Local distribution of collagen fibers determines crack initiation site and its propagation direction during aortic rupture

Although elucidation of the mechanism of aortic aneurysm rupture is important, the characteristics of crack initiation and propagation sites remain unknown. To determine the microscopic properties of these sites, the characteristics of local strains and constituents at crack initiation and propagation sites were investigated during biaxial stretching of porcine thoracic aortas (PTAs). PTAs were sliced into approximately 50-\(\upmu \hbox {m}\)-thick sections, and the center of the sections was made especially thin using our previously developed technique. Alpha-elastin and cell nuclei were fluorescently labeled as indices of local elastin density and as a strain marker, respectively. Birefringence and second harmonic generation (SHG) light images were used to determine local collagen distributions. The specimens were then stretched biaxially with a laboratory-made tensile tester under a fluorescent microscope equipped with a birefringence imaging system. Local strains were calculated from the local displacement of the cell nuclei. The degree of alignment and density of local collagen fibers were measured from retardance and SHG images. The strain distributions, specifically the first and second principal, and maximum shear strains, fluorescent intensity of \(\upalpha \)-elastin, and degree of alignment of collagen fibers, showed insignificant differences between the crack initiation sites and other sites. The retardance and intensity of SHG light at the crack initiation sites were significantly lower than those at other sites for all (\(n = 6\)) specimens. Cracks tended to propagate along the local direction of the collagen fibers. These results indicate that the local density and direction of collagen fibers play an important role in aorta rupture.     

Spatial orientation mapping of fibers using polarization‐sensitive second harmonic generation microscopy

In this work, we present a non‐invasive approach to determine azimuth and elevation angles of collagen fibers capable of generating second harmonic signal. The azimuth angle was determined using the minimum of second harmonic generation (SHG) signal while rotating the plane of polarization of excitation light. The elevation angle was estimated from the ratio of the minimal SHG intensity to the intensity when laser polarization and fiber directions were parallel to each other using experimentally determined calibration curve. Pixel‐resolution images of collagen fiber spatial orientation in tendon from bovine leg, chicken leg, and chicken skin were acquired using our approach of SHG polarization‐resolved microscopy. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Electrophoretic karyotyping ofCandida albicans strains isolated from premature infants and hospital personnel in a neonatal intensive care unit

Electrophoretic karyotyping was used to compare DNA probes of yeasts isolated from blood of preterm neonates (n=66) in a neonatal intensive care unit (NICU) and from the hands of healthy hospital personnel (n=10). The yeasts were identified asCandida albicans using standard laboratory methods. DNA was extracted from yeasts and isolation of identical DNA strains from the pairs nurse-neonate suggested that one nurse transmitted one yeast strain by her hands to three neonates. Four neonates harbored two identical strains originating from two nurses,i.e. each nurse transmitted the same strain to two neonates. In the additional 7 cases transmission of 1 yeast strain by 1 nurse, to 1 neonate was observed. Our data suggest that nonperinatal nosocomial transmission ofC. albicans occurs in neonates. possiblyvia cross-contamination being transferred on hands of health care workers The importance of careful hand washing of staff (health care workers) and other infectioncontrol procedures (to prevent the nosocomial transmission of pathogens in the NICU environment) is emphasizeded.     

A general multi-strain model with environmental transmission: Invasion conditions for the disease-free and endemic states

Although many infectious diseases of humans and wildlife are transmitted via an environmental reservoir, the theory of environmental transmission remains poorly elaborated. Here we introduce an SIR-type multi-strain disease transmission model with perfect cross immunity where environmental transmission is broadly defined by three axioms. We establish the conditions under which a multi-strain endemic state is invaded by another strain which is both directly and environmentally transmitted. We discuss explicit forms for environmental transmission terms and apply our newly derived invasion conditions to a two-strain system. Then, we consider the case of two strains with matching basic reproduction numbers (i.e., R₀), one directly transmitted only and the other both directly and environmentally transmitted, invading each other's endemic state. We find that the strain which is only directly transmitted can invade the endemic state of the strain with mixed transmission. However, the endemic state of the first strain is neutrally stable to invasion by the second strain. Thus, our results suggest that environmental transmission makes the endemic state less resistant to invasion.     

