Wall stress enhanced exocytosis of extracellular vesicles as a possible mechanism of left-right symmetry-breaking in vertebrate development.

In certain vertebrate species, the developing embryo breaks left-right symmetry in a transient organising structure: the "Left-Right Organiser" (LRO) known as the "node" in mice, and "Kupffer's vesicle" in fish. Directional cilia-driven flow is integral to this symmetry-breaking process, however the mechanism by which this flow is translated into an asymmetric signal remains contested; the principal theories are either flow transport of vesicles containing morphogens, or flow mechanosensing by cilia. Whilst some recent work favours the morphogen theory, other findings seem to support mechanosensing. In this study, we consider a hypothesis whereby the cilia themselves drive the release of morphogen-carrying extracellular vesicles (EVs) into the LRO; namely, that fluid stresses on the cell membrane induce/enhance exocytosis of EVs. Using a mathematical model, we calculate significant wall normal and shear stresses for a range of typical cilium parameter values comparable to levels capable of enhancing exocytosis. This mechanism may be able to reconcile the apparently conflicting experimental evidence.

Effects of Fission Yield Data in the Calculation of Antineutrino Spectra for

Fission yields form an integral part of the prediction of antineutrino spectra generated by nuclear reactors, but little attention has been paid to the quality and reliability of the data used in current calculations. Following a critical review of the thermal and fast ENDF/B-VII.1 ^{235}U fission yields, deficiencies are identified and improved yields are obtained, based on corrections of erroneous yields, consistency between decay and fission yield data, and updated isomeric ratios. These corrected yields are used to calculate antineutrino spectra using the summation method. An anomalous value for the thermal fission yield of ^{86}Ge generates an excess of antineutrinos at 5-7 MeV, a feature which is no longer present when the corrected yields are used. Thermal spectra calculated with two distinct fission yield libraries (corrected ENDF/B and JEFF) differ by up to 6% in the 0-7 MeV energy window, allowing for a basic estimate of the uncertainty involved in the fission yield component of summation calculations. Finally, the fast neutron antineutrino spectrum is calculated, which at the moment can only be obtained with the summation method and may be relevant for short baseline reactor experiments using highly enriched uranium fuel.

Fabrication and characterization of injectable hydrogels derived from decellularized skeletal and cardiac muscle.

Biomaterials, which can contain appropriate biomechanical and/or biochemical cues, are increasingly being investigated as potential scaffolds for tissue regeneration and/or repair for treating myocardial infarction, heart failure, and peripheral artery disease. Specifically, injectable hydrogels are touted for their minimally invasive delivery, ability to self-assemble in situ, and capacity to encourage host tissue regeneration. Here we present detailed methods for fabricating and characterizing decellularized injectable cardiac and skeletal muscle extracellular matrix (ECM) hydrogels. The ECM derived hydrogels have low cellular and DNA content, retain sulfated glycosaminoglycans and other extracellular matrix proteins such as collagen, gel at physiologic temperature and pH, and assume a nanofibrous architecture. These injectable hydrogels are amenable to minimally invasive, tissue specific biomaterial therapies for treating myocardial infarction and peripheral artery disease.

Three-dimensional flow in Kupffer's Vesicle.

Whilst many vertebrates appear externally left-right symmetric, the arrangement of internal organs is asymmetric. In zebrafish, the breaking of left-right symmetry is organised by Kupffer's Vesicle (KV): an approximately spherical, fluid-filled structure that begins to form in the embryo 10 hours post fertilisation. A crucial component of zebrafish symmetry breaking is the establishment of a cilia-driven fluid flow within KV. However, it is still unclear (a) how dorsal, ventral and equatorial cilia contribute to the global vortical flow, and (b) if this flow breaks left-right symmetry through mechanical transduction or morphogen transport. Fully answering these questions requires knowledge of the three-dimensional flow patterns within KV, which have not been quantified in previous work. In this study, we calculate and analyse the three-dimensional flow in KV. We consider flow from both individual and groups of cilia, and (a) find anticlockwise flow can arise purely from excess of cilia on the dorsal roof over the ventral floor, showing how this vortical flow is stabilised by dorsal tilt of equatorial cilia, and (b) show that anterior clustering of dorsal cilia leads to around 40 % faster flow in the anterior over the posterior corner. We argue that these flow features are supportive of symmetry breaking through mechano-sensory cilia, and suggest a novel experiment to test this hypothesis. From our new understanding of the flow, we propose a further experiment to reverse the flow within KV to potentially induce situs inversus.

Simulations of particle tracking in the oligociliated mouse node and implications for left-right symmetry-breaking mechanics.

