Sex-related Differences in Stress Reactivity and Cingulum White Matter.

The prefrontal cortex and limbic system are important components of the neural circuit that underlies stress and anxiety. These brain regions are connected by white matter tracts that support neural communication including the cingulum, uncinate fasciculus, and the fornix/stria-terminalis. Determining the relationship between stress reactivity and these white matter tracts may provide new insight into factors that underlie stress susceptibility and resilience. Therefore, the present study investigated sex differences in the relationship between stress reactivity and generalized fractional anisotropy (GFA) of the white matter tracts that link the prefrontal cortex and limbic system. Diffusion weighted images were collected and deterministic tractography was completed in 104 young adults (55 men, 49 women; mean age = 18.87 SEM = 0.08). Participants also completed self-report questionnaires (e.g., Trait Anxiety) and donated saliva (later assayed for cortisol) before, during, and after the Trier Social Stress Test. Results revealed that stress reactivity (area under the curve increase in cortisol) and GFA of the cingulum bundle varied by sex. Specifically, men demonstrated greater cortisol reactivity and greater GFA within the cingulum than women. Further, an interaction between sex, stress reactivity, and cingulum GFA was observed in which men demonstrated a positive relationship while women demonstrated a negative relationship between GFA and cortisol reactivity. Finally, trait anxiety was positively associated with the GFA of the fornix/stria terminalis - the white matter pathways that connect the hippocampus/amygdala to the hypothalamus. These findings advance our understanding of factors that underlie individual differences in stress reactivity.

Functional Connectivity Network Disruption Underlies Domain-Specific Impairments in Attention for Children Born Very Preterm.

Attention problems are common in school-age children born very preterm (VPT; < 32 weeks gestational age), but the contribution of aberrant functional brain connectivity to these problems is not known. As part of a prospective longitudinal study, brain functional connectivity (fc) was assessed alongside behavioral measures of selective, sustained, and executive attention in 58 VPT and 65 full-term (FT) born children at corrected-age 12 years. VPT children had poorer sustained, shifting, and divided attention than FT children. Within the VPT group, poorer attention scores were associated with between-network connectivity in ventral attention, visual, and subcortical networks, whereas between-network connectivity in the frontoparietal, cingulo-opercular, dorsal attention, salience and motor networks was associated with attention functioning in FT children. Network-level differences were also evident between VPT and FT children in specific attention domains. Findings contribute to our understanding of fc networks that potentially underlie typical attention development and suggest an alternative network architecture may help support attention in VPT children.

Sex differences in functional connectivity during fetal brain development.

Sex-related differences in brain and behavior are apparent across the life course, but the exact set of processes that guide their emergence in utero remains a topic of vigorous scientific inquiry. Here, we evaluate sex and gestational age (GA)-related change in functional connectivity (FC) within and between brain wide networks. Using resting-state functional magnetic resonance imaging we examined FC in 118 human fetuses between 25.9 and 39.6 weeks GA (70 male; 48 female). Infomap was applied to the functional connectome to identify discrete prenatal brain networks in utero. A consensus procedure produced an optimal model comprised of 16 distinct fetal neural networks distributed throughout the cortex and subcortical regions. We used enrichment analysis to assess network-level clustering of strong FC-GA correlations separately in each sex group, and to identify network pairs exhibiting distinct patterns of GA-related change in FC between males and females. We discovered both within and between network FC-GA associations that varied with sex. Specifically, associations between GA and posterior cingulate-temporal pole and fronto-cerebellar FC were observed in females only, whereas the association between GA and increased intracerebellar FC was stronger in males. These observations confirm that sexual dimorphism in functional brain systems emerges during human gestation.

Altered functional network connectivity relates to motor development in children born very preterm.