Anisotropic stretch-induced hypertrophy in neonatal ventricular myocytes micropatterned on deformable elastomers

Because cell shape and alignment, cell-matrix adhesion, and cell-cell contact can all affect growth, and because mechanical strains in vivo are multiaxial and anisotropic, we developed an in vitro system for engineering aligned, rod-shaped, neonatal cardiac myocyte cultures. Photolithographic and microfluidic techniques were used to micropattern extracellular matrices in parallel lines on deformable silicone elastomers. Confluent, elongated, aligned myocytes were produced by varying the micropattern line width and collagen density. An elliptical cell stretcher applied 2:1 anisotropic strain statically to the elastic substrate, with the axis of greatest stretch (10%) either parallel or transverse to the myofibrils. After 24 h, the principal strain parallel to myocytes did not significantly alter myofibril accumulation or expression of atrial natriuretic factor (ANF), connexin-43 (Cx-43), or N-cadherin (by indirect immunofluorescent antibody labeling and immunoblotting) compared with unstretched controls. In contrast, 10% transverse principal strain resulted in continuous staining of actin filaments (rhodamine phalloidin); increased immunofluorescent labeling of ANF, Cx-43, and N-cadherin; and upregulation of protein signal intensity by western blotting. By using microfabrication and microfluidics to control cell shape and alignment on an elastic substrate, we found greater effects for transverse than for longitudinal stretch in regulating sarcomere organization, hypertrophy, and cell-to-cell junctions.     

Cell-to-Cell Transmission Can Overcome Multiple Donor and Target Cell Barriers Imposed on Cell-Free HIV

Virus transmission can occur either by a cell-free mode through the extracellular space or by cell-to-cell transmission involving direct cell-to-cell contact. The factors that determine whether a virus spreads by either pathway are poorly understood. Here, we assessed the relative contribution of cell-free and cell-to-cell transmission to the spreading of the human immunodeficiency virus (HIV). We demonstrate that HIV can spread by a cell-free pathway if all the steps of the viral replication cycle are efficiently supported in highly permissive cells. However, when the cell-free path was systematically hindered at various steps, HIV transmission became contact-dependent. Cell-to-cell transmission overcame barriers introduced in the donor cell at the level of gene expression and surface retention by the restriction factor tetherin. Moreover, neutralizing antibodies that efficiently inhibit cell-free HIV were less effective against cell-to-cell transmitted virus. HIV cell-to-cell transmission also efficiently infected target T cells that were relatively poorly susceptible to cell-free HIV. Importantly, we demonstrate that the donor and target cell types influence critically the extent by which cell-to-cell transmission can overcome each barrier. Mechanistically, cell-to-cell transmission promoted HIV spread to more cells and infected target cells with a higher proviral content than observed for cell-free virus. Our data demonstrate that the frequently observed contact-dependent spread of HIV is the result of specific features in donor and target cell types, thus offering an explanation for conflicting reports on the extent of cell-to-cell transmission of HIV.     

Validity of Measurement of Shear Modulus by Ultrasound Shear Wave Elastography in Human Pennate Muscle

Ultrasound shear wave elastography is becoming a valuable tool for measuring mechanical properties of individual muscles. Since ultrasound shear wave elastography measures shear modulus along the principal axis of the probe (i.e., along the transverse axis of the imaging plane), the measured shear modulus most accurately represents the mechanical property of the muscle along the fascicle direction when the probe’s principal axis is parallel to the fascicle direction in the plane of the ultrasound image. However, it is unclear how the measured shear modulus is affected by the probe angle relative to the fascicle direction in the same plane. The purpose of the present study was therefore to examine whether the angle between the principal axis of the probe and the fascicle direction in the same plane affects the measured shear modulus. Shear modulus in seven specially-designed tissue-mimicking phantoms, and in eleven human in-vivo biceps brachii and medial gastrocnemius were determined by using ultrasound shear wave elastography. The probe was positioned parallel or 20° obliquely to the fascicle across the B-mode images. The reproducibility of shear modulus measurements was high for both parallel and oblique conditions. Although there was a significant effect of the probe angle relative to the fascicle on the shear modulus in human experiment, the magnitude was negligibly small. These findings indicate that the ultrasound shear wave elastography is a valid tool for evaluating the mechanical property of pennate muscles along the fascicle direction.     

