Volatile Compound Markers in Beef Irradiated with Accelerated Electrons.

This study focuses on the behavior of volatile organic compounds in beef after irradiation with 1 MeV accelerated electrons with doses ranging from 0.25 kGy to 5 kGy to find reliable dose-dependent markers that could be used for establishing an effective dose range for beef irradiation. GC/MS analysis revealed that immediately after irradiation, the chemical yield and accumulation rate of lipid oxidation-derived aldehydes was higher than that of protein oxidation-derived aldehydes. The nonlinear dose-dependent relationship of the concentration of volatile organic compounds was explained using a mathematical model based on the simultaneous occurrence of two competing processes: decomposition of volatile compounds due to direct and indirect action of accelerated electrons, and accumulation of volatile compounds due to decomposition of other compounds and biomacromolecules. A four-day monitoring of the beef samples stored at 4 °C showed that lipid oxidation-derived aldehydes, protein oxidation-derived aldehydes and alkanes as well as alcohol ethanol as an indicator of bacterial activity were dose-dependent markers of biochemical processes occurring in the irradiated beef samples during storage: oxidative processes during direct and indirect action of irradiation, oxidation due to the action of reactive oxygen species, which are always present in the product during storage, and microbial-enzymatic processes. According to the mathematical model of the change in the concentrations of lipid oxidation-derived aldehydes over time in the beef samples irradiated with different doses, it was found that doses ranging from 0.25 kGy to 1 kGy proved to be most effective for beef irradiation with accelerated electrons, since this dose range decreases the bacterial content without considerable irreversible changes in chemical composition of chilled beef during storage.

Stages of NETosis Development upon Stimulation of Neutrophils with Activators of Different Types.

Before NETs are released, the neutrophil undergoes structural changes. First, it flattens, accompanied by a change in cell shape and rearrangement of the cytoskeleton. Then, nuclear swelling begins, which ends with the ejection of NETs into the extracellular space. We used widefield and confocal fluorescence microscopy to register morphological and structural changes in neutrophils during activation and NETosis. Different types of activators were used, such as NOX-dependent PMA and calcium ionophore A23187. The measurements were performed in a series of sequential stages. In the first stage (30 s after addition of activators and immediately after stimulation of neutrophils), the response of neutrophils to A23187 and PMA exposure was studied. Subsequently, the characteristics of neutrophils in different phases of activation were examined over a longer period of time (30, 60, 120, 180, and 240 min). The specific features of NETosis development were analyzed separately. During the first 30 s, neutrophils appeared to be heterogeneous in shape and structure of the actin cytoskeleton. Characteristic cell shapes included 30″ type 1 cells, similar in shape to the control, with F-actin concentrated in the center of the cytoplasm, and 30″ type 2 cells, which had flattened (spread) shapes with increased frontal dimensions and F-actin distributed throughout the cell. Later, the development of nuclear swelling, the corresponding changes in neutrophil membranes, and NET release into the extracellular space were evaluated. The conditions determining the initiation of chromatin ejection and two characteristic types of decondensed chromatin ejection were revealed. The results obtained contribute to a better understanding of the biophysical mechanisms of neutrophil activation and NETosis development.

Atomic Force Microscopy and High-Resolution Spectrophotometry for Study of Anoxemia and Normoxemia in Model Experiment In Vitro.

The oxygen content in the blood may decrease under the influence of various physicochemical factors and different diseases. The state of hypoxemia is especially dangerous for critically ill patients. In this paper, we describe and analyze the changes in the characteristics of red blood cells (RBCs) with decreasing levels of oxygen in the RBC suspension from normoxemia to hypoxemia/anoxemia in an in vitro model experiment. The RBCs were stored in hypoxemia/anoxemia and normoxemia conditions in closed and open tubes correspondingly. For the quantitative study of RBC parameter changes, we used atomic force microscopy, digital spectrophotometry, and nonlinear curve fitting of the optical spectra. In both closed and open tubes, at the end of the storage period by day 29, only 2% of discocytes remained, and mainly irreversible types, such as microspherocytes and ghosts, were observed. RBC hemolysis occurred at a level of 25-30%. Addition of the storage solution, depending on the concentration, changed the influence of hypoxemia on RBCs. The reversibility of the change in hemoglobin derivatives was checked. Based on the experimental data and model approach, we assume that there is an optimal level of hypoxemia at which the imbalance between the oxidative and antioxidant systems, the rate of formation of reactive oxygen species, and, accordingly, the disturbances in RBCs, will be minimal.

