Dynamic assembly of ultrasoft colloidal networks enables cell invasion within restrictive fibrillar polymers.

In regenerative medicine, natural protein-based polymers offer enhanced endogenous bioactivity and potential for seamless integration with tissue, yet form weak hydrogels that lack the physical robustness required for surgical manipulation, making them difficult to apply in practice. The use of higher concentrations of protein, exogenous cross-linkers, and blending synthetic polymers has all been applied to form more mechanically robust networks. Each relies on generating a smaller network mesh size, which increases the elastic modulus and robustness, but critically inhibits cell spreading and migration, hampering tissue regeneration. Here we report two unique observations; first, that colloidal suspensions, at sufficiently high volume fraction (ϕ), dynamically assemble into a fully percolated 3D network within high-concentration protein polymers. Second, cells appear capable of leveraging these unique domains for highly efficient cell migration throughout the composite construct. In contrast to porogens, the particles in our system remain embedded within the bulk polymer, creating a network of particle-filled tunnels. Whereas this would normally physically restrict cell motility, when the particulate network is created using ultralow cross-linked microgels, the colloidal suspension displays viscous behavior on the same timescale as cell spreading and migration and thus enables efficient cell infiltration of the construct through the colloidal-filled tunnels.

Functional wiring of the yeast kinome revealed by global analysis of genetic network motifs.

A combinatorial genetic perturbation strategy was applied to interrogate the yeast kinome on a genome-wide scale. We assessed the global effects of gene overexpression or gene deletion to map an integrated genetic interaction network of synthetic dosage lethal (SDL) and loss-of-function genetic interactions (GIs) for 92 kinases, producing a meta-network of 8700 GIs enriched for pathways known to be regulated by cognate kinases. Kinases most sensitive to dosage perturbations had constitutive cell cycle or cell polarity functions under standard growth conditions. Condition-specific screens confirmed that the spectrum of kinase dosage interactions can be expanded substantially in activating conditions. An integrated network composed of systematic SDL, negative and positive loss-of-function GIs, and literature-curated kinase-substrate interactions revealed kinase-dependent regulatory motifs predictive of novel gene-specific phenotypes. Our study provides a valuable resource to unravel novel functional relationships and pathways regulated by kinases and outlines a general strategy for deciphering mutant phenotypes from large-scale GI networks.

Ultrasoft, highly deformable microgels.

Microgels are colloidally stable, hydrogel microparticles that have previously been used in a range of (soft) material applications due to their tunable mechanical and chemical properties. Most commonly, thermo and pH-responsive poly(N-isopropylacrylamide) (pNIPAm) microgels can be fabricated by precipitation polymerization in the presence of the co-monomer acrylic acid (AAc). Traditionally pNIPAm microgels are synthesized in the presence of a crosslinking agent, such as N,N'-methylenebisacrylamide (BIS), however, microgels can also be synthesized under 'crosslinker free' conditions. The resulting particles have extremely low (<0.5%), core-localized crosslinking resulting from rare chain transfer reactions. AFM nanoindentation of these ultralow crosslinked (ULC) particles indicate that they are soft relative to crosslinked microgels, with a Young's modulus of ∼10 kPa. Furthermore, ULC microgels are highly deformable as indicated by a high degree of spreading on glass surfaces and the ability to translocate through nanopores significantly smaller than the hydrodynamic diameter of the particles. The size and charge of ULCs can be easily modulated by altering reaction conditions, such as temperature, monomer, surfactant and initiator concentrations, and through the addition of co-monomers. Microgels based on the widely utilized, biocompatible polymer polyethylene glycol (PEG) can also be synthesized under crosslinker free conditions. Due to their softness and deformability, ULC microgels are a unique base material for a wide variety of biomedical applications including biomaterials for drug delivery and regenerative medicine.

Synergistic effects of particulate matter and substrate stiffness on epithelial-to-mesenchymal transition.

