiological cross sectional area, etc.) and calculated the relative muscle masses and belly lengths of the major muscle groups to make interspecific comparisons. An extensive description of all foot and ankle muscles is given and differences between gibbons, bonobos and humans are discussed. No major differences were found between the foot and ankle musculature of both apes; however, marked differences were found between the ape and human foot. The human foot is specialized for solely one type of locomotion, whereas ape feet are extremely adaptable to a wide variety of locomotor modes. Apart from providing interesting anatomical data, this study can also be helpful for the interpretation of fossil (pre)hominids.