Binary Colloidal Nanoparticle Concentration Gradients in a Centrifugal Field at High Concentration.

Binary colloidal nanoparticles have been found to form different types of crystalline phases at varied radial positions in a centrifugal field by Chen et al. ( ACS Nano 2015, 9, 6944-50). The variety of binary phase behaviors resulted from the two different nanoparticle concentration gradients, but to date, the gradients can only be empirically controlled. For the first time, we are able to measure, fit, and simulate binary hard-sphere colloidal nanoparticle concentration gradients at high particle concentrations up to 30 vol %, which enables tailor-made gradients in a centrifugal field. By this means, a continuous range of binary particle concentration ratios can be accessed in one single experiment to obtain an extended phase diagram. By dispersing two differently sized silica nanoparticles labeled with two different fluorescence dyes in a refractive index matching solvent, we can use a multi-wavelength analytical ultracentrifuge (MWL-AUC) to measure the individual concentration gradient for each particle size in sedimentation-diffusion equilibrium. The influence of the remaining slight turbidity at high concentration can be corrected using the MWL spectra from the AUC data. We also show that the experimental concentration gradients can be fitted using a noninteracting nonideal sedimentation model. By using these fitted parameters, we are able to simulate nanoparticle concentration gradients, which agreed with the subsequent experiments at a high concentration of 10 vol % and thus allowed for the simulation of binary concentration gradients of hard-sphere nanoparticles in preparative ultracentrifuges (PUCs). Finally we demonstrated that by simulating the concentration gradients in PUCs, a continuous and extended binary nanoparticle phase diagram can be obtained by simply studying the structure evolution along the centrifugal field for one single sample instead of a large number of experiments with discrete compositions as in conventional studies.

Estimation of tile drainage contribution to streamflow and nutrient loads at the watershed scale based on continuously monitored data.

Nitrogen losses from artificially drained watersheds degrade water quality at local and regional scales. In this study, we used an end-member mixing analysis (EMMA) together with high temporal resolution water quality and streamflow data collected in the 122 km2 Otter Creek watershed located in northeast Iowa. We estimated the contribution of three end-members (groundwater, tile drainage, and quick flow) to streamflow and nitrogen loads and tested several combinations of possible nitrate concentrations for the end-members. Results indicated that subsurface tile drainage is responsible for at least 50% of the watershed nitrogen load between April 15 and November 1, 2015. Tiles delivered up to 80% of the stream N load while providing only 15-43% of the streamflow, whereas quick flows only marginally contributed to N loading. Data collected offer guidance about areas of the watershed that should be targeted for nitrogen export mitigation strategies.

Nuclear deformation and neutron excess as competing effects for dipole strength in the pygmy region.

The electromagnetic dipole strength below the neutron-separation energy has been studied for the xenon isotopes with mass numbers A=124, 128, 132, and 134 in nuclear resonance fluorescence experiments using the γELBE bremsstrahlung facility at Helmholtz-Zentrum Dresden-Rossendorf and the HIγS facility at Triangle Universities Nuclear Laboratory Durham. The systematic study gained new information about the influence of the neutron excess as well as of nuclear deformation on the strength in the region of the pygmy dipole resonance. The results are compared with those obtained for the chain of molybdenum isotopes and with predictions of a random-phase approximation in a deformed basis. It turned out that the effect of nuclear deformation plays a minor role compared with the one caused by neutron excess. A global parametrization of the strength in terms of neutron and proton numbers allowed us to derive a formula capable of predicting the summed E1 strengths in the pygmy region for a wide mass range of nuclides.

Foodborne outbreaks of shigellosis in the USA, 1998-2008.

We examined reported outbreaks of foodborne shigellosis in the USA from 1998 to 2008 and summarized demographic and epidemiological characteristics of 120 confirmed outbreaks resulting in 6208 illnesses. Most reported foodborne shigellosis outbreaks (n = 70, 58%) and outbreak-associated illnesses (n = 3383, 54%) were restaurant-associated. The largest outbreaks were associated with commercially prepared foods distributed in multiple states and foods prepared in institutional settings. Foods commonly consumed raw were implicated in 29 (24%) outbreaks and infected food handlers in 28 (23%) outbreaks. Most outbreaks (n = 86, 72%) were caused by Shigella sonnei. Targeted efforts to reduce contamination during food handling at multiple points in the food processing and distribution system, including food preparation in restaurants and institutional settings, could prevent many foodborne disease outbreaks and outbreak-related illnesses including those due to Shigella.