Dysfunctional pulmonary homeostasis and repair, including diseases such as pulmonary fibrosis, chronic obstructive pulmonary disease (COPD*), and tumorigenesis, have been increasing steadily over the past decade, a fact that heavily implicates environmental influences. Several investigations have suggested that the lung "precursor cell"--the alveolar type II (ATII) epithelial cell--is central in the initiation and progression of pulmonary fibrosis. Specifically, ATII cells have been shown (Iwano et al. 2002) to be capable of undergoing an epithelial-to-mesenchymal transition (EMT). EMT, the de-differentiation of an epithelial cell into a mesenchymal cell, has been theorized to increase the number of extracellular matrix (ECM)-secreting mesenchymal cells, perpetuating fibrotic conditions and resulting in increased lung tissue stiffness. In addition, increased exposure to pollution and inhalation of particulate matter (PM) have been shown to be highly correlated with an increased incidence of pulmonary fibrosis. Although both of these events are involved in the progression of pulmonary fibrosis, the relationship between tissue stiffness, exposure to PM, and the initiation and course of EMT remains unclear. The hypothesis of this study was twofold: 1. That alveolar epithelial cells cultured on increasingly stiff substrates become increasingly contractile, leading to enhanced transforming growth factor beta (TGF-ß) activation and EMT; and 2. That exposure of alveolar epithelial cells to PM with an aerodynamic diameter ≤ 2.5 µm (PM2.5; also known as fine PM) results in enhanced cell contractility and EMT. Our study focused on the relationship between the micromechanical environment and external environmental stimuli on the phenotype of alveolar epithelial cells. This relationship was explored by first determining how increased tissue stiffness affects the regulation of fibronectin (Fn)-mediated EMT in ATII cells in vitro. We cultured ATII cells on substrates of increasing stiffness and evaluated changes in cell contractility and EMT. We found that stiff, but not soft, Fn substrates were able to induce EMT and that this event depended on a contractile phenotype of the cell and the subsequent activation of TGF-ß. In addition, we were able to show that activation or suppression of cell contractility by way of exogenous factors was sufficient to overcome the effect of substrate stiffness. Pulse-chase experiments indicated that the effect on cell contractility is dose- and time-dependent. In response to low levels of TGF-ß on soft surfaces, either added exogenously or produced through contraction induced by the stiffness agonist thrombin, cells initiate EMT; on removal of the TGF-ß, they revert to an epithelial phenotype. Overall, the results from this first part of our study identified matrix stiffness or cell contractility as critical targets for the control of EMT in fibrotic diseases. For the second part of our study, we wanted to investigate whether exposure to PM2.5, which might have higher toxicity than coarser PM because of its small size and large surface-to-mass ratio, altered the observed stiffness-mediated EMT. Again, we cultured ATII cells on increasingly stiff substrates with or without the addition of three concentrations of PM2.5. We found that exposure to PM2.5 was involved in increased stiffness-mediated EMT, as shown by increases in mesenchymal markers, cell contractility, and TGF-ß activation. Most notably, on substrates with an elastic modulus (E) of 8 kilopascals (kPa), a physiologically relevant range for pulmonary fibrosis, the addition of PM2.5 resulted in increased mesenchymal cells and EMT; these were not seen in the absence of the PM2.5. Overall, this study showed that there is a delicate balance between substrate stiffness, TGF-ß, and EMT. Furthermore, we showed that exposure to PM2.5 is able to further mediate this interaction. The higher levels of EMT seen with exposure to PM2.5 might have been a result of a positive feedback loop, in which enhanced exposure to PM2.5 through the loss of cell-cell junctions during the initial stages of EMT led to the cells being more susceptible to the effects of surrounding immune cells and inflammatory signals that can further activate TGF-ß and drive additional EMT progression. Overall, our work--showing increased cell contractility, TGF-ß activation, and EMT in response to substrate stiffness and PM2.5 exposure--highlights the importance of both the micromechanical and biochemical environments in lung disease. These findings suggest that already-fibrotic tissue might be more susceptible to further damage than healthy tissue when exposed to PM2.5.