Individuals born very preterm (<32 weeks gestation) are at increased risk for neuromotor impairments. The ability to characterize the structural and functional mechanisms underlying these impairments remains limited using existing neuroimaging techniques. Resting state-functional magnetic resonance imaging (rs-fMRI) holds promise for defining the functional network architecture of the developing brain in relation to typical and aberrant neurodevelopment. In 58 very preterm and 65 term-born children studied from birth to age 12 years, we examined relations between functional connectivity measures from low-motion rs-fMRI data and motor skills assessed using the Movement Assessment Battery for Children, 2nd edition. Across all subscales, motor performance was better in term than very preterm children. Examination of relations between functional connectivity and motor measures using enrichment analysis revealed between-group differences within cerebellar, frontoparietal, and default mode networks, and between basal ganglia-motor, thalamus-motor, basal ganglia-auditory, and dorsal attention-default mode networks. Specifically, very preterm children exhibited weaker associations between motor scores and thalamus-motor and basal ganglia-motor network connectivity. These findings highlight key functional brain systems underlying motor development. They also demonstrate persisting developmental effects of preterm birth on functional connectivity and motor performance in childhood, providing evidence for an alternative network architecture supporting motor function in preterm children.

Honey Bee Viruses in Wild Bees: Viral Prevalence, Loads, and Experimental Inoculation.

Evidence of inter-species pathogen transmission from managed to wild bees has sparked concern that emerging diseases could be causing or exacerbating wild bee declines. While some pathogens, like RNA viruses, have been found in pollen and wild bees, the threat these viruses pose to wild bees is largely unknown. Here, we tested 169 bees, representing 4 families and 8 genera, for five common honey bee (Apis mellifera) viruses, finding that more than 80% of wild bees harbored at least one virus. We also quantified virus titers in these bees, providing, for the first time, an assessment of viral load in a broad spectrum of wild bees. Although virus detection was very common, virus levels in the wild bees were minimal-similar to or lower than foraging honey bees and substantially lower than honey bees collected from hives. Furthermore, when we experimentally inoculated adults of two different bee species (Megachile rotundata and Colletes inaequalis) with a mixture of common viruses that is lethal to honey bees, we saw no effect on short term survival. Overall, we found that honey bee RNA viruses can be commonly detected at low levels in many wild bee species, but we found no evidence that these pathogens cause elevated short-term mortality effects. However, more work on these viruses is greatly needed to assess effects on additional bee species and life stages.

Defining the Insect Pollinator Community Found in Iowa Corn and Soybean Fields: Implications for Pollinator Conservation.

Although corn (Zea mays L.) and soybeans (Glycine max L.) do not require pollination, they offer floral resources used by insect pollinators. We asked if a similar community of insect pollinators visits these crops in central Iowa, a landscape dominated by corn and soybean production. We used modified pan traps (i.e., bee bowls) in both corn and soybean fields during anthesis and used nonmetric multidimensional scaling (NMS) to compare the communities found in the two crops. Summed across both crops, 6,704 individual insects were captured representing at least 60 species, morphospecies, or higher-level taxa. Thirty-four species were collected in both crops, 19 collected only in corn and seven were collected only in soybean. The most abundant taxa were Lasioglossum [Dialictus] spp., Agapostemon virescens Cresson, Melissodes bimaculata (Lepeletier), and Toxomerus marginatus (Say), which accounted for 65% of the insect pollinators collected from both crops. Although social bees (Apis mellifera L. and Bombus spp.) were found in both crops, they accounted for only 0.5% of all insects captured. The NMS analysis revealed a shared community of pollinators composed of mostly solitary, ground nesting bees. Many of these species have been found in other crop fields throughout North America. Although corn and soybean are grown in landscapes that are often highly disturbed, these data suggest that a community of pollinators can persist within them. We suggest approaches to conserving this community based on partnering with activities that aim to lessen the environmental impact of annual crop production.

Insect Pollinators in Iowa Cornfields: Community Identification and Trapping Method Analysis.