The concept of internal anatomical asymmetry is familiar-usually in humans the heart is on the left and the liver is on the right; however, how does the developing embryo know to produce this consistent laterality? Symmetry-breaking initiates with left-right asymmetric cilia-driven fluid mechanics in a small fluid-filled structure called the ventral node in mice. However, the question of what converts this flow into left-right asymmetric development remains unanswered. A leading hypothesis is that flow transports morphogen-containing vesicles within the node, the absorption of which results in asymmetrical gene expression. To investigate how vesicle transport might result in the situs patterns observe in wild-type and mutant experiments, we extend the open-source Stokes flow package, NEAREST, to consider the hydrodynamic and Brownian motion of particles in a mouse model with flow driven by one, two and 112 beating cilia. Three models for morphogen-containing particle released are simulated to assess their compatibility with observed results in oligociliated and wild-type mouse embryos: uniformly random release, localized cilium stress-induced release and localized release from motile cilia themselves. Only the uniformly random release model appears consistent with the data, with neither localized release model resulting in significant transport in the oligociliated embryo. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.

A universal scaling law of mammalian touch.

For most mammals, touch is the first sense to develop. They must feel vibrations on the surface of their skin to enable them to respond to various stimuli in their environment, a process called vibrotaction. But how do mammals perceive these vibrations? Through mathematical modeling of the skin and touch receptors, we show that vibrotaction is dominated by "surface" Rayleigh waves traveling cooperatively through all layers of the skin and bone. Applying our model to experimental data, we identify a universal scaling law for the depth of touch receptors across multiple species, indicating an evolutionarily conserved constant in the sensation of vibrations.

Epidermal growth factor (EGF)-like repeats of human tenascin-C as ligands for EGF receptor.

Signaling through growth factor receptors controls such diverse cell functions as proliferation, migration, and differentiation. A critical question has been how the activation of these receptors is regulated. Most, if not all, of the known ligands for these receptors are soluble factors. However, as matrix components are highly tissue-specific and change during development and pathology, it has been suggested that select growth factor receptors might be stimulated by binding to matrix components. Herein, we describe a new class of ligand for the epidermal growth factor (EGF) receptor (EGFR) found within the EGF-like repeats of tenascin-C, an antiadhesive matrix component present during organogenesis, development, and wound repair. Select EGF-like repeats of tenascin-C elicited mitogenesis and EGFR autophosphorylation in an EGFR-dependent manner. Micromolar concentrations of EGF-like repeats induced EGFR autophosphorylation and activated extracellular signal-regulated, mitogen-activated protein kinase to levels comparable to those induced by subsaturating levels of known EGFR ligands. EGFR-dependent adhesion was noted when the ligands were tethered to inert beads, simulating the physiologically relevant presentation of tenascin-C as hexabrachion, and suggesting an increase in avidity similar to that seen for integrin ligands upon surface binding. Specific binding to EGFR was further established by immunofluorescence detection of EGF-like repeats bound to cells and cross-linking of EGFR with the repeats. Both of these interactions were abolished upon competition by EGF and enhanced by dimerization of the EGF-like repeat. Such low affinity behavior would be expected for a matrix-"tethered" ligand; i.e., a ligand which acts from the matrix, presented continuously to cell surface EGF receptors, because it can neither diffuse away nor be internalized and degraded. These data identify a new class of "insoluble" growth factor ligands and a novel mode of activation for growth factor receptors.

Enhanced myocardial function in transgenic mice overexpressing the beta 2-adrenergic receptor.

Transgenic mice were created with cardiac-specific overexpression of the beta 2-adrenergic receptor. This resulted in increased basal myocardial adenylyl cyclase activity, enhanced atrial contractility, and increased left ventricular function in vivo; these parameters at baseline in the transgenic animals were equal to those observed in control animals maximally stimulated with isoproterenol. These results illustrate a useful approach for studying the effect of gene expression on cardiac contractility. Because chronic heart failure in humans is accompanied by a reduction in the number of myocardial beta-adrenergic receptors and in inotropic responsiveness, these results suggest a potential gene therapy approach to this disease state.

Respiratory assessment in child and adolescent residential treatment settings: reducing restraint-associated risks.

TOPIC: Crisis situations of youth in treatment settings may require restraints. Restraints should only be used in situations where there is imminent danger to the child and when there is no alternative. They are meant to maintain the child's safety, but there is risk for respiratory compromise. PURPOSE: Nursing care of children in restraints must include respiratory assessment and, when indicated, immediate intervention to prevent disastrous outcomes. SOURCES: Review using PubMed and established texts confirms that clinical skills and knowledge is essential to child and adolescent psychiatric nursing. CONCLUSIONS: Clinical assessment and awareness of risks in physical restraints is essential for the safety and well-being of the child.