Advanced Multicompartment Diffusion MRI Models and Their Application in Multiple Sclerosis.

Conventional MR imaging techniques are sensitive to pathologic changes of the brain and spinal cord seen in MS, but they lack specificity for underlying axonal and myelin integrity. By isolating the signal contribution from different tissue compartments, newly developed advanced multicompartment diffusion MR imaging models have the potential to detect specific tissue subtypes and associated injuries with increased pathologic specificity. These models include neurite orientation dispersion and density imaging, diffusion basis spectrum imaging, multicompartment microscopic diffusion MR imaging with the spherical mean technique, and models enabled through high-gradient diffusion MR imaging. In this review, we provide an appraisal of the current literature on the physics principles, histopathologic validation, and clinical applications of each of these techniques in both brains and spinal cords of patients with MS. We discuss limitations of each of the methods and directions that future research could take to provide additional validation of their roles as biomarkers of axonal and myelin injury in MS.

Basic molecular fingerprinting of immature cerebellar cortical inhibitory interneurons and their precursors.

While the development of cerebellar granule and Purkinje neurons has been extensively studied, little is known about the developmental mechanisms that lead to the generation and diversification of inhibitory GABAergic interneurons of the cerebellar cortex. To address this issue, we compared gene expression in complete, early postnatal murine cerebella to that in cerebella from which immature inhibitory interneurons and their precursors had been stripped based on their expression of green fluorescent protein (GFP) from the Pax2 locus. We identified some 300 candidate genes selectively enriched within immature cerebellar cortical inhibitory interneurons and/or their precursors, many of which were also expressed in their adult descendants and/or the embryonic cerebellar ventricular epithelium that gives rise to these cells. None of the genes identified, among them Tcfap2alpha, Tcfap2beta, Lbxcor1 and Lbx1, was cell-type specific. Rather, gene expression, and also splicing, changed dynamically during development and rather reflects stage of differentiation than lineage. Consistently, cluster analysis of transcriptional regulators and genes specific for adult cerebellar GABAergic cells does not suggest a hierarchical lineage relationship or an early commitment of subtypes of cerebellar cortical inhibitory interneurons. Together, these data support the notion that diversification of cerebellar inhibitory interneurons is highly regulative and subject to local signaling to postmigratory precursors.

Dynamic organization of developing Purkinje cells revealed by transgene expression.

The cerebellum has many properties that make it a useful model for investigating neural development. Purkinje cells, the major output neurons of the cerebellar cortex, have drawn special attention because of the availability of biochemical markers and mutants that affect their development. The spatial expression of L7, a protein specific for Purkinje cells, and L7 beta Gal, a gene expressed in transgenic mice that was constructed from the L7 promoter and the marker beta-galactosidase, delineated bands of Purkinje cells that increased in number during early postnatal development. Expression of the transgene in adult reeler mutant mice, which show inverted cortical lamination, and in primary culture showed that the initial expression of L7 is intrinsic to Purkinje cells and does not depend on extracellular signals. This may reflect an underlying developmental map in cerebellum.

Electrical activity in cerebellar cultures determines Purkinje cell dendritic growth patterns.

In primary dissociated cultures of mouse cerebellum a number of Purkinje cell-specific marker proteins and characteristic ionic currents appear at the appropriate developmental time. During the first week after plating, Purkinje cell dendrites elongate, but as electrical activity emerges the dendrites stop growing and branch. If endogenous electrical activity is inhibited by chronic tetrodotoxin or high magnesium treatment, dendrites continue to elongate, as if they were still immature. At the time that branching begins, intracellular calcium levels become sensitive to tetrodotoxin, suggesting that this cation may be involved in dendrite growth. Even apparently mature Purkinje cells alter their dendritic growth in response to changes in activity, suggesting long-term plasticity.

fos-lacZ transgenic mice: mapping sites of gene induction in the central nervous system.

A transgenic mouse line containing a fos-lacZ fusion gene was derived in which beta-galactosidase activity identified cell populations expressing fos either constitutively or after stimulation. Seizures and light pulses induced nuclear lacZ activity in defined populations of neurons in vivo, and an array of neurotransmitters, including glutamate, induced the transgene in primary brain cultures. In unstimulated mice, the major sites of fos-lacZ expression were skin, hair follicle, and bone. fos-lacZ mice provide a new avenue for activity mapping studies based on gene expression.

Control of segment-like patterns of gene expression in the mouse cerebellum.