Availability of mass flowering plants in landscapes dominated by agriculture can have a strong positive impact on the density of generalist, native pollinators. Row-crop production in Iowa accounts for 75% of the arable acres, with corn, Zea mays, representing the majority of hectares planted. To date, there has been no description of the insect pollinator community found within Iowa cornfields. We report a field study to determine the optimal sampling methodology to characterize the community of insect pollinators within cornfields. During 2012 and 2013, 3,616 insect pollinators representing 51 species were captured using bee bowls, and 945 individuals representing 10 species were captured using sticky cards. We examined the effects of trap type, height, and bowl color on the described community. Bee bowls captured a more abundant and species rich community than sticky cards with all species captured on sticky cards also present in bee bowls. Traps deployed at the height of the tassels describe a more abundant and species rich community of pollinators than traps at ear height (2x as many individuals) or ground height (4x as many individuals). Blue bowls captured more bees than white (2.75x as many individuals) or yellow bowls (3.5x as many individuals); and yellow bowls captured more flies than white (2x as many individuals) or blue (2.3x as many individuals). To provide the most complete description of the community of insect pollinators using cornfields as a resource, we suggest sampling-using bee bowls at the height of the tassels using all three bee bowl colors.

Threat-related learning relies on distinct dorsal prefrontal cortex network connectivity.

Conditioned changes in the emotional response to threat (e.g. aversive unconditioned stimulus; UCS) are mediated in part by the prefrontal cortex (PFC). Unpredictable threats elicit large emotional responses, while the response is diminished when the threat is predictable. A better understanding of how PFC connectivity to other brain regions varies with threat predictability would provide important insights into the neural processes that mediate conditioned diminution of the emotional response to threat. The present study examined brain connectivity during predictable and unpredictable threat exposure using a fear conditioning paradigm (previously published in Wood et al., 2012) in which unconditioned functional magnetic resonance imaging data were reanalyzed to assess effective connectivity. Granger causality analysis was performed using the time series data from 15 activated regions of interest after hemodynamic deconvolution, to determine regional effective connectivity. In addition, connectivity path weights were correlated with trait anxiety measures to assess the relationship between negative affect and brain connectivity. Results indicate the dorsomedial PFC (dmPFC) serves as a neural hub that influences activity in other brain regions when threats are unpredictable. In contrast, the dorsolateral PFC (dlPFC) serves as a neural hub that influences the activity of other brain regions when threats are predictable. These findings are consistent with the view that the dmPFC coordinates brain activity to take action, perhaps in a reactive manner, when an unpredicted threat is encountered, while the dlPFC coordinates brain regions to take action, in what may be a more proactive manner, to respond to predictable threats. Further, dlPFC connectivity to other brain regions (e.g. ventromedial PFC, amygdala, and insula) varied with negative affect (i.e. trait anxiety) when the UCS was predictable, suggesting that stronger connectivity may be required for emotion regulation in individuals with higher levels of negative affect.

Expression of inappropriate cadherins by epithelial tumor cells promotes endocytosis and degradation of E-cadherin via competition for p120(ctn).

Cadherin cell-cell adhesion proteins play an important role in modulating the behavior of tumor cells. E-cadherin serves as a suppressor of tumor cell invasion, and when tumor cells turn on the expression of a non-epithelial cadherin, they often express less E-cadherin, enhancing the tumorigenic phenotype of the cells. Here, we show that when A431 cells are forced to express R-cadherin, they dramatically downregulate the expression of endogenous E- and P-cadherin. In addition, we show that this downregulation is owing to increased turnover of the endogenous cadherins via clathrin-dependent endocytosis. p120(ctn) binds to the juxtamembrane domain of classical cadherins and has been proposed to regulate cadherin adhesive activity. One way p120(ctn) may accomplish this is to serve as a rheostat to regulate the levels of cadherin. Here, we show that the degradation of E-cadherin in response to expression of R-cadherin is owing to competition for p120(ctn).

Cadherin junctions in mammary tumors.

Cadherins are the transmembrane component of adherens junctions found between interacting cells in tissues. The cadherins bind cells to one another in a specific manner and link to the actin cytoskeleton through intracellular catenins. In addition to promoting strong cell-cell adhesion, cadherins appear to initiate and modify intracellular signaling pathways. The loss of E-cadherin function in epithelial cells is thought to be an important step in tumorigenesis. Moreover, anomalous expression of inappropriate cadherins in epithelial cells alters their behavior and may contribute to the tumorigenic phenotype. For breast cancer the decreased expression of E-cadherin alone may have limited value as a prognostic indicator; however, examining the repertoire of cadherins and catenins expressed by tumors may provide useful prognostic information.