Mechanochemical Synergism of Reactive Oxygen Species Influences on RBC Membrane.

The influences of various factors on blood lead to the formation of extra reactive oxygen species (ROS), resulting in the disruption of morphology and functions of red blood cells (RBCs). This study considers the mechanisms of the mechanochemical synergism of OH• free radicals, which are most active in the initiation of lipid peroxidation (LPO) in RBC membranes, and H2O2 molecules, the largest typical diffusion path. Using kinetic models of differential equations describing CH2O2t and COH•t, we discuss two levels of mechanochemical synergism that occur simultaneously: (1) synergism that ensures the delivery of highly active free radicals OH• to RBC membranes and (2) a positive feedback system between H2O2 and OH•, resulting in the partial restoration of spent molecules. As a result of these ROS synergisms, the efficiency of LPO in RBC membranes sharply increases. In blood, the appearance of OH• free radicals is due to the interaction of H2O2 molecules with free iron ions (Fe2+) which arise as a result of heme degradation. We experimentally established the quantitative dependences of COH• CH2O2 using the methods of spectrophotometry and nonlinear curve fitting. This study extends the analysis of the influence of ROS mechanisms in RBC suspensions.

Hemoglobin Derivatives in Beef Irradiated with Accelerated Electrons.

The efficiency of food irradiation depends on the accuracy of the irradiation dose range that is sufficient for inhibiting microbiological growth without causing an irreversible change to the physical and chemical properties of foods. This study suggests that the concentration of hemoglobin derivatives can be used as a criterion for establishing the limit for chilled beef irradiation at which irradiation-induced oxidation becomes irreversible. The express spectrophotometry method for estimating the hemoglobin derivative concentration shows a nonlinear increase in methemoglobin concentration from 15% to 50% in beef irradiated by accelerated electrons with the doses ranging from 250 Gy to 10,000 Gy. The monitoring of the hemoglobin derivative concentration for three days after irradiation shows nonmonotonous dependencies of methemoglobin concentration in beef in the storage time since the oxidation of hemoglobin occur as a result of irradiation and biochemical processes in beef during storage. The proposed method based on the quantitative analysis of the hemoglobin derivative concentration can be used to estimate the oxidation level for irradiation of foods containing red blood cells. The study proposes a model that describes the change in hemoglobin derivative concentration in beef after irradiation considering that oxidation of hemoglobin can be triggered by the direct ionization caused by accelerated electrons, biochemical processes as a result of bacterial activity, and reactive oxygen species appearing during irradiation and storage. This research throws light on the mechanisms behind food irradiation during storage that should be taken into account for selecting the optimal parameters of irradiation.

Persistent autism-relevant behavioral phenotype and social neuropeptide alterations in female mice offspring induced by maternal transfer of PBDE congeners in the commercial mixture DE-71.