Digital image correlation analysis of the load transfer by implant-supported restorations

This study compared splinted and non-splinted implant-supported prosthesis with and without a distal proximal contact using a digital image correlation method. An epoxy resin model was made with acrylic resin replicas of a mandibular first premolar and second molar and with threaded implants replacing the second premolar and first molar. Splinted and non-splinted metal-ceramic screw-retained crowns were fabricated and loaded with and without the presence of the second molar. A single-camera measuring system was used to record the in-plane deformation on the model surface at a frequency of 1.0Hz under a load from 0 to 250N. The images were then analyzed with specialist software to determine the direct (horizontal) and shear strains along the model. Not splinting the crowns resulted in higher stress transfer to the supporting implants when the second molar replica was absent. The presence of a second molar and an effective interproximal contact contributed to lower stress transfer to the supporting structures even for non-splinted restorations. Shear strains were higher in the region between the molars when the second molar was absent, regardless of splinting. The opposite was found for the region between the implants, which had higher shear strain values when the second molar was present. When an effective distal contact is absent, non-splinted implant-supported restorations introduce higher direct strains to the supporting structures under loading. Shear strains appear to be dependent also on the region within the model, with different regions showing different trends in strain changes in the absence of an effective distal contact.     

Experimental transmission of Trypanosoma cruzi through the genitalia of albino mice

Trypanosoma cruzi is usually transmitted by contact with the excreta of infected Triatominae; among non-vectorial infections, direct transmission through coitus has been proposed. We investigated this possibility by instilling, through the external meatus of the vagina and the penis of previously anesthetized NMRI albino mice, blood of mice infected with strains isolated from Didelphis marsupialis (opossum, strain CO57), Rattus rattus (rat, strain CO22) and human (strain EP). Some animals were allowed to copulate the same day of the instillation. In other experiments, the strains were inoculated in the scrotum. To determine the effect of immunosuppression, some mice were treated with cyclophosphamide 30 days post-instillation. Controls were instilled orally and ocularly. Vaginal instillation with strain CO22 produced systemic infection with tropism to the heart, skeletal muscle, skin, duodenum, pancreas, ovary and sternum. Scrotal inoculation with strain EP likewise invaded liver, spleen, lung, lymph nodes and urogenital organs; while strain CO57 invaded skeletal and cardiac muscle, pancreas, testis, and vas deferens. Penile infection with strain CO22 was detected by xenodiagnosis. Immunosuppression did not increase parasitemia of vaginally infected mice or controls. Mating did not produce infection. Our results show that contact of blood trypomastigotes of T. cruzi with genital mucosa can produce blood and tissue infections. These results are discussed in relation to reports of frequent experimental tropism of T. cruzi toward urogenital organs.     

Roles of Cell Signaling Pathways in Cell-to-Cell Contact-Mediated Epstein-Barr Virus Transmission