Dynamics of cilia length in left-right development.

Reduction in the length of motile cilia in the zebrafish left-right organizer (LRO), also known as Kupffer's vesicle, has a large impact on left-right development. Here we demonstrate through genetic overexpression in zebrafish embryos and mathematical modelling that the impact of increased motile cilia length in embryonic LRO fluid flow is milder than that of short cilia. Through Arl13b overexpression, which increases cilia length without impacting cilia beat frequency, we show that the increase in cilium length is associated with a decrease in beat amplitude, resulting in similar flow strengths for Arl13b overexpression and wild-type (WT) embryos, which were not predicted by current theory. Longer cilia exhibit pronounced helical beat patterns and, consequently, lower beat amplitudes relative to WT, a result of an elastohydrodynamic shape transition. For long helical cilia, fluid dynamics modelling predicts a mild (approx. 12%) reduction in the torque exerted on the fluid relative to the WT, resulting in a proportional reduction in flow generation. This mild reduction is corroborated by experiments, providing a mechanism for the mild impact on organ situs.

Advantages of concurrent biochemotherapy modified by decrescendo interleukin-2, granulocyte colony-stimulating factor, and tamoxifen for patients with metastatic melanoma.

PURPOSE: Concurrent biochemotherapy results in high response rates but also significant toxicity in patients with metastatic melanoma. We attempted to improve its efficacy and decrease its toxicity by using decrescendo dosing of interleukin-2 (IL-2), posttreatment granulocyte colony-stimulating factor (G-CSF), and low-dose tamoxifen. PATIENTS AND METHODS: Forty-five patients with poor prognosis metastatic melanoma were treated at a community hospital inpatient oncology unit affiliated with the John Wayne Cancer Institute (Santa Monica, CA) between July 1995 and September 1997. A 5-day modified concurrent biochemotherapy regimen of dacarbazine, vinblastine, cisplatin, decrescendo IL-2, interferon alfa-2b, and tamoxifen was repeated at 21-day intervals. G-CSF was administered beginning on day 6 for 7 to 10 days. RESULTS: The overall response rate was 57% (95% confidence interval, 42% to 72%), the complete response rate was 23%, and the partial response rate was 34%. Complete remissions were achieved in an additional 11% of patients by surgical resection of residual disease after biochemotherapy. The median time to progression was 6.3 months and the median duration of survival was 11.4 months. At a maximum follow-up of 36 months (range, 10 to 36 months), 32% of patients are alive and 14% remain free of disease. Decrescendo IL-2 dosing and administration of G-CSF seemed to reduce toxicity, length of hospital stay, and readmission rates. No patient required intensive care unit monitoring, and there were no treatment-related deaths. CONCLUSION: The data from this study indicate that the modified concurrent biochemotherapy regimen reduces the toxicity of concurrent biochemotherapy with no apparent decrease in response rate in patients with poor prognosis metastatic melanoma.

Modulation of channel function by polyamines.

Polyamines are found in every cell of the body and the intricate enzymatic reactions responsible for their metabolism and transport in mammalian cells are now well understood. Despite intense efforts, elucidation of the role of polyamines has suffered in that little information of physiological relevance has surfaced. Recently, recombinant receptor techniques and increased availability of polyamine analogues have revealed, as discussed here by David Johnson, modulation of NMDA receptors by polyamines by reversal of tonic proton inhibition, and a function for these compounds as 'intrinsic rectifier factors' for K+ channels.

Spermatozoa scattering by a microchannel feature: an elastohydrodynamic model.

Sperm traverse their microenvironment through viscous fluid by propagating flagellar waves; the waveform emerges as a consequence of elastic structure, internal active moments and low Reynolds number fluid dynamics. Engineered microchannels have recently been proposed as a method of sorting and manipulating motile cells; the interaction of cells with these artificial environments therefore warrants investigation. A numerical method is presented for large-amplitude elastohydrodynamic interaction of active swimmers with domain features. This method is employed to examine hydrodynamic scattering by a model microchannel backstep feature. Scattering is shown to depend on backstep height and the relative strength of viscous and elastic forces in the flagellum. In a 'high viscosity' parameter regime corresponding to human sperm in cervical mucus analogue, this hydrodynamic contribution to scattering is comparable in magnitude to recent data on contact effects, being of the order of 5°-10°. Scattering can be positive or negative depending on the relative strength of viscous and elastic effects, emphasizing the importance of viscosity on the interaction of sperm with their microenvironment. The modulation of scattering angle by viscosity is associated with variations in flagellar asymmetry induced by the elastohydrodynamic interaction with the boundary feature.