A Purkinje cell-specific transgene, L7-lacZ, is expressed in a series of parasagitally oriented stripes in the mouse cerebellum. This banding pattern can be perturbed by promoter mutation, showing that a combination of positive and negative control elements contributes to the temporal and spatial map of L7 gene expression. In addition to the parasagittal stripes, certain mutations reveal Purkinje cells organized into compartments oriented in the transverse plane of the cerebellum. Transcription factors of the POU or homeobox families appear to be involved in controlling L7 expression in the transverse orientation. Strikingly, some of the domains of gene expression revealed by the mutations appear to correspond to functional compartments of Purkinje cells, thereby suggesting an underlying genetic principle used to orchestrate functional organization in the nervous system.

Suppression of tumor-derived Semaphorin 7A and genetic ablation of host-derived Semaphorin 7A impairs tumor progression in a murine model of advanced breast carcinoma.

Solid tumors can generate a plethora of neurogenesis-related molecules that enhance their growth and metastasis. Among them, we have identified axonal guidance molecule Semaphorin 7A (SEMA7A) in breast cancer. The goal of this study was to determine the therapeutic effect of suppressing SEMA7A levels in the 4T1 murine model of advanced breast carcinoma. We used anti-SEMA7A short hairpin RNA (shRNA) to gene silence SEMA7A in 4T1 mammary tumor cells. When implanted into the mammary fat pads of syngeneic mice, SEMA7A shRNA-expressing 4T1 tumors exhibited decreased growth rates, deferred metastasis and reduced mortality. In vitro, SEMA7A shRNA-expressing 4T1 cells had weakened proliferative, migratory and invasive abilities, and decreased levels of mesenchymal factors. Atomic force microscopy studies showed that SEMA7A shRNA-expressing 4T1 cells had an increase in cell stiffness that corresponded with their decreased malignant potential. Genetic ablation of host-derived SEMA7A further enhanced the antitumor effects of SEMA7A shRNA gene silencing in 4T1 cells. Our preclinical findings demonstrate a critical role for SEMA7A in mediating mammary tumor progression.

From zebra stripes to postal zones: deciphering patterns of gene expression in the cerebellum.

The analysis of patterned gene expression has been an important tool for dissecting the molecular and developmental bases of functional compartmentalization in the mammalian cerebellum. In particular, sagittally-oriented cellular aggregates arranged along the mediolateral axis are the patterning element most commonly invoked to illustrate cerebellar compartmentalization, and these are revealed both by patterns of afferent projection and by a number of classical biochemical markers that are distributed in a pattern of'zebra stripes'. Compartmentation along both the mediolateral and rostrocaudal axes might be linked mechanistically to segmentation in the fruit fly, since early cerebellar development is especially dependent upon the expression of mammalian homologs of Drosophila segmentation genes. In addition, as has been demonstrated in the retinotectal system, some of these genes are likely to control positional information required for the sagittal organization of cerebellar afferent projections. However, in contrast to these global or macro zones, the cerebellum is also compartmentalized at the subcellular or micro level. This can be visualized by differential patterns of mRNA distribution within the sole cerebellar efferent system, the Purkinje cell, defining within such cells a number of distinct subcellular domains or 'postal zones'. The global versus subcellular levels of cerebellar compartmentalization are related since they both appear to be linked to patterns of afferent innervation.A major goal of cerebellar research will be to unravel the true nature of such a relationship, and its relevance to function and behavior.

Functional expression of the new gap junction gene connexin47 transcribed in mouse brain and spinal cord neurons.

A new mouse gap junction gene that codes for a protein of 46,551 Da has been identified and designated connexin47 (Cx47). It mapped as a single-copy gene to mouse chromosome 11. In human HeLa cells and Xenopus oocytes, expression of mouse Cx47 or a fusion protein of Cx47 and enhanced green fluorescent protein induced intercellular channels that displayed strong sensitivity to transjunctional voltage. Tracer injections in Cx47-transfected HeLa cells revealed intercellular diffusion of neurobiotin, Lucifer yellow, and 4',6-diamidino-2-phenylindole. Recordings of single channels yielded a unitary conductance of 55 pS main state and 8 pS substate. Cx47 mRNA expression was high in spinal cord and brain but was not found in retina, liver, heart, and lung. A low level of Cx47 expression was detected in ovaries. In situ hybridizations demonstrated high expression in alpha motor neurons of the spinal cord, pyramidal cells of the cortex and hippocampus, granular and molecular layers of the dentate gyrus, and Purkinje cells of the cerebellum as well as several nuclei of the brainstem. This expression pattern is distinct from, although partially overlapping with, that of the neuronally expressed connexin36 gene. Thus, electrical synapses in adult mammalian brain are likely to consist of different connexin proteins depending on the neuronal subtype.