Epstein-Barr virus (EBV), a human gamma herpesvirus, establishes a life-long latent infection in B lymphocytes and epithelial cells following primary infection. Several lines of evidence indicate that the efficiency of EBV infection in epithelial cells is accelerated up to 10(4)-fold by coculturing with EBV-infected Burkitt's lymphoma (BL) cells compared to infection with cell-free virions, indicating that EBV infection into epithelial cells is mainly mediated via cell-to-cell contact. However, the molecular mechanisms involved in this pathway are poorly understood. Here, we establish a novel assay to assess cell-to-cell contact-mediated EBV transmission by coculturing an EBV-infected BL cell line with an EBV-negative epithelial cell line under stimulation for lytic cycle induction. By using this assay, we confirmed that EBV was transmitted from BL cells to epithelial cells via cell-to-cell contact but not via cell-to-cell fusion. The inhibitor treatments of extracellular signal-regulated kinase (ERK) and nuclear factor (NF)-κB pathways blocked EBV transmission in addition to lytic induction. The blockage of the phosphoinositide 3-kinase (PI3K) pathway impaired EBV transmission coupled with the inhibition of lytic induction. Knockdown of the RelA/p65 subunit of NF-κB reduced viral transmission. Moreover, these signaling pathways were activated in cocultured BL cells and in epithelial cells. Finally, we observed that viral replication was induced in cocultured BL cells. Taken together, our data suggest that cell-to-cell contact induces multiple cell signaling pathways in BL cells and epithelial cells, contributing to the induction of the viral lytic cycle in BL cells and the enhancement of viral transmission to epithelial cells.     

Orientation of apical and basal actin stress fibers in isolated and subconfluent endothelial cells as an early response to cyclic stretching

We investigated the response of apical and basal actin stress fibers (SFs) and its dependency on cell confluency for endothelial cells subjected to cyclic stretching. Porcine aortic endothelial cells from the 2nd and 5th passages were transferred to a fibronectin-coated silicone chamber with 5000-8000 cells/cm2 (isolated condition), positioning the cells apart, or with 25,000-27,000 cells/cm2 (subconfluent condition), allowing cell-to-cell contact. The substrate was stretched cyclically by 0.5 Hz for 2 h with a peak strain on the substrate that was 15% in the stretch direction and -4% in the transverse direction. The actin filaments (AFs) were stained with rhodamine phalloidin and their orientations were examined under a confocal laser scanning microscope. In the basal region, SFs formed in all of the cells under both the isolated and subconfluent conditions. We observed an average of 5 and 9 SFs per cell under the isolated and subconfluent conditions, respectively, in the fluorescent images of the apical region. We also observed cells that were bush-like without apical AFs or apical SFs. On average, the SFs in the subconfluent cells oriented in the direction of minimal strain, while the SFs in the isolated cells oriented in the direction of a 2% compressive strain. These results suggest that such differential response may be due to differences in the transmission of mechanical stretching to the central and apical regions of the cell through the SFs. We also speculate that cell-to-cell contact might change the strength, orientation, and anchorage of apical AFs and play a critical role in mechanical signal transduction.     

Photosynthetic unit size and electron-transport chain in a photoreaction center-depleted mutant of Rhodospirillum rubrum

The aim of this work was to explain the relatively fast growth of a mutant of Rhodospirillum rubrum (F24.1) which contains 7–8% of an apparently normal photoreaction center. We explored the double hypothesis that the size of its photosynthetic unit is larger than that of the wild type and that its electron-transport chain is organized in a network rather than in isolated loops. The first feature would allow faster growth under less than saturating light intensities and the second would allow faster maximal electron fluxes than would be predicted from the photoreaction center content. With respect to the first possibility, measurements of absorbance changes at 793 nm induced by short flashes of increasing intensity indicate that the photosynthetic unit of strain F24.1 is 5.6-fold larger than that of strain S1. The second possibility was verified by measuring relative electron fluxes at the photoreaction center in the two strains. This was established in the steady state from the amount of primary donor oxidized by a continuous light beam of increasing intensity. This electron flux was found to be about 70% as high in strain F24.1 as in strain S1. A more detailed study of the electron-transport chain indicated that cytochrome c₂ is by far the main secondary electron donor in strain F24.1. No evidence could be obtained for the existence of another secondary donor in that strain. The mole ratio of cytochrome c₂ to photoreaction center is about 6 in strain F24.1 as conpared to about 0.5 in strain S1. In strain 24.1, the pool of secondary donor appears to be collectively involved in the reduction of the oxidized primary donor. The replacement time at the photoreaction center of a first equivalent of oxidized cytochrome c₂ by a second equivalent of reduced cytochrome c₂ is less than or equal to 0.2 ms. The effect of the photoreaction center content on the size of the photosynthetic unit is discussed in terms of the different models proposed for the organisation of the photosynthetic unit. We propose that the electron-transport chain is organized in a network, perhaps by virtue of the lateral mobility of some of the electron carriers such as ubiquinone and cytochrome c₂.     