Upregulation of P-cadherin expression in the lesional skin of pemphigus, Hailey-Hailey disease and Darier's disease.

BACKGROUND: Autoimmune blistering diseases, pemphigus vulgaris (PV) and pemphigus foliaceus (PF), are known to be caused by binding of autoantibodies to the desmosomal cadherins, desmoglein 3 and desmoglein 1, respectively. Recently, mutations in the genes coding Ca2+ pumps leads to inherited blistering diseases, Hailey-Hailey disease (HHD) and Darier's disease (DD). Cadherins are a family of Ca2+-dependent cell adhesion molecules and P-cadherin is one of the major cadherins expressed in the epidermis. Although detailed mechanisms of acantholysis of these blistering diseases have not been fully clarified, abnormal expression of cadherins caused by altered Ca2+ concentration due to the binding of autoantibodies to cell surface or by mutations in Ca2+ pumps is suggested to be involved in mechanisms of acantholysis of these autoimmune and inherited blistering diseases. The purpose of the present study was to determine whether altered P-cadherin expression is present in these diseases. METHOD: Distribution patterns of P-cadherin in skin specimens from patients with PV (n=2), PF (n=2), HHD (n=4) and DD (n=3), were examined with confocal laser scanning microscopy using two anti-P-cadherin antibodies, 6A9 and NCC-CAD-299. RESULTS: In normal control skin, P-cadherin expression was restricted to the basal layer. In contrast, positive immunostaining of P-cadherin was observed not only in the basal cells, but also in the suprabasal cells in lesional skin of all the acantholytic diseases. CONCLUSIONS: The present results clearly demonstrated that upregulation of P-cadherin expression occurs in the acantholysis in all the four blistering diseases PV, PF, HHD and DD. Upregulation of P-cadherin may be involved in the pathomechanism of both the autoimmune blistering diseases and the inherited blistering diseases.

N-cadherin is developmentally regulated and functionally involved in early hematopoietic cell differentiation.

The cadherins, an important family of cell adhesion molecules, are known to play major roles during embryonic development and in the maintenance of solid tissue architecture. In the hematopoietic system, however, little is known of the role of this cell adhesion family. By RT-PCR, western blot analysis and immunofluorescence staining we show that N-cadherin, a classical type I cadherin mainly expressed on neuronal, endothelial and muscle cells, is expressed on the cell surface of resident bone marrow stromal cells. FACS analysis of bone marrow mononuclear cells revealed that N-cadherin is also expressed on a subpopulation of early hematopoietic progenitor cells. Triple-color FACS analysis defined a new CD34(+) CD19(+) N-cadherin(+) progenitor cell population. During further differentiation, however, N-cadherin expression is lost. Treatment of CD34(+) progenitor cells with function-perturbing N-cadherin antibodies drastically diminished colony formation, indicating a direct involvement of N-cadherin in the differentiation program of early hematopoietic progenitors. N-cadherin can also mediate adhesive interactions within the bone marrow as demonstrated by inhibition of homotypic interactions of bone-marrow-derived cells with N-cadherin antibodies. Together, these data strongly suggest that N-cadherin is involved in the development and retention of early hematopoietic progenitors within the bone marrow microenvironment.

Truncated DCC reduces N-cadherin/catenin expression and calcium-dependent cell adhesion in neuroblastoma cells.

The deleted in colorectal cancer (DCC) protein is important in the pathway guidance of cells and cell processes during neural development, and DCC has also been implicated in the aberrant cellular migrations of neuroblastoma dissemination. We attempted to further define DCC protein function by the overexpression of full-length and truncated DCC constructs in a human neuroblastoma cell line. Overexpression of the truncated DCC protein resulted in a less epithelioid morphology. This was accompanied by decreases in expression of N-cadherin and alpha- and beta-catenin by immunoblot and Northern blot analysis. Levels of desmoglein were relatively less affected, whereas endogenous DCC protein levels were increased in the truncated transfectants. N-cadherin immunofluorescence was consistent with the immunoblot studies and localized the protein to the cytoplasm and sites of cell-cell contact. Cell aggregation studies demonstrated diminished calcium-dependent aggregation in the truncated transfectants. In conclusion, overexpression of a truncated DCC protein in neuroblastoma cells resulted in the loss of an epithelioid morphology, diminished expression of N-cadherin and alpha- and beta-catenin, and diminished calcium-dependent cell adhesion. These studies provide the first evidence of an apparent functional link between DCC and N-cadherin/catenin-dependent cell adhesion.