Immunologic effects of combined protease inhibitor and reverse transcriptase inhibitor therapy in previously treated chronic HIV-1 infection.

OBJECTIVE: To evaluate the efficacy of combination protease and reverse transcriptase inhibitor therapy in correcting HIV-1-induced lymphocyte subset abnormalities in previously treated adults. DESIGN: A 48-week observational study of lymphocyte subsets in 12 participants in the Multicenter AIDS Cohort Study who were already taking at least one reverse transcriptase inhibitor and added a protease inhibitor to their treatment regimen. Comparison groups were HIV-seronegative homosexual men, HIV-seronegative heterosexual men, and homosexual HIV-1-infected men who were long-term non-progressors. METHODS: Three-color immunofluorescence and monoclonal antibodies were used to assess HIV-1-induced lymphocyte subset alterations related to immune deficiency and immune activation. Plasma HIV-1 RNA levels were monitored to assess suppression of viral replication. RESULTS: CD4+ cell counts significantly increased and lymphocyte activation measured as CD38 and HLA-DR expression on CD8+ T cells significantly decreased by 48 weeks. CD4+ cell values remained abnormal even in those who were fully suppressed. Some T-cell activation markers decreased to levels observed in long-term non-progressors. The increase in CD4+ T-cell numbers reached a plateau by week 24, but the increase in resting HLA-DR- CD38-T cells was sustained through week 48. Proportions of CD45RA+ CD62L-selectin+ and CD28+ CD4+ T-cell subsets and Fas expression were not abnormal at baseline compared with seronegative homosexual controls. CONCLUSIONS: The most significant impact of suppression of viral replication was reversal of T-cell activation. However, normalization of lymphocyte subset perturbations associated with chronic HIV-1 infection was not achieved after 1 year of treatment with current combination antiretroviral regimens. More profound viral suppression, therapy for longer than 1 year, or immunologic augmentation may be needed to fully reverse the abnormalities.

IgM anti-ganglioside antibodies induced by melanoma cell vaccine correlate with survival of melanoma patients.

Melanoma cells express ganglioside antigens GM3, GD3, GM2, and GD2 on their surface. This study examined whether immunization with a melanoma cell vaccine induced anti-ganglioside antibody responses in melanoma patients and whether these responses were correlated with survival. Sixty-six patients who had received melanoma cell vaccine immunotherapy after surgical removal of regional metastatic melanoma were identified. Cryopreserved serum samples from these patients were used in an enzyme-linked immunsorbent assay to determine the IgM antibody levels to GM2, GD2, GM3, and GD3 prior to melanoma cell vaccine treatment and 4 wk after the first melanoma cell vaccine immunization. All antibody levels significantly increased by week 4 (p < 0.001 for all four antibodies) and all increases were significantly associated with survival (anti-GD2, p < 0.001; anti-GM2, p = 0.001; anti-GD3, p < 0.001; anti-GM3, p < 0.001). Anti-tumor activity of these antibodies was proved using five representative antibody-positive sera in a complement-dependent cytotoxicity assay with cultured melanoma cell lines. These studies suggest that GM2, GD2, GM2, and GD3 expressed by melanoma cells can induce specific IgM antibodies and that high levels of these antibodies might have a beneficial impact on survival.

Metabolic and weight-loss effects of a long-term dietary intervention in obese patients.

BACKGROUND: Obesity is a chronic disease that has become one of the most serious health problems in Western society. OBJECTIVE: We assessed the long-term effects of an energy-restricted diet combined with 1 or 2 daily meal replacements on body weight and biomarkers of disease risk in 100 obese patients. DESIGN: Phase 1 consisted of a 3-mo, prospective, randomized, parallel intervention study of 2 dietary interventions to reduce weight. The energy-restricted diet (5.2-6.3 MJ/d) consisted of conventional foods (group A) or an isoenergetic diet with 2 meals and 2 snacks replaced daily by energy-controlled, vitamin-and-mineral-supplemented prepared foods (group B). Phase 2 consisted of a 24-mo, case-control, weight-maintenance study with an energy-restricted diet and 1 meal and 1 snack replaced daily for all patients. RESULTS: Total weight loss (as a percentage of initial body weight) was 5.9+/-5.0% in group A and 11.3+/-6.8% in group B (P < 0.0001). During phase 1, mean weight loss in group B (n = 50) was 7.1+/-3.5 kg, with significant reductions in plasma triacylglycerol, glucose, and insulin concentrations (P < 0.0001). Group A patients (n = 50) lost an average of 1.3+/-2.2 kg with no significant improvements in these biomarkers. During phase 2, both groups lost on average an additional 0.07% of their initial body weight every month (P < 0.01). During the 27-mo study, both groups experienced significant reductions in systolic blood pressure and plasma concentrations of triacylglycerol, glucose, and insulin (P < 0.01). CONCLUSION: These findings support the hypothesis that defined meal replacements can be used for successful, long-term weight control and improvements in certain biomarkers of disease risk.