Polybrominated diphenyl ethers (PBDEs) are ubiquitous persistent organic pollutants (POPs) that are known neuroendocrine disrupting chemicals with adverse neurodevelopmental effects. PBDEs may act as risk factors for autism spectrum disorders (ASD), characterized by abnormal psychosocial functioning, although direct evidence is currently lacking. Using a translational exposure model, we tested the hypothesis that maternal transfer of a commercial mixture of PBDEs, DE-71, produces ASD-relevant behavioral and neurochemical deficits in female offspring. C57Bl6/N mouse dams (F0) were exposed to DE-71 via oral administration of 0 (VEH/CON), 0.1 (L-DE-71) or 0.4 (H-DE-71) mg/kg bw/d from 3 wk prior to gestation through end of lactation. Mass spectrometry analysis indicated in utero and lactational transfer of PBDEs (in ppb) to F1 female offspring brain tissue at postnatal day (PND) 15 which was reduced by PND 110. Neurobehavioral testing of social novelty preference (SNP) and social recognition memory (SRM) revealed that adult L-DE-71 F1 offspring display deficient short- and long-term SRM, in the absence of reduced sociability, and increased repetitive behavior. These effects were concomitant with reduced olfactory discrimination of social odors. Additionally, L-DE-71 exposure also altered short-term novel object recognition memory but not anxiety or depressive-like behavior. Moreover, F1 L-DE-71 displayed downregulated mRNA transcripts for oxytocin (Oxt) in the bed nucleus of the stria terminalis (BNST) and supraoptic nucleus, and vasopressin (Avp) in the BNST and upregulated Avp1ar in BNST, and Oxtr in the paraventricular nucleus. Our work demonstrates that developmental PBDE exposure produces ASD-relevant neurochemical, olfactory processing and behavioral phenotypes that may result from early neurodevelopmental reprogramming within central social and memory networks.

Topological Relationships Cytoskeleton-Membrane Nanosurface-Morphology as a Basic Mechanism of Total Disorders of RBC Structures.

The state of red blood cells (RBCs) and their functional possibilities depend on the structural organization of the membranes. Cell morphology and membrane nanostructure are compositionally and functionally related to the cytoskeleton network. In this work, the influence of agents (hemin, endogenous oxidation during storage of packed RBCs, ultraviolet (UV) radiation, temperature, and potential of hydrogen (pH) changes) on the relationships between cytoskeleton destruction, membrane nanostructure, and RBC morphology was observed by atomic force microscope. It was shown that the influence of factors of a physical and biochemical nature causes structural rearrangements in RBCs at all levels of organization, forming a unified mechanism of disturbances in relationships "cytoskeleton-membrane nanosurface-cell morphology". Filament ruptures and, consequently, large cytoskeleton pores appeared. The pores caused membrane topological defects in the form of separate grain domains. Increasing loading doses led to an increase in the number of large cytoskeleton pores and defects and their fusion at the membrane nanosurfaces. This caused the changes in RBC morphology. Our results can be used in molecular cell biology, membrane biophysics, and in fundamental and practical medicine.

Towards 3D Bioprinted Spinal Cord Organoids.

Three-dimensional (3D) cultures, so-called organoids, have emerged as an attractive tool for disease modeling and therapeutic innovations. Here, we aim to determine if boundary cap neural crest stem cells (BC) can survive and differentiate in gelatin-based 3D bioprinted bioink scaffolds in order to establish an enabling technology for the fabrication of spinal cord organoids on a chip. BC previously demonstrated the ability to support survival and differentiation of co-implanted or co-cultured cells and supported motor neuron survival in excitotoxically challenged spinal cord slice cultures. We tested different combinations of bioink and cross-linked material, analyzed the survival of BC on the surface and inside the scaffolds, and then tested if human iPSC-derived neural cells (motor neuron precursors and astrocytes) can be printed with the same protocol, which was developed for BC. We showed that this protocol is applicable for human cells. Neural differentiation was more prominent in the peripheral compared to central parts of the printed construct, presumably because of easier access to differentiation-promoting factors in the medium. These findings show that the gelatin-based and enzymatically cross-linked hydrogel is a suitable bioink for building a multicellular, bioprinted spinal cord organoid, but that further measures are still required to achieve uniform neural differentiation.

Molecular genetic analysis of neural stem cells after space flight and simulated microgravity on earth.

Understanding how stem cells adapt to space flight conditions is fundamental for human space missions and extraterrestrial settlement. We analyzed gene expression in boundary cap neural crest stem cells (BCs), which are attractive for regenerative medicine by their ability to promote proliferation and survival of cocultured and co-implanted cells. BCs were launched to space (space exposed cells) (SEC), onboard sounding rocket MASER 14 as free-floating neurospheres or in a bioprinted scaffold. For comparison, BCs were placed in a random positioning machine (RPM) to simulate microgravity on earth (RPM cells) or were cultured under control conditions in the laboratory. Using next-generation RNA sequencing and data post-processing, we discovered that SEC upregulated genes related to proliferation and survival, whereas RPM cells upregulated genes associated with differentiation and inflammation. Thus, (i) space flight provides unique conditions with distinctly different effects on the properties of BC compared to earth controls, and (ii) the space flight exposure induces postflight properties that reinforce the utility of BC for regenerative medicine and tissue engineering.