Validity of predischarge measures for predicting time to harm in older adults.

BACKGROUND: Concern is often expressed about the ability of persons with cognitive impairment to manage safely after discharge home from hospital. Measures validated for predicting safety are required. PURPOSE: The purpose of this study was to determine whether two predischarge functional measures were valid for predicting time to incident of harm after discharge. METHOD: Participants (n = 47) were recruited from an inpatient rehabilitation unit. The Assessment of Motor and Process Skills (AMPS) and Cognitive Performance Test (CPT) were administered in hospital. Incident-of-harm outcome was measured by caregiver telephone questionnaire monthly for 6 months. FINDINGS: Compared with all independent variables, AMPS Process scale was the best single predictor of time to incident of harm (p = .01). CPT had a high specificity (91%) for identifying persons who did not have harm. IMPLICATIONS: Both AMPS and CPT demonstrated predictive validity for harm outcome over less predictive variables, such as comorbidities and activities-of-daily-living burden of care.

Baby on board: do responses to stress in the maternal brain mediate adverse pregnancy outcome?

Stress and adverse environmental surroundings result in suboptimal conditions in a pregnant mother such that she may experience poor pregnancy outcome including complete pregnancy failure and preterm labor. Furthermore her developing baby is at risk of adverse programming, which confers susceptibility to long term ill health. While some mechanisms at the feto-maternal interface underlying these conditions are understood, the underlying cause for their adverse adaptation is often not clear. Progesterone plays a key role at many levels, including control of neuroendocrine responses to stress, procuring the required immune balance and controlling placental and decidual function, and lack of progesterone can explain many of the unwanted consequences of stress. How stress that is perceived by the mother inhibits progesterone secretion and action is beginning to be investigated. This overview of maternal neuroendocrine responses to stress throughout pregnancy analyses how they interact to compromise progesterone secretion and precipitate undesirable effects in mother and offspring.

Oxytocin and the maternal brain.

Oxytocin released within the brain from both magnocellular and parvocellular systems of the hypothalamus has diverse effects on behavior. When oxytocin is released within the brain, its effects are to diminish fearfulness; this not only encourages social investigation of newcomers, but also may enhance a tendency to express aggression toward an intruder. Oxytocin supports social recognition, redirects behavior away from feeding directed behavior toward sexual behavior, and facilitates the formation of social bonds. This system, which depends not only upon release of oxytocin but also on oxytocin receptor distribution within the brain, becomes particularly important at parturition, when a bond is first formed between mother and offspring.

Early risk factors for miscarriage: a prospective cohort study in pregnant women.

Many pregnancies are lost during early gestation, but clinicians still lack tools to recognize risk factors for miscarriage. Thus, the identification of risk factors for miscarriage during the first trimester in women with no obvious risk for a pregnancy loss was the aim of this prospective cohort trial. A total of 1098 women between gestation weeks 4 and 12 in whom no apparent signs of a threatened pregnancy could be diagnosed were recruited. Demographic, anamnestic, psychometric and biological data were documented at recruitment and pregnancy outcomes were registered subsequently. Among the cases with sufficiently available data, 809 successfully progressing pregnancies and 55 subsequent miscarriages were reported. In this cohort, risk of miscarriage was significantly increased in women at higher age (>33 years), lower body mass index (< or =20 kg/ m(2)) and lower serum progesterone concentrations (< or =12 ng/ml) prior to the onset of the miscarriage. Women with subsequent miscarriage also perceived higher levels of stress/demands (supported by higher concentrations of corticotrophin-releasing hormone) and revealed reduced concentrations of progesterone-induced blocking factor. These risk factors were even more pronounced in the subcohort of women (n = 335) recruited between gestation weeks 4 and 7. The identification of these risk factors and development of an interaction model of these factors, as introduced in this article, will help clinicians to recognize pregnant women who require extra monitoring and who might benefit from therapeutic interventions such as progestogen supplementation, especially during the first weeks of pregnancy, to prevent a miscarriage.