N-Cadherin extracellular repeat 4 mediates epithelial to mesenchymal transition and increased motility.

E- and N-cadherin are members of the classical cadherin family of proteins. E-cadherin plays an important role in maintaining the normal phenotype of epithelial cells. Previous studies from our laboratory and other laboratories have shown that inappropriate expression of N-cadherin by tumor cells derived from epithelial tissue results in conversion of the cell to a more fibroblast-like cell, with increased motility and invasion. Our present study was designed to determine which domains of N-cadherin make it different from E-cadherin, with respect to altering cellular behavior, such as which domains are responsible for the epithelial to mesenchymal transition and increased cell motility and invasion. To address this question, we constructed chimeric cadherins comprised of selected domains of E- and N-cadherin. The chimeras were transfected into epithelial cells to determine their effect on cell morphology and cellular behavior. We found that a 69-amino acid portion of EC-4 of N-cadherin was necessary and sufficient to promote both an epithelial to mesenchymal transition in squamous epithelial cells and increased cell motility. Here, we show that different cadherin family members promote different cellular behaviors. In addition, we identify a novel activity that can be ascribed to the extracellular domain of N-cadherin.

Lack of correlation between serum levels of E- and P-cadherin fragments and the presence of breast cancer.

Breast cancers often show reduced expression of the transmembrane cell-cell adhesion protein, E-cadherin. In addition, approximately half of breast carcinomas express P-cadherin, which correlates with poor survival. A large fragment of the E-cadherin extracellular domain can be detected in serum, and it has been proposed that an increase in serum E-cadherin can denote the presence of a tumor. In this study, we tested the possibility that serum E- or P-cadherin levels might be useful diagnostic or prognostic indicators in breast cancer. However, we found no indication that the level of serum E-cadherin correlated with the presence of breast cancer. In addition, although we successfully detected a fragment of P-cadherin in serum, we found that its level was considerably lower than that of E-cadherin and did not correlate with the presence of P-cadherin-positive breast cancer.

Dissociation of intra- and extracellular domains of desmosomal cadherins and E-cadherin in Hailey-Hailey disease and Darier's disease.

In order to clarify the pathomechanism of acantholysis in Hailey-Hailey disease (HHD) and Darier's disease (DD), the distribution of desmosomal and adherens junction-associated proteins was studied in the skin of patients with HHD (n = 4) and DD (n = 3). Domain-specific antibodies were used to determine the cellular localization of the desmosomal transmembrane glycoproteins (desmogleins 1 and 3 and desmocollin), desmosomal plaque proteins (desmoplakin, plakophilin and plakoglobin) and adherens junction-associated proteins (E-cadherin, alpha-catenin, beta-catenin and actin). A significant difference in staining patterns between intra- and extracellular domains of desmosomal cadherins and E-cadherin was demonstrated in acantholytic cells in both HHD and DD, but not in those in pemphigus vulgaris and pemphigus foliaceus samples used as controls. In acantholytic cells in HHD and DD, antibodies against attachment plaque proteins and intracellular epitopes of desmosomal cadherins exhibited diffuse cytoplasmic staining, whereas markedly reduced staining was observed with antibodies against extracellular epitopes of the desmogleins. Similarly, membrane staining of an intracellular epitope of E-cadherin was preserved, while immunoreactivity of an extracellular epitope of E-cadherin was destroyed. While the DD gene has been identified as ATP2A2, the gene for HHD has not been clarified. The dissociation of intra- and extracellular domains of desmosomal cadherin and E-cadherin is characteristic of the acantholytic cells in HHD and DD, and not of pemphigus. This common phenomenon in HHD and DD might be closely related to the pathophysiological mechanisms in both conditions.

