Efficient,specific, developmentally appropriate cre‐mediated recombination in anterior pituitary gonadotropes and thyrotropes

Tissue‐specific expression of cre recombinase is a well‐established genetic tool to analyze gene function, and it is limited only by the efficiency and specificity of available cre mouse strains. Here, we report the generation of a transgenic line containing a cre cassette with codon usage optimized for mammalian cells (iCre) under the control of a mouse glycoprotein hormone α‐subunit (αGSU) regulatory sequences in a bacterial artificial chromosome genomic clone. Initial analysis of this transgenic line, Tg(αGSU‐iCre), with cre reporter strains reveals onset of cre activity in the differentiating cells of the developing anterior pituitary gland at embryonic day 12.5, with a pattern characteristic of endogenous αGSU. In adult mice, αGSU‐iCre was active in the anterior lobe of the pituitary gland and in the cells that produce αGSU (gonadotropes and thyrotropes) with high penetrance. Little or no activity was observed in other tissues, including skeletal and cardiac muscle, brain, kidney, lungs, testis, ovary, and liver. This αGSU‐iCre line is suitable for efficient, specific, and developmentally regulated deletion of floxed alleles in anterior pituitary gonadotropes and thyrotropes. genesis 51:785–792. © 2013 Wiley Periodicals, Inc.     

Endothelial Cell Permeability during Hantavirus Infection Involves Factor XII-Dependent Increased Activation of the Kallikrein-Kinin System

Hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) are diseases caused by hantavirus infections and are characterized by vascular leakage due to alterations of the endothelial barrier. Hantavirus-infected endothelial cells (EC) display no overt cytopathology; consequently, pathogenesis models have focused either on the influx of immune cells and release of cytokines or on increased degradation of the adherens junction protein, vascular endothelial (VE)-cadherin, due to hantavirus-mediated hypersensitization of EC to vascular endothelial growth factor (VEGF). To examine endothelial leakage in a relevant in vitro system, we co-cultured endothelial and vascular smooth muscle cells (vSMC) to generate capillary blood vessel-like structures. In contrast to results obtained in monolayers of cultured EC, we found that despite viral replication in both cell types as well as the presence of VEGF, infected in vitro vessels neither lost integrity nor displayed evidence of VE-cadherin degradation. Here, we present evidence for a novel mechanism of hantavirus-induced vascular leakage involving activation of the plasma kallikrein-kinin system (KKS). We show that incubation of factor XII (FXII), prekallikrein (PK), and high molecular weight kininogen (HK) plasma proteins with hantavirus-infected EC results in increased cleavage of HK, higher enzymatic activities of FXIIa/kallikrein (KAL) and increased liberation of bradykinin (BK). Measuring cell permeability in real-time using electric cell-substrate impedance sensing (ECIS), we identified dramatic increases in endothelial cell permeability after KKS activation and liberation of BK. Furthermore, the alterations in permeability could be prevented using inhibitors that directly block BK binding, the activity of FXIIa, or the activity of KAL. Lastly, FXII binding and autoactivation is increased on the surface of hantavirus-infected EC. These data are the first to demonstrate KKS activation during hantavirus infection and could have profound implications for treatment of hantavirus infections.     

In Vivo Adaptation and Persistence of Neisseria meningitidis within the Nasopharyngeal Mucosa

Neisseria meningitidis (Nme) asymptomatically colonizes the human nasopharynx, yet can initiate rapidly-progressing sepsis and meningitis in rare instances. Understanding the meningococcal lifestyle within the nasopharyngeal mucosa, a phase of infection that is prerequisite for disease, has been hampered by the lack of animal models. Herein, we compare mice expressing the four different human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) that can bind the neisserial Opa protein adhesins, and find that expression of human CEACAM1 is necessary and sufficient to establish intranasal colonization. During infection, in vivo selection for phase variants expressing CEACAM1-specific Opa proteins occurs, allowing mucosal attachment and entry into the subepithelial space. Consistent with an essential role for Opa proteins in this process, Opa-deficient meningococci were unable to colonize the CEACAM1-humanized mice. While simple Opa-mediated attachment triggered an innate response regardless of meningococcal viability within the inoculum, persistence of viable Opa-expressing bacteria within the CEACAM1-humanized mice was required for a protective memory response to be achieved. Parenteral immunization with a capsule-based conjugate vaccine led to the accumulation of protective levels of Nme-specific IgG within the nasal mucus, yet the sterilizing immunity afforded by natural colonization was instead conferred by Nme-specific IgA without detectable IgG. Considered together, this study establishes that the availability of CEACAM1 helps define the exquisite host specificity of this human-restricted pathogen, displays a striking example of in vivo selection for the expression of desirable Opa variants, and provides a novel model in which to consider meningococcal infection and immunity within the nasopharyngeal mucosa.     