Lead neuropathy. 1) Morphometry, nerve conduction, and choline acetyltransferase transport: new finding of endoneurial edema associated with segmental demyelination.

Morphometric and pathologic studies along the length of the peripheral nervous system were obtained in groups of rats fed 4% lead carbonate for 3 and 6 months and in match-fed controls. The number and diameter histograms of L6 cytons of spinal ganglia and of myelinated fibers of proximal and distal portions of peroneal and sural nerve were not significantly different from the control groups. On the other hand, segmental demyelination occurred approximately as frequently in proximal as in distal parts of nerves. At 3 months approximately 1/3 of teased myelinated fibers showed changes of segmental demyelination (Condition C), or of remyelination after segmental demyelination (Condition F) or of both segmental demyelination and of remyelination (Condition D), while at 6 months more than 4/5ths of fibers showed these changes. As expected, regression lines of axonal area on number of lamellae of myelin, were less steep in nerves of rats fed on lead for 6 months as compared to controls. Axonal transport of choline acetyltransferase in lead neuropathy did not differ from that in control rats. As expected from the studies of others, conduction velocity of myelinated fibers of caudal nerve were low. A new finding was the often quite striking increase of transverse fascicular area of peripheral nerves. This was due to edema which appeared to develop at about the time of onset os segmental demyelination. Although the edema may be an epiphenomenon, it could be an important observation bearing on the development of lead neuropathy. It would be important to know next whether or not the blood nerve barrier is altered in lead neuropathy.

Quantification of p38/synaptophysin in highly purified adrenal medullary chromaffin vesicles.

Chromaffin vesicles were first purified by differential and density gradient centrifugation in isotonic (Percoll) gradients. In subsequent sucrose gradients p38/synaptophysin exhibited the same distribution as established marker substances of chromaffin vesicles. Quantification of immunoblots revealed that 750 ng p38/synaptophysin per mg of protein were present in the chromaffin vesicles recovered from the sucrose gradient. Thus the amount of p38/synaptophysin per mg protein of chromaffin vesicles is about 100 times lower than that observed in clear (synaptic) vesicles. However, because of the large difference in surface area and protein content, the amount of p38/synaptophysin per single vesicle is the same in both types of organelles.

The alpha1/2 helical backbone of the prodomains defines the intrinsic inhibitory specificity in the cathepsin L-like cysteine protease subfamily.

Proregions of papain-like cysteine proteases are potent and often highly selective inhibitors of their parental enzymes. The molecular basis of their selectivity is poorly understood. For two closely related members of the cathepsin L-like subfamily we established strong selectivity differences. The propeptide of cathepsin S was observed to inhibit cathepsin L with a K(i) of 0.08 nM, yet cathepsin L propeptide inhibited cathepsin S only poorly. To identify the respective structural correlates we engineered chimeric propeptides and compared their inhibitory specificity with the wild-types. Specificity resided in the N-terminal part, strongly suggesting that the backbone of the prodomain was the underlying structure.

Clinical vs quantitative evaluation of cutaneous sensation.

A comparison of the evaluation of cutaneous sensation by neurologic examination and by quantitative assessment in 107 patients with various neuromuscular disease has shown that there is a reasonably good correlation between the recognition of abnormalities of cutaneous sensation by clinical and by quantitative methods. Clinical neurologists tend to underestimate abnormalities of touch-pressure sensation. The quantitative evaluation of cutaneous sensation is useful in quality control of the clinical examination, in correlating the nature of sensation loss with the compound action potential and morphometry of biopsied nerve, in providing a score of sensation to follow the course and the effect of treatment protocols, and in monitoring for an adverse effect on sensation by an environmental or industrial poison. There is a good correlation between abnormality of touch-pressure sensation and loss of large myelinated fibers of sural nerve in neuropathy.

Myoclonus dystonia: possible association with obsessive-compulsive disorder and alcohol dependence.