Dynamic changes in oxytocin receptor expression and activation at parturition in the rat brain.

Oxytocin plays a pivotal role in rat parturition, acting within the brain to facilitate its own release in the supraoptic nucleus (SON) and paraventricular nucleus, and to stimulate maternal behavior. We investigated oxytocin receptor (OTR) expression and activation perinatally. Using a (35)S-labeled riboprobe complementary to OTR mRNA, OTR expression was quantified in proestrus virgin, 21- and 22-day pregnant, parturient (90 min. from pup 1 birth), and postpartum (4-12 h from parturition) rats. Peak OTR mRNA expression was observed at parturition in the SON, brainstem regions, medial preoptic area (mPOA), bed nucleus of the stria terminalis (BnST), and olfactory bulbs, but there was no change in the paraventricular nucleus and lateral septum. OTR mRNA expression was increased on the day of expected parturition in the SON and brainstem, suggesting that oxytocin controls the pathway mediating input from uterine signals. Likewise, OTR mRNA expression was increased in the mPOA and BnST during labor/birth. In the olfactory bulbs and medial amygdala, parturition induced increased OTR mRNA expression compared with pre-parturition, reflecting their immediate response to new stimuli at birth. Postpartum OTR expression in all brain regions returned to levels observed in virgin rats. Parturition significantly increased the number of double-immunolabeled cells for Fos and OTR within the SON, brainstem, BnST, and mPOA regions compared with virgin rats. Thus, there are dynamic region-dependent changes in OTR-expressing cells at parturition. This altered OTR distribution pattern in the brain perinatally reflects the crucial role oxytocin plays in orchestrating both birth and maternal behavior.

Neuroendocrine mechanisms of change in food intake during pregnancy: a potential role for brain oxytocin.

During pregnancy body weight, and particularly adiposity, increase, due to hyperphagia rather than decreased energy metabolism. These physiological adaptations provide the growing fetus(es) with nutrition and prepare the mother for the metabolically-demanding lactation period following birth. Mechanisms underlying the hyperphagia are still poorly understood. Although the peripheral signals that drive appetite and satiety centers of the brain are increased in pregnancy, the brain may become insensitive to their effects. For example, leptin secretion increases but hypothalamic resistance to leptin actions develops. However, several adaptations in hypothalamic neuroendocrine systems may converge to increase ingestive behavior. Oxytocin is one of the anorectic hypothalamic neuropeptides. Oxytocin neurons, both centrally-projecting parvocellular oxytocin neurons and central dendritic release of oxytocin from magnocellular neurons, may play a key role in regulating energy intake. During feeding in non-pregnant rats, magnocellular oxytocin neurons, especially those in the supraoptic nucleus, become strongly activated indicating their imminent role in meal termination. However, in mid-pregnancy the excitability of these neurons is reduced, central dendritic oxytocin release is inhibited and patterns of oxytocin receptor binding in the brain alter. Our recent data suggest that lack of central oxytocin action may partly contribute to maternal hyperphagia. However, although opioid inhibition is a major factor in oxytocin neuron restraint during pregnancy and opioids enhance food intake, an increase in opioid orexigenic actions were not observed. While changes in several central input pathways to oxytocin neurons are likely to be involved, the high level of progesterone secretion during pregnancy is probably the ultimate trigger for the adaptations.

Cognitive assessments for older adults: which ones are used by Canadian therapists and why.

BACKGROUND: Occupational therapists routinely evaluate cognition in older adults, yet little is known about which assessments they use and for what purposes. PURPOSE: To examine the standardised and non-standardised assessments used by occupational therapists to evaluate cognition. METHOD: A random sample of 1042 Canadian occupational therapists completed the questionnaire by e-mail, post, or Internet website (n=247, response rate: 24.5%). RESULTS: Respondents reported using 75 standardised and non-standardised measures. The assessments were grouped according to theoretical approach: bottom-up (assessment of cognitive components), top-down (assessment of function) and combined (either of above, plus interview). Theoretical approaches were used similarly across regions, despite differences in reporting of particular assessments. Therapists used more bottom-up assessments that were standardised, identified deficits, and easy to administer. They used more top-down assessments that were non-standardised, predicted function, and fit with their theoretical approach. CONCLUSION: It is recommended that standardised top-down assessments be developed to support evidence-based occupational therapy.