Fathers' Influence on Their Children's Cognitive and Emotional Development: From Toddlers to Pre-K

The Color of My Skin is an instrument developed to assess children's internalized idea (abstraction) of the color of their skin; their satisfaction with that color; the desire, if any, to change the color of their skin; and their affect regarding their skin color. The assessment is part of a questionnaire utilized in a 3-year longitudinal study that examines psychosocial development, physical health, and behavioral adjustment of Puerto Rican children (N = 257) reared in the Greater Boston area. The results demonstrate that children's internalized representation of their skin color is a construct that can be reliably and validly measured. The children's ratings of their skin color were not associated with their sex, school grade, ethnic identity, the child's or the parent's nativity, or the racial or ethnic compositions of 3 social contexts: their neighborhood, their classmates, and their closest friends. Puerto Rican children did not show a preference for light-colored skin. Moreover, there were no significant differences in self-esteem based on the child's self-reported skin color. The lack of association between self-esteem and skin color was interpreted in light of a developmental tendency prevalent in early to middle childhood to place a positive value on different aspects of one's self. Whereas almost all children (96%) reported being happy or very happy with their color, 16% of the children would like to change their skin color if they could (51% to a lighter and 46% to a darker color).     

Linking anti‐predator behaviour to prey demography reveals limited risk effects of an actively hunting large carnivore

Ecological theory predicts that the diffuse risk cues generated by wide‐ranging, active predators should induce prey behavioural responses but not major, population‐ or community‐level consequences. We evaluated the non‐consumptive effects (NCEs) of an active predator, the grey wolf (Canis lupus), by simultaneously tracking wolves and the behaviour, body fat, and pregnancy of elk (Cervus elaphus), their primary prey in the Greater Yellowstone Ecosystem. When wolves approached within 1 km, elk increased their rates of movement, displacement and vigilance. Even in high‐risk areas, however, these encounters occurred only once every 9 days. Ultimately, despite 20‐fold variation in the frequency of encounters between wolves and individual elk, the risk of predation was not associated with elk body fat or pregnancy. Our findings suggest that the ecological consequences of actively hunting large carnivores, such as the wolf, are more likely transmitted by consumptive effects on prey survival than NCEs on prey behaviour.     

Spatial and temporal mapping of the PfEMP1 export pathway in Plasmodium falciparum

The human malaria parasite, Plasmodium falciparum, modifies the red blood cells (RBCs) that it infects by exporting proteins to the host cell. One key virulence protein, P. falciparum Erythrocyte Membrane Protein‐1 (PfEMP1), is trafficked to the surface of the infected RBC, where it mediates adhesion to the vascular endothelium. We have investigated the organization and development of the exomembrane system that is used for PfEMP1 trafficking. Maurer's cleft cisternae are formed early after invasion and proteins are delivered to these (initially mobile) structures in a temporally staggered and spatially segregated manner. Membrane‐Associated Histidine‐Rich Protein‐2(MAHRP2)‐containing tether‐like structures are generated as early as 4 h post invasion and become attached to Maurer's clefts. The tether/Maurer's cleft complex docks onto the RBC membrane at ～ 20 h post invasion via a process that is not affected by cytochalasin D treatment. We have examined the trafficking of a GFP chimera of PfEMP1 expressed in transfected parasites. PfEMP1B‐GFP accumulates near the parasite surface, within membranous structures exhibiting a defined ultrastructure, before being transferred to pre‐formed mobile Maurer's clefts. Endogenous PfEMP1 and PfEMP1B‐GFP are associated with Electron‐Dense Vesicles that may be responsible for trafficking PfEMP1 from the Maurer's clefts to the RBC membrane.     

Infection of macrophages with Mycobacterium tuberculosis induces global modifications to phagosomal function

The phagosome is a central mediator of both the homeostatic and microbicidal functions of a macrophage. Following phagocytosis, Mycobacterium tuberculosis (Mtb) is able to establish infection through arresting phagosome maturation and avoiding the consequences of delivery to the lysosome. The infection of a macrophage by Mtb leads to marked changes in the behaviour of both the macrophage and the surrounding tissue as the bacterium modulates its environment to promote its survival. In this study, we use functional physiological assays to probe the biology of the phagosomal network in Mtb‐infected macrophages. The resulting data demonstrate that Mtb modifies phagosomal function in a TLR2/TLR4‐dependent manner, and that most of these modifications are consistent with an increase in the activation status of the cell. Specifically, superoxide burst is enhanced and lipolytic activity is decreased upon infection. There are some species‐ or cell type‐specific differences between human and murine macrophages in the rates of acidification and the degree of proteolysis. However, the most significant modification is the marked reduction in intra‐phagosomal lipolysis because this correlates with the marked increase in the retention of host lipids in the infected macrophage, which provides a potential source of nutrients that can be accessed by Mtb.     

Epigenetic regulation by DNA methyltransferases during torpor in the thirteen-lined ground squirrel Ictidomys tridecemlineatus

Molecular and Cellular Biochemistry - The thirteen-lined ground squirrel, Ictidomys tridecemlineatus, is a mammal capable of lowering its Tb to almost 0 °C while undergoing deep...