The relationship of membrane stiffness, cytoskeleton structure and storage time of pRBCs.

BACKGROUND AND OBJECTIVES: In clinical practice, it has been shown that transfusion of packed red blood cells (pRBCs) with late shelf life increases the risk of post-transfusion complications. OBJECTIVE: To study relationship of membrane stiffness, cytoskeleton structure and storage time of pRBCs. MATERIALS AND METHODS: pRBCs were processed and stored according to blood bank procedure, for 42 days, at +4°C; pRBC samples were taken on days 3, 12, 19, 21, 24, 28, 35 and 42. Cytoskeleton images and membrane stiffness were studied using atomic force microscope. RESULTS: In the course of the pRBC storage, the cytoskeleton network configuration underwent structural changes. Simultaneously, pRBC membrane stiffness was increasing, with the correlation coefficient 0·88. Until 19 days, the stiffness grew slowly, in 19-24 days there occurred a transition period, after which its growth rate was three times higher than the initial. A chain of pathological processes developed in pRBC during long storage: pH reduction (linked to increased oxidative stress), then cytoskeletal destruction and an associated increase in pRBC membrane stiffness. CONCLUSION: During prolonged storage of pRBCs and their acidification, there is a progression of pRBC cytoskeletal changes and associated increase of membrane stiffness, observed to increase in rate after days 19-24. Mutual measurements of cytoskeletal integrity and membrane stiffness may be useful quality assessment tool to study the molecular mechanisms of RBC structural degradation during storage.

Spin-Orbital Conversion of a Strongly Focused Light Wave with High-Order Cylindrical-Circular Polarization.

We discuss interesting effects that occur when strongly focusing light with mth-order cylindrical-circular polarization. This type of hybrid polarization combines properties of the mth-order cylindrical polarization and circular polarization. Reluing on the Richards-Wolf formalism, we deduce analytical expressions that describe E- and H-vector components, intensity patterns, and projections of the Poynting vector and spin angular momentum (SAM) vector at the strong focus. The intensity of light in the strong focus is theoretically and numerically shown to have an even number of local maxima located along a closed contour centered at an on-axis point of zero intensity. We show that light generates 4m vortices of a transverse energy flow, with their centers located between the local intensity maxima. The transverse energy flow is also shown to change its handedness an even number of times proportional to the order of the optical vortex via a full circle around the optical axis. It is interesting that the longitudinal SAM projection changes its sign at the focus 4m times. The longitudinal SAM component is found to be positive, and the polarization vector is shown to rotate anticlockwise in the focal spot regions where the transverse energy flow rotates anticlockwise, and vice versa-the longitudinal SAM component is negative and the polarization vector rotates clockwise in the focal spot regions where the transverse energy flow rotates clockwise. This spatial separation at the focus of left and right circularly polarized light is a manifestation of the optical spin Hall effect. The results obtained in terms of controlling the intensity maxima allow the transverse mode analysis of laser beams in sensorial applications. For a demonstration of the proposed application, the metalens is calculated, which can be a prototype for an optical microsensor based on sharp focusing for measuring roughness.

Tight focusing of a cylindrical vector beam by a hyperbolic secant gradient index lens.

In this Letter, we investigate the tight focusing of a second-order cylindrical vector beam by a hyperbolic secant gradient index lens with a thickness of 10 µm, a radius of 9.43 µm, and a refractive index on the axis of 3.47 (silicon). It is shown that the lens forms the reverse energy flow near its shadow surface. Moreover, it was obtained that the spherical hole in the center of the shadow plane with a diameter of 0.3 µm allows us to localize the direct energy flow inside the lens material and with the reverse energy flow in an area of free space.

