Two-photon imaging of cancer cell extravasation in live mice

Abstract MDA-MB-231 breast cancer cells were engineered to express cytoplasmic paxillin-GFP and nuclear H2B-mCherry. In order to image extravasation, the cancer cells were injected in the blood stream of nude mice. Using 2-photon excitation microscopy we can simultaneously excite the two probes and also visualize the autofluorescence of tissues. A skin flap was opened to visualize blood vessels and recognize the position of the cancer cells. Two-photon imaging showed that after an initial phase in which the cells are non-adherent, some cells spread on the internal surface of the capillaries. Days later some cells started to appear on the external side of the capillary. The extravasated cells extend very long protrusions into the tissue. The goal was to determine if at the end of the long protrusion, if it is possible to observe the formation of focal adhesions by imaging paxillin-GFP. Preliminary results show that when cells start to adhere to the blood vessel wall they form focal adhesions as determined by the characteristic elongated features observed in the paxillin-GFP channel. New approaches will allow the tracking of the tip of the protrusion to determine if focal adhesions are forming there as the cells extravasate. This is important in establishing the mechanism of cell extravasation and migration in tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1412. doi:10.1158/1538-7445.AM2011-141

Shedding light on melanins within in situ human eye melanocytes using 2-photon microscopy profiling techniques.

Choroidal melanocytes (HCMs) are melanin-producing cells in the vascular uvea of the human eye (iris, ciliary body and choroid). These cranial neural crest-derived cells migrate to populate a mesodermal microenvironment, and display cellular functions and extracellular interactions that are biologically distinct to skin melanocytes. HCMs (and melanins) are important in normal human eye physiology with roles including photoprotection, regulation of oxidative damage and immune responses. To extend knowledge of cytoplasmic melanins and melanosomes in label-free HCMs, a non-invasive 'fit-free' approach, combining 2-photon excitation fluorescence lifetimes and emission spectral imaging with phasor plot segmentation was applied. Intracellular melanin-mapped FLIM phasors showed a linear distribution indicating that HCM melanins are a ratio of two fluorophores, eumelanin and pheomelanin. A quantitative histogram of HCM melanins was generated by identifying the image pixel fraction contributed by phasor clusters mapped to varying eumelanin/pheomelanin ratio. Eumelanin-enriched dark HCM regions mapped to phasors with shorter lifetimes and longer spectral emission (580-625 nm) and pheomelanin-enriched lighter pigmented HCM regions mapped to phasors with longer lifetimes and shorter spectral emission (550-585 nm). Overall, we demonstrated that these methods can identify and quantitatively profile the heterogeneous eumelanins/pheomelanins within in situ HCMs, and visualize melanosome spatial distributions, not previously reported for these cells

Real-time imaging of 3-dimensional cancer cell movement in tissues

Abstract Our knowledge of how cells move in 3D in tissues is limited due to the lack of imaging methods that can produce 3D images fast enough and with sufficient resolution. Cancer cells migrate in 3D by forming adhesion points at the end of very long cellular protrusions. These protrusions are very thin and it is difficult to visualize adhesions along the protrusion surface. Conventional 3D stack reconstruction has relatively low resolution unless it is done using many frames. This results in a very slow acquisition in 3D confocal microscopy. Faster methods of 3D data acquisition (spinning disk microscopy) cannot be easily implemented since there is significant amount of scatter in tissues. A major obstacle in imaging adhesions is to find and track them so that they will not go out of focus. We are developing a new method which is based on orbiting imaging around cellular protrusions to visualize protein dynamics during extravasation. A feedback mechanism controls the center of the orbit to be at the center of the fluorescence distribution. A program reconstructs the shape of the protrusions in 3D. The fluorescence intensity in one or more channels is also simultaneously measured. The fluorescence intensity of one channel is used to paint the protrusion shape, which results in the 3D reconstruction of the protrusion. During the orbit, the second channel of the microscope measures the second harmonic generation (SHG) signal. We then correlated the appearance of bright fluorescence spots on the protrusion surface with the points of contact of the protrusion. This method will enable imaging of cancer cell invasion in 3-dimentions in live mice in real time. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4750. doi:10.1158/1538-7445.AM2011-475

Raster-image-correlation spectroscopy of paxillin-GFP-expressing breast cancer cell in vitro and in vivo