Vimentin expression in human squamous carcinoma cells: relationship with phenotypic changes and cadherin-based cell adhesion.

Phenotypic changes resembling an epithelial-to-mesenchymal transition often occur as epithelial cells become tumorigenic. Two proteins that have been implicated in this process are vimentin and N-cadherin. In this study, we sought to establish a link between expression of vimentin and N-cadherin as oral squamous epithelial cells undergo a morphologic change resembling an epithelial-to-mesenchymal transition. We found that N-cadherin and vimentin did not influence the expression of one another.

The amino- and carboxyl-terminal tails of (beta)-catenin reduce its affinity for desmoglein 2.

beta-catenin and plakoglobin are members of the armadillo family of proteins and were first identified as components of intercellular adhering junctions. In the adherens junction beta-catenin and plakoglobin serve to link classical cadherins to the actin-based cytoskeleton. In the desmosome plakoglobin links the desmosomal cadherins, the desmogleins and the desmocollins, to the intermediate filament cytoskeleton. beta-catenin is not a component of the desmosome. Previously we have shown that the central armadillo repeat region of plakoglobin is the site for desmosomal cadherin binding. We hypothesized that the unique amino- and/or carboxyl-terminal ends of beta-catenin may regulate its exclusion from the desmosomal plaque. To test this hypothesis we used chimeras between beta-catenin and plakoglobin to identify domain(s) that modulate association with desmoglein 2. Chimeric constructs, each capable of associating with classical cadherins, were assayed for association with the desmosomal cadherin desmoglein 2. Addition of either the N- or C-terminal tail of beta-catenin to the armadillo repeats of plakoglobin did not interfere with desmoglein 2 association. However, when both beta-catenin amino terminus and carboxyl terminus were added to the plakoglobin armadillo repeats, association with desmoglein 2 was diminished. Removal of the first 26 amino acids from this construct restored association. We show evidence for direct protein-protein interactions between the amino- and carboxyl-terminal tails of beta-catenin and propose that a sequence in the first 26 amino acids of beta-catenin along with its carboxyl-terminal tail decrease its affinity for desmoglein and prevent its inclusion in the desmosome.

Development of chimeric molecules for recognition and targeting of antigen-specific B cells in pemphigus vulgaris.

Pemphigus vulgaris (PV) is an autoimmune blistering disease characterized by circulating pathogenic IgG antibodies against desmoglein 3 (Dsg3). The purpose of this study was to develop chimeric molecules for specific recognition and elimination of autoimmune B cells in PV. Mouse hybridoma cell lines producing anti-Dsg3 antibody (5H10, 12A2) were developed as an in vitro model system for targeting B cells. Dsg3-GFP, a baculoprotein containing the entire extracellular domain of Dsg3 fused with green fluorescence protein, recognized and targeted the hybridoma cells through their surface immunoglobulin receptors in an antigen-specific way. The epitopes of these monoclonal antibodies were mapped on the amino terminal EC1 and part of EC2, a region considered functionally important in cadherins. Chimeric toxin molecules containing the mapped region (Dsg3deltaN1) and modified Pseudomonas exotoxin were produced in bacteria (Dsg3deltaN1-PE40-KDEL, PE3 7-Dsg3deltaN1-KDEL) and tested in vitro on hybridoma cell lines. The chimeric toxins, but not Dsg3deltaN1 alone, showed dose-dependent toxic activity with a reduction in hybridoma cell number to 40-60% of toxin-negative control cultures, compared with little or no effect on anti-Dsg3-negative hybridoma cells. Furthermore, these toxins showed toxic effects on anti-Dsg3 IgG-producing B cells from Dsg3deltaN1-immunized mice, with a 60% reduction in cell number compared with Dsg3deltaN1 alone. Thus, specific recognition and targeting of antigen-specific B cells in PV was demonstrated; this strategy may hold promise as a future therapeutic option for PV and other autoimmune diseases.