Extracellular Vesicles as Innovative Tool for Diagnosis, Regeneration and Protection against Neurological Damage.

Extracellular vesicles (EVs) have recently attracted a great deal of interest as they may represent a new biosignaling paradigm. According to the mode of biogenesis, size and composition, two broad categories of EVs have been described, exosomes and microvesicles. EVs have been shown to carry cargoes of signaling proteins, RNA species, DNA and lipids. Once released, their content is selectively taken up by near or distant target cells, influencing their behavior. Exosomes are involved in cell-cell communication in a wide range of embryonic developmental processes and in fetal-maternal communication. In the present review, an outline of the role of EVs in neural development, regeneration and diseases is presented. EVs can act as regulators of normal homeostasis, but they can also promote either neuroinflammation/degeneration or tissue repair in pathological conditions, depending on their content. Since EV molecular cargo constitutes a representation of the origin cell status, EVs can be exploited in the diagnosis of several diseases. Due to their capability to cross the blood-brain barrier (BBB), EVs not only have been suggested for the diagnosis of central nervous system disorders by means of minimally invasive procedures, i.e., "liquid biopsies", but they are also considered attractive tools for targeted drug delivery across the BBB. From the therapeutic perspective, mesenchymal stem cells (MSCs) represent one of the most promising sources of EVs. In particular, the neuroprotective properties of MSCs derived from the dental pulp are here discussed.

Relations of child effortful control to personality, well-being and parenting.

This study examined effortful control and its relations to personality, parenting and well-being in a community sample of Russian preschool children (N = 365, 46% girls) using parent-reported effortful control scale from the very short form of the children's behaviour questionnaire, the inventory of child individual differences-short version, the Alabama parenting questionnaire-preschool revision, the self-reporting questionnaire, and parent and teacher reports on the strengths and difficulties questionnaire. The findings confirmed the four-factor structure of effortful control, including inhibitory control, attentional control, low-intensity pleasure and perceptual sensitivity. Girls demonstrated higher scores than boys on effortful control, perceptual sensitivity, inhibitory control and low-intensity pleasure. Older children scored higher than younger on inhibitory control and perceptual sensitivity and lower on low-intensity pleasure. Gender and age accounted for less than 3% of the variance in effortful control. Effortful control was associated with personality traits of conscientiousness, agreeableness and openness, and with positive emotions and low-negative affect. Higher effortful control was associated with higher levels of prosocial behaviour and lower levels of externalising and internalising problems. Structural modelling showed that the effect of parenting on child prosocial and problem behaviours may be mediated by effortful control.

Orbital angular momentum of a laser beam behind an off-axis spiral phase plate.

Here we obtain a simple formula for the relative total orbital angular momentum (OAM) of a paraxial light beam with arbitrary rotationally symmetric complex amplitude, passed through an off-axis spiral phase plate (SPP). The formula shows that the OAM is zero if the incident light is bounded by a diaphragm, and the SPP center is outside it. Another consequence of the obtained formula is that if the incident light is limited by an annular diaphragm then the full angular momentum is the same, regardless of the position of the SPP center inside the shaded circle of the annular diaphragm. This means that it is more appropriate to illuminate the SPP by beams with an annular intensity distribution because, in this case, the inaccurate alignment of the SPP center and the center of the annular intensity distribution do not affect the total OAM of the beam. In addition, we obtain a formula for the OAM density of such a light beam in the initial plane. The experimentally measured OAM is in good agreement with that calculated by the obtained formula.

Cross-talk among flesh-eating Aeromonas hydrophila strains in mixed infection leading to necrotizing fasciitis.