Abstract Raster-image-correlation spectroscopy (RICS) is a noninvasive technique to detect and quantify events in the living cell, including concentrations of molecules and their diffusion coefficients. Any cell containing a fluorophore that can be imaged with a laser scanning microscope can be analyzed with RICS. We obtained RICS images with an Olympus FluoView FV1000 confocal microscope using Olympus FluoView software to acquire data and SimFCS software to perform RICS analysis. Paxillin is involved in the assembly of focal adhesions, which was linked to green fluorescent protein (GFP) for the current study. In this study, we describe RICS of paxillin-GFP expression in breast cancer cells (MDA-MB-231) in vitro and in vivo. Slow-moving membrane-bound paxillin proteins were measured in live breast cancer cells in vitro. Paxillin-GFP-expressing breast cancer cells (1×106) were injected in the epigastric cranials vein of the nude mouse. Paxillin-GFP-expressing breast cancer cells became attached to the inner vessel wall within 3 hours after injection. Rapidly-moving cytosolic paxillin-GFP molecules were imaged with RICS. With the ability to measure the molecular dynamics of paxillin in cancer cells in vitro and in vivo by RICS, we are now capable of studying the role of both slow-moving paxillin in the cell membrane and rapidly-moving cytosolic paxillin in cancer-cell behavior. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5183. doi:1538-7445.AM2012-518

A Loop Region in the N-Terminal Domain of Ebola Virus VP40 Is Important in Viral Assembly, Budding, and Egress

Ebola virus (EBOV) causes viral hemorrhagic fever in humans and can have clinical fatality rates of ~60%. The EBOV genome consists of negative sense RNA that encodes seven proteins including viral protein 40 (VP40). VP40 is the major Ebola virus matrix protein and regulates assembly and egress of infectious Ebola virus particles. It is well established that VP40 assembles on the inner leaflet of the plasma membrane of human cells to regulate viral budding where VP40 can produce virus like particles (VLPs) without other Ebola virus proteins present. The mechanistic details, however, of VP40 lipid-interactions and protein-protein interactions that are important for viral release remain to be elucidated. Here, we mutated a loop region in the N-terminal domain of VP40 (Lys127, Thr129, and Asn130) and find that mutations (K127A, T129A, and N130A) in this loop region reduce plasma membrane localization of VP40. Additionally, using total internal reflection fluorescence microscopy and number and brightness analysis we demonstrate these mutations greatly reduce VP40 oligomerization. Lastly, VLP assays demonstrate these mutations significantly reduce VLP release from cells. Taken together, these studies identify an important loop region in VP40 that may be essential to viral egress

A precision functional atlas of personalized network topography and probabilities

Although the general location of functional neural networks is similar across individuals, there is vast person-to-person topographic variability. To capture this, we implemented precision brain mapping functional magnetic resonance imaging methods to establish an open-source, method-flexible set of precision functional network atlases-the Masonic Institute for the Developing Brain (MIDB) Precision Brain Atlas. This atlas is an evolving resource comprising 53,273 individual-specific network maps, from more than 9,900 individuals, across ages and cohorts, including the Adolescent Brain Cognitive Development study, the Developmental Human Connectome Project and others. We also generated probabilistic network maps across multiple ages and integration zones (using a new overlapping mapping technique, Overlapping MultiNetwork Imaging). Using regions of high network invariance improved the reproducibility of executive function statistical maps in brain-wide associations compared to group average-based parcellations. Finally, we provide a potential use case for probabilistic maps for targeted neuromodulation. The atlas is expandable to alternative datasets with an online interface encouraging the scientific community to explore and contribute to understanding the human brain function more precisely

Nutrition and exercise prevent excess weight gain in overweight pregnant women

Purpose: To determine the effect of a Nutrition and Exercise Lifestyle Intervention Program (NELIP) for overweight (OW) and obese (OB) pregnant women on pregnancy weight gain, birth weight, and maternal weight retention at 2 months postpartum. Methods: This is a single-arm intervention matched by prepregnant body mass index, age, and parity to a historical cohort (4:1). Women with a prepregnancy body mass index of\u3e25.0 kg•m (N = 65) participated in a NELIP starting at 16-20 wk of pregnancy, continuing until delivery. NELIP consisted of an individualized nutrition plan with total energy intake of approximately 2000 kcal•d (8360 kJ•d) and 40%-55% of total energy intake from carbohydrate. Exercise consisted of a walking program (30% HR reserve), three to four times per week, using a pedometer to count steps. Matched historical cohort (MC; N = 260) was from a large local perinatal database. Results: Weight gained by women on the NELIP was 6.8 ± 4.1 kg (0.38 ± 0.2 kg•wk), with a total pregnancy weight gain of 12.0 ± 5.7 kg. Excessive weight gain occurred before NELIP began at 16 wk of gestation. Eighty percent of the women did not exceed recommended pregnancy weight gain on NELIP. Weight retention at 2 months postpartum was 2.2 ± 5.6 kg with no difference between the OW and the OB women on NELIP. Mean birth weight was not different between NELIP (3.59 ± 0.5 kg) and MC (3.56 ± 0.6 kg, P \u3e 0.05). Conclusions: NELIP reduces the risk of excessive pregnancy weight gain with minimal weight retention at 2 months postpartum in OW and OB women. This intervention may assist OW and OB women in successful weight control after childbirth. Copyright © 2010 by the American College of Sports Medicine