Plasma release-cell layering theory for blood flow

Significant internal structural changes occur in flowing blood when shear strain exceeds the critical value of 1 (unit strain), forcing alignment of the erythrocytes and releasing trapped plasma, which in turn leads to the formation of multiple layers of plasma on which oriented and compacted cells slide. These effects are identified in the inflections in the shear rate dependence of viscoelasticity of normal blood and in the viscous and elastic stress-to-strain relationships. Theoretical factors for plasma release and cell compaction allow calculation of the viscous and elastic properties of the cell layers from measured whole blood viscoelasticity and plasma viscosity. The new plasma release-cell layering theory encompasses, reinterprets and unifies many diverse previous observations relating to how blood flows, and provides a new understanding of the roles of red cell deformability and aggregation tendency.     

A simple low-cost microcontroller-based photometric instrument for monitoring chloroplast movement

A new microcontroller-based photometric instrument for monitoring blue light dependent changes in leaf transmission (chloroplast movement) was developed based on a modification of the double-beam technique developed by Walzcak and Gabrys [(1980) Photosynthetica 14: 65–72]. A blue and red bicolor light emitting diode (LED) provided both a variable intensity blue actinic light and a low intensity red measuring beam. A phototransistor detected the intensity of the transmitted measuring light. An inexpensive microcontroller independently and precisely controlled the light emission of the bicolor LED. A typical measurement event involved turning off the blue actinic light for 100 μs to create a narrow temporal window for turning on and measuring the transmittance of the red light. The microcontroller was programmed using LogoChip Logo (http://www.wellesley.edu/Physics/Rberg/logochip/) to record fluence rate response curves. Laser scanning confocal microscopy was utilized to correlate the changes in leaf transmission with intercellular chloroplast position. In the dark, the chloroplasts in the spongy mesophyll exhibited no evident asymmetries in their distribution, however, in the palisade layer the cell surface in contact with the overlying epidermis was devoid of chloroplasts. The low light dependent decrease in leaf transmittance in dark acclimated leaves was correlated with the movement of chloroplasts within the palisade layer into the regions previously devoid of chloroplasts. Changes in leaf transmittance were evident within one minute following the onset of illumination. Minimal leaf transmittance was correlated with chloroplasts having retreated from cell surfaces perpendicular to the incident light (avoidance reaction) in both spongy and palisade layers.Electronic Supplementary Material Supplementary material is available for this article at     

Effect of shear flow on the phase behavior of an aqueous gelatin-dextran emulsion

A rheo-optical methodology, based on small angle light scattering and transmitted light intensity measurements, has been used to study in situ and on a time resolved basis the shear induced morphology in ternary two-phase water-gelatin-dextran mixtures. Emulsions close to the binodal line as well as far from it have been investigated. It is shown that above a critical shear rate, shear-induced mixing occurs at the length scales probed by the laser light. It is hypothesized that the shear-induced homogenization is due to the shear forces that exceed the intermolecular forces of the self-association process of the gelatin. The isothermal phase diagram at a fixed shear rate has been determined. In addition, the structure evolution after cessation of flow has been studied. When flow is stopped after homogenization, phase separation occurs almost instantaneously. When subsequently applying a low shear rate, the structure coarsens due to coalescence of the dispersed droplets. The kinetics of this coalescence process is strain controlled.     