Necrotizing fasciitis (NF) caused by flesh-eating bacteria is associated with high case fatality. In an earlier study, we reported infection of an immunocompetent individual with multiple strains of Aeromonas hydrophila (NF1-NF4), the latter three constituted a clonal group whereas NF1 was phylogenetically distinct. To understand the complex interactions of these strains in NF pathophysiology, a mouse model was used, whereby either single or mixed A. hydrophila strains were injected intramuscularly. NF2, which harbors exotoxin A (exoA) gene, was highly virulent when injected alone, but its virulence was attenuated in the presence of NF1 (exoA-minus). NF1 alone, although not lethal to animals, became highly virulent when combined with NF2, its virulence augmented by cis-exoA expression when injected alone in mice. Based on metagenomics and microbiological analyses, it was found that, in mixed infection, NF1 selectively disseminated to mouse peripheral organs, whereas the other strains (NF2, NF3, and NF4) were confined to the injection site and eventually cleared. In vitro studies showed NF2 to be more effectively phagocytized and killed by macrophages than NF1. NF1 inhibited growth of NF2 on solid media, but ExoA of NF2 augmented virulence of NF1 and the presence of NF1 facilitated clearance of NF2 from animals either by enhanced priming of host immune system or direct killing via a contact-dependent mechanism.

Vaginal ecosystem modeling of growth patterns of anaerobic bacteria in microaerophilic conditions.

The human vagina constitutes a complex ecosystem created through relationships established between host mucosa and bacterial communities. In this ecosystem, classically defined bacterial aerobes and anaerobes thrive as communities in the microaerophilic environment. Levels of CO2 and O2 present in the vaginal lumen are impacted by both the ecosystem's physiology and the behavior and health of the human host. Study of such complex relationships requires controlled and reproducible causational approaches that are not possible in the human host that, until recently, was the only place these bacterial communities thrived. To address this need we have utilized our ex vivo human vaginal mucosa culture system to support controlled, reproducible colonization by vaginal bacterial communities (VBC) collected from healthy, asymptomatic donors. Parallel vaginal epithelial cells (VEC)-VBC co-cultures were exposed to two different atmospheric conditions to study the impact of CO2 concentrations upon the anaerobic bacteria associated with dysbiosis and inflammation. Our data suggest that in the context of transplanted VBC, increased CO2 favored specific lactobacilli species defined as microaerophiles when grown as monocultures. In preliminary studies, the observed community changes also led to shifts in host VEC phenotypes with significant changes in the host transcriptome, including altered expression of select molecular transporter genes. These findings support the need for additional study of the environmental changes associated with behavior and health upon the symbiotic and adversarial relationships that are formed in microbial communities present in the human vaginal ecosystem.

High-throughput, signature-tagged mutagenic approach to identify novel virulence factors of Yersinia pestis CO92 in a mouse model of infection.

The identification of new virulence factors in Yersinia pestis and understanding their molecular mechanisms during an infection process are necessary in designing a better vaccine or to formulate an appropriate therapeutic intervention. By using a high-throughput, signature-tagged mutagenic approach, we created 5,088 mutants of Y. pestis strain CO92 and screened them in a mouse model of pneumonic plague at a dose equivalent to 5 50% lethal doses (LD50) of wild-type (WT) CO92. From this screen, we obtained 118 clones showing impairment in disseminating to the spleen, based on hybridization of input versus output DNA from mutant pools with 53 unique signature tags. In the subsequent screen, 20/118 mutants exhibited attenuation at 8 LD50 when tested in a mouse model of bubonic plague, with infection by 10/20 of the aforementioned mutants resulting in 40% or higher survival rates at an infectious dose of 40 LD50. Upon sequencing, six of the attenuated mutants were found to carry interruptions in genes encoding hypothetical proteins or proteins with putative functions. Mutants with in-frame deletion mutations of two of the genes identified from the screen, namely, rbsA, which codes for a putative sugar transport system ATP-binding protein, and vasK, a component of the type VI secretion system, were also found to exhibit some attenuation at 11 or 12 LD50 in a mouse model of pneumonic plague. Likewise, among the remaining 18 signature-tagged mutants, 9 were also attenuated (40 to 100%) at 12 LD50 in a pneumonic plague mouse model. Previously, we found that deleting genes encoding Braun lipoprotein (Lpp) and acyltransferase (MsbB), the latter of which modifies lipopolysaccharide function, reduced the virulence of Y. pestis CO92 in mouse models of bubonic and pneumonic plague. Deletion of rbsA and vasK genes from either the Δlpp single or the Δlpp ΔmsbB double mutant augmented the attenuation to provide 90 to 100% survivability to mice in a pneumonic plague model at 20 to 50 LD50. The mice infected with the Δlpp ΔmsbB ΔrbsA triple mutant at 50 LD50 were 90% protected upon subsequent challenge with 12 LD50 of WT CO92, suggesting that this mutant or others carrying combinational deletions of genes identified through our screen could potentially be further tested and developed into a live attenuated plague vaccine(s).