Charge segregation in weakly ionized microgels.

We investigate microgels synthesized from N-isopropylacrylamide (NIPAM) copolymerized with a large mol% of acrylic acid, finding that when the acid groups are partially ionized at high temperatures, competition between ion-induced swelling and hydrophobic deswelling of poly(NIPAM) chains results in microphase separation. In cross-linked microgels, this manifests as a dramatic decrease in the ratio between the radius of gyration and the hydrodynamic radius to ∼0.2, indicating that almost all the mass of the microgel is concentrated near the particle center. We also observe a concurrent decrease of the polymer network length scale via small-angle neutron scattering, confirming the presence of a dense, deswollen core surrounded by a diffuse, charged periphery. We compare these results to those obtained for a system of charged ultralow-cross-linked microgels; the form factor shows a distinct peak at high q when the temperature exceeds a threshold value. We successfully fit the form factor to theory developed to describe scattering from weakly charged gels in poor solvents, and we tie this behavior to charge segregation in the case of the cross-linked microgels.

Endogenous opioids and attenuated hypothalamic-pituitary-adrenal axis responses to immune challenge in pregnant rats.

In late pregnant rats, the hypothalamic-pituitary-adrenal (HPA) axis is hyporesponsive to psychogenic stressors. Here, we investigated attenuated HPA responses to an immune challenge and a role for endogenous opioids. ACTH and corticosterone were assayed in blood samples from virgin and 21 d pregnant rats before and after endotoxin [lipopolysaccharide (LPS); 1 microg/kg, i.v.], interleukin-1beta (IL-1beta; 500 ng/kg, i.v.), or vehicle. In virgins, plasma ACTH concentrations increased 1 h after LPS and 15 min after IL-1beta, as did corticosterone, with no responses in pregnant rats. In situ hybridization revealed increased corticotrophin releasing hormone (CRH) mRNA expression in the dorsomedial parvocellular paraventricular nucleus (pPVN) and increased anterior pituitary pro-opiomelanocortin mRNA expression 4 h after IL-1beta in virgins; these responses were absent in pregnant rats. In contrast, immunocytochemistry showed that Fos expression was similarly increased in the nucleus tractus solitarius (NTS) A2 region in virgin and pregnant rats 90 min and 4 h after IL-1beta. Naloxone pretreatment (5 mg/kg, i.v.) restored ACTH and pPVN CRH mRNA responses after IL-1beta in pregnant rats but reduced the CRH mRNA response in virgins without affecting ACTH. Proenkephalin-A and mu-opioid receptor mRNA expression in the NTS was significantly increased in the pregnant rats, indicating upregulated brainstem opioid mechanisms. IL-1beta increased noradrenaline release in the PVN of virgin, but not pregnant, rats. However, naloxone infused directly into the PVN increased noradrenaline release after IL-1beta in pregnant rats. Thus, the HPA axis responses to immune signals are suppressed in pregnancy at the level of pPVN CRH neurons through an opioid mechanism, possibly acting by preterminal autoinhibition of NTS projections to the pPVN.

Brain oxytocin correlates with maternal aggression: link to anxiety.