Preparation of [14C]colestipol hydrochloride and its disposition in the human, dog and rat.

Colestipol hydrochloride, a polymeric, ion-exchange type, hypocholesterolemic agent, acting by sequestering bile acids, was labeled with carbon-14. The disposition of the labeled material was studied in the human, dog and rat. The extent of absorption from the gastrointestinal tract, as judged by urinary excretion of radioactivity, was very small and correlated well with the contents of water-soluble and dialyzable materials in the colestipol hydrochloride. Results were consistent with the dialyzable material in the drug being the absorbable species.

Marked enhancement in myocardial function resulting from overexpression of a human beta-adrenergic receptor gene.

Transgenic mice with intense cardiac expression of a human beta-adrenergic receptor gene were engineered and shown to display marked improvements in baseline myocardial and left ventricular function. Heart/body weight ratios and histologic appearance were not found to be significantly altered, suggesting that receptor gene expression did not induce pathologic changes. Given the substantial reduction in beta-adrenergic receptor density and resultant reduction in inotropic responsiveness observed in chronic heart failure, these findings represent a novel approach for increasing myocardial function with important clinical implications.

The effects of potassium on the rat middle cerebral artery.

After traumatic brain injury, extracellular K(+) in brain can dramatically increase. We studied the effects of increased K(+) on the isolated pressurized rat middle cerebral artery (MCA). MCAs (200-250 microm OD) were isolated, cannulated with glass micropipettes, and pressurized. K(+) was increased in the extraluminal bath using three paradigms: (1) isotonic K(+) (K(iso)) where increases in K(+) were offset by decreases in Na(+), (2) hypertonic K(+) (K(hyper)) where K(+) was increased without a concomitant adjustment of Na(+), and (3) K(suc), a solution using K(iso) but with the addition of sucrose to obtain a hypertonic solution. Increases in K(+) in the extraluminal bath produced significant dilations (approximately 20%) at 21 mM K(+) in all three groups (K(iso), K(hyper), and K(suc)). With the K(hyper) and K(suc) groups, the magnitude of the dilation diminished with further increases in K(+). L-NAME (10(-5) M), an inhibitor of nitric oxide synthase, had no effect on the response of the K(hyper) and K(suc) groups at 21 mM but significantly enhanced constrictions of the MCAs above 40 mM K(+) compared to the control. The K(iso) group was not affected by L-NAME at any K(+) concentration and showed profound constrictions above 40 mM K(+). We conclude that changes in the K(+) concentration and osmolality of the extracellular fluid may have profound effects on the cerebral vasculature.

Nitric oxide in the potassium-induced response of the rat middle cerebral artery: a possible permissive role.

In the middle cerebral artery (MCA), the presence of nitric oxide (NO) is responsible for maintaining a more dilated state than in its absence during increases in extracellular K(+) and osmolality. The purpose of the present study was to determine whether the involvement of NO was due to (a) a direct effect of the K(+)/osmolality (K(hyper)) on the endothelium or (b) a 'permissive' role of NO. MCAs (approximately 210 microm o.d.) were isolated, cannulated with glass micropipettes, and pressurized to 85 mmHg. When K(+) (KCl) in the extraluminal bath was increased to 21 mM, the diameter increased by 15-20% with the magnitude of dilation diminishing with further increases in K(hyper). The addition of N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-5) mM), an inhibitor of nitric oxide synthase, had no significant effect on dilations at lower K(hyper) concentrations but constricted the arteries relative to the control at 51, 66, and 81 mM K(hyper). In the presence of L-NAME, the addition of an exogenous NO donor, S-nitroso-N-acetylpenicillamine (SNAP, 10(-8) M) or an analog of cGMP, 8-bromo-cGMP (6x10(-5) M), tended to restore the response of K(hyper)to near the original response. We conclude that the basal release of NO from the endothelium plays a permissive role in the K(hyper)-induced response.