BACKGROUND: Inherited myoclonus-dystonia (M-D) is a disorder that is characterized primarily by myoclonic jerks and is often accompanied by dystonia. In addition to motor features, psychiatric disease is reported in some families. METHODS: To determine whether the same genetic etiology underlies both neurologic and psychiatric signs, the authors studied psychiatric symptoms in nonmanifesting carriers (NMC), noncarriers (NC), and manifesting carriers (MC) in three families demonstrating linkage of M-D to the 7q21 locus. Interviewers administered the computerized version of the Composite International Diagnostic Interview. Algorithms for the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition diagnosis of obsessive-compulsive disorder (OCD), generalized anxiety disorder, major affective disorder, alcohol abuse, alcohol dependence, drug abuse, and drug dependence were used. Rates of disorders among the MC, NMC, and NC were compared. RESULTS: Of 55 participating individuals, 16 were MC, 11 were NMC, and 28 were NC. The rate of OCD was greater in carriers (5/27) compared with NC (0/28) (p = 0.023). It was also greater in the symptomatic gene carriers (4/16) compared with the asymptomatic group (1/11) (p = 0.022). Alcohol dependence was increased in the symptomatic carriers (7/16) (p = 0.027), but not in the carrier group overall (7/27). CONCLUSION: OCD may be associated with the DYT11 M-D gene; however, a larger sample is necessary to confirm this finding. Alcohol dependence is highly associated with expressing symptoms of M-D. This may be explained by self-medication with alcohol to improve motor symptoms of M-D.

Phenotypic features of myoclonus-dystonia in three kindreds.

BACKGROUND: Myoclonus-dystonia (M-D) is a movement disorder with involuntary jerks and dystonic contractions. Autosomal dominant alcohol-responsive M-D is associated with mutations in the epsilon-sarcoglycan gene (SGCE) (six families) and with a missense change in the D2 dopamine receptor (DRD2)gene (one family). OBJECTIVE: To investigate the clinical phenotype associated with M-D including motor symptoms, psychiatric disorders, and neuropsychological deficits. METHODS: Fifty individuals in three M-D families were evaluated and a standardized neurologic examination and DNA analysis were performed. Psychiatric profiles were established with the Diagnostic Interviews for Genetic Studies (DIGS) and the Yale-Brown Obsessive-Compulsive Scale (YBOCS). Cognition was evaluated with standardized neuropsychological tests. RESULTS: Distinct truncating mutations in the SGCE gene were identified in each family. Additionally, a missense alteration in the DRD2 gene was previously found in one family. Motor expression was variable, with onset of myoclonus or dystonia or both affecting the upper body and progression to myoclonus and dystonia in most cases. Psychiatric profiles revealed depression, obsessive-compulsive disorder, substance abuse, anxiety/panic/phobic disorders, and psychosis in two families, and depression only in the third family. Averaged scores from cognitive testing showed impaired verbal learning and memory in one family, impaired memory in the second family, and no cognitive deficits in the third family. CONCLUSIONS: Cognitive deficits may be associated with M-D. Psychiatric abnormalities correlate with the motor symptoms in affected individuals. Assessment of additional M-D families with known mutations is needed to determine whether these are characteristic phenotypic manifestations of M-D.

Growth factor-induced c-fos expression defines distinct subsets of midbrain dopaminergic neurons.

Growth factors are considered pivotal for the development, maintenance, and function of mesencephalic dopaminergic neurons. Recent studies have identified a plethora of growth factors which support the survival and differentiation of embryonic dopaminergic neurons. However, the exact cellular targets of these growth factors, and, thus, their precise mechanisms of action, remain largely unknown. To identify these cellular targets, we analysed, at the single cell level, growth factor-induced c-fos expression in dissociated mesencephalic cell cultures derived from a fos-lac Z transgenic mouse line. Pharmacological interference with cell-cell communication was utilized to control for direct growth factor effects. beta-Galactosidase-expressing cells were phenotypically characterized by immunocytochemistry to specific neural cell markers. Glia cell line-derived neurotrophic factor, basic fibroblast growth factor, brain-derived neurotrophic factor, and neurotrophin-3 directly induced Fos expression in differently sized, yet overlapping, populations of tyrosine hydroxylase-immunoreactive dopaminergic neurons. In an additional subpopulation of dopaminergic neurons, neurotrophin-3 induced fos-lac Z expression indirectly through a glutamate-mediated activation of N-methyl-D-aspartate receptors. Consistent with their proposed glial-mediated mode of action, transforming growth factor alpha and platelet-derived growth factor induced Fos expression predominantly in glia but only in a very small number of dopaminergic neurons. These findings demonstrate that individual dopaminergic neurons represent the direct targets of different sets of extracellular growth factors. Our findings further establish that growth factors affect dopaminergic neurons by indirect mechanisms which require specific cell-cell communication. These data also suggest a potential role for growth factors in the establishment of the morphological and functional diversity of midbrain dopaminergic neurons.