The oxytocinergic system is critically involved in the regulation of maternal behavior, which includes maternal aggression. Because aggression has been linked to anxiety, we investigated the maternal aggression and the role of brain oxytocin in lactating Wistar rats selectively bred for high anxiety-related behavior (HAB) or low anxiety-related behavior (LAB) during the 10 min maternal defense test. HAB dams displayed more maternal aggression against a virgin intruder compared with LAB dams, resulting in more defensive behavior and higher anxiety of HAB-defeated virgins. The different levels of aggression were accompanied by opposite oxytocin release patterns within the paraventricular nucleus (PVN; HAB, increase; LAB, decrease). Furthermore, oxytocin release was higher within the central nucleus of the amygdala (CeA) of HAB dams compared with LABs. A direct correlation between the offensive behavior displayed during the maternal defense test and local oxytocin release was found in both the PVN and CeA. Using retrodialysis, blockade of endogenous oxytocin action by infusion of an oxytocin receptor antagonist (des-Gly-NH2,d(CH2)5[Tyr(Me)2,Thr4]OVT) into the PVN or CeA reduced maternal aggression of HAB dams, whereas infusion of synthetic oxytocin into the PVN tended to increase aggression toward the intruder in LAB dams. There were no significant differences in oxytocin receptor mRNA expression or oxytocin receptor binding between lactating HAB and LAB dams. Therefore, differences in intracerebral release patterns of oxytocin, rather than differences at the level of oxytocin receptors, are critical for the regulation of maternal aggressive behavior.

A novel platelet lysate hydrogel for endothelial cell and mesenchymal stem cell-directed neovascularization.

UNLABELLED: Mesenchymal stem cells (MSC) hold promise in promoting vascular regeneration of ischemic tissue in conditions like critical limb ischemia of the leg. However, this approach has been limited in part by poor cell retention and survival after delivery. New biomaterials offer an opportunity to localize cells to the desired tissue after delivery, but also to improve cell survival after delivery. Here we characterize the mechanical and microstructural properties of a novel hydrogel composed of pooled human platelet lysate (PL) and test its ability to promote MSC angiogenic activity using clinically relevant in vitro and in vivo models. This PL hydrogel had comparable storage and loss modulus and behaved as a viscoelastic solid similar to fibrin hydrogels despite having 1/4-1/10th the fibrin content of standard fibrin gels. Additionally, PL hydrogels enabled sustained release of endogenous PDGF-BB for up to 20days and were resistant to protease degradation. PL hydrogel stimulated pro-angiogenic activity by promoting human MSC growth and invasion in a 3D environment, and enhancing endothelial cell sprouting alone and in co-culture with MSCs. When delivered in vivo, the combination of PL and human MSCs improved local tissue perfusion after 8days compared to controls when assessed with laser Doppler perfusion imaging in a murine model of hind limb ischemia. These results support the use of a PL hydrogel as a scaffold for MSC delivery to promote vascular regeneration. STATEMENT OF SIGNIFICANCE: Innovative strategies for improved retention and viability of mesenchymal stem cells (MSCs) are needed for cellular therapies. Human platelet lysate is a potent serum supplement that improves the expansion of MSCs. Here we characterize our novel PL hydrogel's desirable structural and biologic properties for human MSCs and endothelial cells. PL hydrogel can localize cells for retention in the desired tissue, improves cell viability, and augments MSCs' angiogenic activity. As a result of these unique traits, PL hydrogel is ideally suited to serve as a cell delivery vehicle for MSCs injected into ischemic tissues to promote vascular regeneration, as demonstrated here in a murine model of hindlimb ischemia.

Alpha-melanocyte-stimulating hormone stimulates oxytocin release from the dendrites of hypothalamic neurons while inhibiting oxytocin release from their terminals in the neurohypophysis.