Exhaled aerosol transmission of pandemic and seasonal H1N1 influenza viruses in the ferret

Person-to-person transmission of influenza viruses occurs by contact (direct and fomites) and non-contact (droplet and small particle aerosol) routes, but the quantitative dynamics and relative contributions of these routes are incompletely understood. The transmissibility of influenza strains estimated from secondary attack rates in closed human populations is confounded by large variations in population susceptibilities. An experimental method to phenotype strains for transmissibility in an animal model could provide relative efficiencies of transmission. We developed an experimental method to detect exhaled viral aerosol transmission between unanesthetized infected and susceptible ferrets, measured aerosol particle size and number, and quantified the viral genomic RNA in the exhaled aerosol. During brief 3-hour exposures to exhaled viral aerosols in airflow-controlled chambers, three strains of pandemic 2009 H1N1 strains were frequently transmitted to susceptible ferrets. In contrast one seasonal H1N1 strain was not transmitted in spite of higher levels of viral RNA in the exhaled aerosol. Among three pandemic strains, the two strains causing weight loss and illness in the intranasally infected 'donor' ferrets were transmitted less efficiently from the donor than the strain causing no detectable illness, suggesting that the mucosal inflammatory response may attenuate viable exhaled virus. Although exhaled viral RNA remained constant, transmission efficiency diminished from day 1 to day 5 after donor infection. Thus, aerosol transmission between ferrets may be dependent on at least four characteristics of virus-host relationships including the level of exhaled virus, infectious particle size, mucosal inflammation, and viral replication efficiency in susceptible mucosa.     

Mapping the dynamics of shear stress-induced structural changes in endothelial cells

Hemodynamic shear stress regulates endothelial cell biochemical processes that govern cytoskeletal contractility, focal adhesion dynamics, and extracellular matrix (ECM) assembly. Since shear stress causes rapid strain focusing at discrete locations in the cytoskeleton, we hypothesized that shear stress coordinately alters structural dynamics in the cytoskeleton, focal adhesion sites, and ECM on a time scale of minutes. Using multiwavelength four-dimensional fluorescence microscopy, we measured the displacement of rhodamine-fibronectin and green fluorescent protein-labeled actin, vimentin, paxillin, and/or vinculin in aortic endothelial cells before and after onset of steady unidirectional shear stress. In the cytoskeleton, the onset of shear stress increased actin polymerization into lamellipodia, altered the angle of lateral displacement of actin stress fibers and vimentin filaments, and decreased centripetal remodeling of actin stress fibers in subconfluent and confluent cell layers. Shear stress induced the formation of new focal complexes and reduced the centripetal remodeling of focal adhesions in regions of new actin polymerization. The structural dynamics of focal adhesions and the fibronectin matrix varied with cell density. In subconfluent cell layers, shear stress onset decreased the displacement of focal adhesions and fibronectin fibrils. In confluent monolayers, the direction of fibronectin and focal adhesion displacement shifted significantly toward the downstream direction within 1 min after onset of shear stress. These spatially coordinated rapid changes in the structural dynamics of cytoskeleton, focal adhesions, and ECM are consistent with focusing of mechanical stress and/or strain near major sites of shear stress-mediated mechanotransduction.     

H9N2型禽流感病毒传播途径分子机制的研究

选择禽流感病毒(avian influenza virus,AIV)A/chicken/Zhuhai/154/2003(H9N2)(AZHl54)株作为骨架病毒,与来自A/chicken/Guangdong/SS/94(H9N2)(SS94)AIV的NA基因和HA基因进行H9N2亚型重排.动物传播性实验发现:AZH154、SS94和3株重排的H9N2亚型AIV都可以经直接接触途径传播;5株H9N2 AIV都不经过粪便接触传播;且AZH154株和重组的H9N2亚型AZH154/SSHA能经过气溶胶传播.实验结果表明:AIV(H9N2)的NA基因与该病毒气溶胶传播途径有重要关系,即2003年珠海地区AIV(H9N2)的大流行可能是因为病毒获得气溶胶传播途径的特性,且病毒的NA基因发挥了重要的作用.