Neural crest stem cells from hair follicles and boundary cap have different effects on pancreatic islets in vitro.

PURPOSE: Neural crest stem cells derived from the boundary cap (bNCSCs), markedly promote survival, proliferation and function of insulin producing ß-cells in vitro and in vivo after coculture/transplantation with pancreatic islets [ 1, 2 ]. Recently, we have shown that beneficial effects on ß-cells require cadherin contacts between bNCSCs and ß-cells [ 3, 4 ]. Here we investigated whether hair follicle (HF) NCSCs, a potential source for human allogeneic transplantation, exert similar positive effects on ß-cells. MATERIALS AND METHODS: We established cocultures of HF-NCSCs or bNCSCs from mice expressing enhanced green fluorescent protein together with pancreatic islets from DxRed expressing mice or NMRI mice and compared their migration towards islet cells and effect on proliferation of ß-cells as well as intracellular relations between NCSCs and islets using qRT-PCR analysis and immunohistochemistry. RESULTS: Whereas both types of NCSCs migrated extensively in the presence of islets, only bNCSCs demonstrated directed migration toward islets, induced ß-cell proliferation and increased the presence of cadherin at the junctions between bNCSCs and ß-cells. Even in direct contact between ß-cells and HF-NCSCs, no cadherin expression was detected. CONCLUSIONS: These observations indicate that HF-NCSCs do not confer the same positive effect on ß-cells as demonstrated for bNCSCs. Furthermore, these data suggest that induction of cadherin expression by HF-NCSCs may be useful for their ability to support ß-cells in coculture and after transplantation.

Deletion of Braun lipoprotein and plasminogen-activating protease-encoding genes attenuates Yersinia pestis in mouse models of bubonic and pneumonic plague.

Currently, there is no FDA-approved vaccine against Yersinia pestis, the causative agent of bubonic and pneumonic plague. Since both humoral immunity and cell-mediated immunity are essential in providing the host with protection against plague, we developed a live-attenuated vaccine strain by deleting the Braun lipoprotein (lpp) and plasminogen-activating protease (pla) genes from Y. pestis CO92. The Δlpp Δpla double isogenic mutant was highly attenuated in evoking both bubonic and pneumonic plague in a mouse model. Further, animals immunized with the mutant by either the intranasal or the subcutaneous route were significantly protected from developing subsequent pneumonic plague. In mice, the mutant poorly disseminated to peripheral organs and the production of proinflammatory cytokines concurrently decreased. Histopathologically, reduced damage to the lungs and livers of mice infected with the Δlpp Δpla double mutant compared to the level of damage in wild-type (WT) CO92-challenged animals was observed. The Δlpp Δpla mutant-immunized mice elicited a humoral immune response to the WT bacterium, as well as to CO92-specific antigens. Moreover, T cells from mutant-immunized animals exhibited significantly higher proliferative responses, when stimulated ex vivo with heat-killed WT CO92 antigens, than mice immunized with the same sublethal dose of WT CO92. Likewise, T cells from the mutant-immunized mice produced more gamma interferon (IFN-γ) and interleukin-4. These animals had an increasing number of tumor necrosis factor alpha (TNF-α)-producing CD4(+) and CD8(+) T cells than WT CO92-infected mice. These data emphasize the role of TNF-α and IFN-γ in protecting mice against pneumonic plague. Overall, our studies provide evidence that deletion of the lpp and pla genes acts synergistically in protecting animals against pneumonic plague, and we have demonstrated an immunological basis for this protection.