The peptides alpha-melanocyte stimulating hormone (alpha-MSH) and oxytocin, when administered centrally, produce similar behavioral effects. alpha-MSH induces Fos expression in supraoptic oxytocin neurons, and alpha-MSH melanocortin-4 receptors (MC4Rs) are highly expressed in the supraoptic nucleus, suggesting that alpha-MSH and oxytocin actions are not independent. Here we investigated the effects of alpha-MSH on the activity of supraoptic neurons. We confirmed that alpha-MSH induces Fos expression in the supraoptic nucleus when injected centrally and demonstrated that alpha-MSH also stimulates Fos expression in the nucleus when applied locally by retrodialysis. Thus alpha-MSH-induced Fos expression is not associated with electrophysiological excitation of supraoptic neurons because central injection of alpha-MSH or selective MC4 receptor agonists inhibited the electrical activity of oxytocin neurons in the supraoptic nucleus recorded in vivo. Consistent with these observations, oxytocin secretion into the bloodstream decreased after central injection of alpha-MSH. However, MC4R ligands induced substantial release of oxytocin from dendrites in isolated supraoptic nuclei. Because dendritic oxytocin release can be triggered by changes in [Ca2+]i, we measured [Ca2+]i responses in isolated supraoptic neurons and found that MC4R ligands induce a transient [Ca2+]i increase in oxytocin neurons. This response was still observed in low extracellular Ca2+ concentration and probably reflects mobilization of [Ca2+]i from intracellular stores rather than entry via voltage-gated channels. Taken together, these results show for the first time that a peptide, here alpha-MSH, can induce differential regulation of dendritic release and systemic secretion of oxytocin, accompanied by dissociation of Fos expression and electrical activity.

Molecular interference of fibrin's divalent polymerization mechanism enables modulation of multiscale material properties.

Protein based polymers provide an exciting and complex landscape for tunable natural biomaterials through modulation of molecular level interactions. Here we demonstrate the ability to modify protein polymer structural and mechanical properties at multiple length scales by molecular 'interference' of fibrin's native polymerization mechanism. We have previously reported that engagement of fibrin's polymerization 'hole b', also known as 'b-pockets', through PEGylated complementary 'knob B' mimics can increase fibrin network porosity but also, somewhat paradoxically, increase network stiffness. Here, we explore the possible mechanistic underpinning of this phenomenon through characterization of the effects of knob B-fibrin interaction at multiple length scales from molecular to bulk polymer. Despite its weak monovalent binding affinity for fibrin, addition of both knob B and PEGylated knob B at concentrations near the binding coefficient, Kd, increased fibrin network porosity, consistent with the reported role of knob B-hole b interactions in promoting lateral growth of fibrin fibers. Addition of PEGylated knob B decreases the extensibility of single fibrin fibers at concentrations near its Kd but increases extensibility of fibers at concentrations above its Kd. The data suggest this bimodal behavior is due to the individual contributions knob B, which decreases fiber extensibility, and PEG, which increase fiber extensibility. Taken together with laser trap-based microrheological and bulk rheological analyses of fibrin polymers, our data strongly suggests that hole b engagement increases in single fiber stiffness that translates to higher storage moduli of fibrin polymers despite their increased porosity. These data point to possible strategies for tuning fibrin polymer mechanical properties through modulation of single fiber mechanics.

Neuroendocrine responses to stress in mice: hyporesponsiveness in pregnancy and parturition.

Hypothalamo-pituitary-adrenal axis secretory responses to stress were compared in female virgin, late pregnant, parturient, and lactating mice. The basal plasma ACTH concentration was not different in pregnancy or lactation compared with virgins, but corticosterone concentration and corticosteroid-binding globulin capacity were greatly elevated in late pregnancy. Secretory responses to novel environment were attenuated in pregnant, but not lactating, mice compared with virgin females, whereas ACTH responses to forced swimming were attenuated in both groups. The expression of immediate early gene (nur77) mRNA increased in paraventricular nucleus neurons after stress exposure in virgin and lactating, but not pregnant, mice. During parturition, the basal ACTH concentration was similar to virgin and pregnant controls and did not increase with stress. Oxytocin secretion in response to either novel environment or forced swimming was unchanged in any reproductive group, whereas vasopressin secretion was decreased by both stressors, but only in virgins. Pretreatment with oxytocin receptor antagonist centrally had no effect on ACTH responses to stress in either virgin or pregnant mice. Pretreatment with an opioid receptor antagonist increased ACTH responses to stress in virgin mice, indicating opioid inhibition, but had no effect in pregnancy. Thus, in mice hypothalamo-pituitary-adrenal hyporesponsiveness in late pregnancy is a consequence of reduced responsiveness of paraventricular neurons, but inhibition by opioids or intracerebral oxytocin does not appear to be involved.