Risk of pregnancy-related hypertension within 5 years of exposure to drinking water contaminated with Escherichia coli O157:H7

The authors evaluated the risk for pregnancy-related hypertension among previously healthy women who conceived within 5 years of exposure to drinking water contaminated with Escherichia coli O157.H7 in Walkerton, Canada (2000). Chronic hypertension was defined as systolic/diastolic blood pressure ≥140/90 mm Hg before 20 weeks gestation; gestational hypertension was defined as new onset systolic/diastolic blood pressure ≥140/90 mm Hg ≥20 weeks gestation. The incidence of hypertension was compared between women who were asymptomatic during the outbreak to those who experienced acute gastroenteritis. Blood pressure data were available for 135 of 148 eligible pregnancies. The adjusted relative risks for chronic and gestational hypertension were 1.5 (95% confidence interval [CI]: 0.3-7.7) and 1.0 (95% CI: 0.4-2.5), respectively. Mean arterial pressure before 20 weeks gestation was 2.7 mm Hg higher in women who had acute gastroenteritis (95% CI: 0.05-5.4). A trend toward higher chronic hypertension and mean arterial pressure in early pregnancy was observed among women who experienced gastroenteritis after exposure to bacterially-contaminated drinking water. © 2010 Wiley Periodicals, Inc

Line Broadening in Field Metal-poor Red Giant and Red Horizontal Branch Stars

We report 349 radial velocities for 45 metal-poor field red giant and red horizontal branch stars. We have have identified one new spectroscopic binary, HD 4306, and one possible such system, HD 184711. We also report 57 radial velocities for 11 of the 91 stars reported on previously by Carney et al. (2003). As was found in the previous study, radial velocity "jitter" is present in many of the most luminous stars. Excluding stars showing spectroscopic binary orbital motion, all 7 of the red giants with M(V) <= -2.0 display jitter, as well as 3 of the 14 stars with -2.0 <= M(V) <= -1.4. We have also measured line broadening in all of the new spectra, using synthetic spectra as templates. The most luminous red giants show significant line broadening, as do many of the red horizontal branch stars, and we discuss briefly possible causes.Comment: To appear in the Astronomical Journa

Atonic Postpartum Hemorrhage: Blood Loss, Risk Factors, and Third Stage Management

AbstractObjectiveAtonic postpartum hemorrhage rates have increased in many industrialized countries in recent years. We examined the blood loss, risk factors, and management of the third stage of labour associated with atonic postpartum hemorrhage.MethodsWe carried out a case-control study of patients in eight tertiary care hospitals in Canada between January 2011 and December 2013. Cases were defined as women with a diagnosis of atonic postpartum hemorrhage, and controls (without postpartum hemorrhage) were matched with cases by hospital and date of delivery. Estimated blood loss, risk factors, and management of the third stage labour were compared between cases and controls. Conditional logistic regression was used to adjust for confounding.ResultsThe study included 383 cases and 383 controls. Cases had significantly higher mean estimated blood loss than controls. However, 16.7% of cases who delivered vaginally and 34.1% of cases who delivered by Caesarean section (CS) had a blood loss of < 500 mL and < 1000 mL, respectively; 8.2% of controls who delivered vaginally and 6.7% of controls who delivered by CS had blood loss consistent with a diagnosis of postpartum hemorrhage. Factors associated with atonic postpartum hemorrhage included known protective factors (e.g., delivery by CS) and risk factors (e.g., nulliparity, vaginal birth after CS). Uterotonic use was more common in cases than in controls (97.6% vs. 92.9%, P < 0.001). Delayed cord clamping was only used among those who delivered vaginally (7.7% cases vs. 14.6% controls, P = 0.06).ConclusionThere is substantial misclassification in the diagnosis of atonic postpartum hemorrhage, and this could potentially explain the observed temporal increase in postpartum hemorrhage rates