A NOMARSKI INTERFERENCE STUDY OF MEGASPOROGENESIS AND MEGAGAMETOGENESIS IN SMELOWSKIA CALYCINA (CRUCIFERAE)

Ovule development, megasporogenesis and megagametogenesis in an aneuploid population of the arctic-alpine crucifer, Smelowskia calycina, were examined to test for the possibility of apomictic seed production. Whole mounts of ovules cleared in Herr's “4½” clearing fluid were examined using Nomarski differential interference microscopy. The campylotropous ovule was bitegmic, with a micropyle formed by both integuments. The single archesporial cell of a crassinucellar nucellus functioned directly as a megasporocyte, dividing to form a linear tetrad of megaspores. The chalazal megaspore divided to form an 8-nucleate, 7-celled gametophyte of the Polygonum type, having hooked synergids with a well-developed filiform apparatus and polar nuclei that fused prior to fertilization. In the absence of any anomalous development indicative of agamospermy, seed production was assumed to be sexual.     

A Method of Permanently Mounting Biological Tissue Cleared in Herr's Four-And-A-Half Clearing Fluid

A technique which should be generally applicable for preparing permanent mounts of tissue cleared in Herr's four-and-a-half clearing fluid is described. This technique involves transferring plant or animal tissues through a series of solutions consisting of Pienarr's fixative, Herr's clearing fluid, chloral hydrate, acetone and finally polyester resin for mounting. Material prepared using this method is exceptionally transparent and well preserved, and is suitable for either phase contrast or Nomarski interference microscopy.     

Use of the Four-and-a-Half Clearing Technique to Study Gymnosperm EmbryoIogy: Cunninghamia lanceolata

Since its introduction in 1971, the four-and-a-half clearing technique has been widely applied to the study of ovule and female gametophyte development in flowering plants as an alternative to the more arduous paraffin section methods. The technique has undergone several modifications that have broadened its application in studies of Angiosperm embryology. To date, however, the technique has not been successfully applied to embryological features of Gymnosperms. Dark coloration caused by naturally occurring substances and by-products of fixation render the clearing fluid ineffective, and special pretreatment methods used to remove dark substances in Angiosperm ovules have little or no effect on Gymnosperm material. In the technique reported here, paraffin sections of ovules and young seeds of Cunninghamia lanceolata 80-120 μm thick are cleared in benzyl benzoate-412 clearing fluid and examined with phase contrast optics. Observations of the mature female gametophyte in these cleared preparations are compared with those obtained from 10 μm sections, stained with safranin and fast green, and examined with bright-field optics. Although contrast and definition are more pronounced in stained sections than in cleared ones, the differences would not alter one's interpretation of characteristic structural features. The thick, cleared section offers an advantage over the thin, stained one in that many structural entities are contained within a single section rather than spread through several serial sections. The time required for clearing thick sections is much shorter than that required for making permanent stained preparations.     

Assessing land clearing potential in the Canadian agriculture–forestry interface with a multi-attribute frontier approach

The pattern of forest land clearing in a region can be viewed as a gauge of sustainable (or unsustainable) use of agricultural and forest resources. In this study we examine the geographical distribution of land clearing potential in the Canadian agriculture–forestry interface and propose a new landscape-scale indicator that quantifies this potential. We consider the possibility that forested land will be cleared for agriculture as a trade-off between the land's capacity to support agriculture and its comparative value if it remains forested. However, this trade-off is complicated by the land's susceptibility to fragmentation (and subsequent conversion), which derives from the local pattern of forest, agriculture and other land cover types. We find the locations in the agriculture–forestry interface with the highest land clearing potential by delineating nested multi-attribute frontiers in the dimensions of the land's agricultural capacity, its estimated forest productivity and its fragmentation potential. The multi-attribute frontier concept addresses our lack of knowledge about the relative importance of these multiple drivers of land conversion by objectively combining them into a single-dimensional land clearing pressure metric in a geographical setting. Overall, our approach provides a simple yet informative indicator which reveals the geographical stratification of land clearing pressures across large regions. In general, the spatial delineation of areas with high land clearing potential agrees well with recent evidence of land clearing and deforestation events in Canada.     

A NEW CLEARING-SQUASH TECHNIQUE FOR THE STUDY OF OVULE DEVELOPMENT IN ANGIOSPERMS

The simple, efficient method described here for the study of ovule and megagametophyte development in angiosperms provides for the extension of investigation beyond the limits imposed by the traditional but arduous section technique. Excised pistils previously fixed in FPA₅₀ and stored in 70 % ethanol are placed in a clearing fluid composed of lactic acid (85 %), chloral hydrate, phenol, clove oil, and xylene (2:2:2:2:1, by weight). After 24 hr, ovules dissected from the ovularies are transferred with some of the fluid to a slide, covered so that the cover glass is supported laterally by two permanently affixed covers, and examined with phase contrast optics. The unique action of the clearing fluid permits the study of cellular structure with the phase oil objective focused at any focal plane within the ovule. Downward focusing thus reveals a series of optical sections in the sagittal, frontal, or transverse plane depending on the orientation of the ovule. Orientation can be altered by a slight shifting of the cover glass on the lateral support mounts. The ovules become quite fragile in the clearing fluid. Pressure applied to the cover glass gradually breaks the ovule apart without disrupting the structural integrity of individual cells. This squash procedure provides for extending observations to cytological features of megasporocytes, megaspores, and megagametophytes previously identified in intact ovules. The new method is applied here to the study of ovule development in two unrelated species, Cassia abbreviata Oliver var. granitica Bak. f. (Leguminosae) and Ludwigia uruguayensis (Camb.) Hara. (Onagraceae). For best results, the ovules of Ludwigia must be pretreated in lactic acid (85 %) for 24 hr prior to application of the clearing fluid. Other methods for pretreatment likely will be required as the technique is applied to a wider range of flowering plant species.     

An Analysis of Methods for Permanently Mounting Ovules Cleared in Four-and-a-Half Type Clearing Fluids

Ovules cleared in benzyl benzoate-4 1/2 clearing fluid can be permanently mounted in Piccolyte or Permount by replacing the clearing fluid with absolute ethanol, upgrading the ovules in mixtures of ethanol and xylene (3:1, 2:2, 1:3, and xylene), and mounting them in either mountant under the supported coverglass of a Raj slide. Optical sagittal sections through the ovules resemble microtome sections in that the protoplasts are slightly shrunken away from the cell walls. The artifact is common in permanently mounted sections; fixation and paraffin infiltration are usually cited as the causes—its appearance in the whole-mounted ovules is caused by xylene. Although miscible with the clearing fluid, Euparal is the least satisfactory of the standard mountants for permanent preparations of cleared ovules and is best used with an equal quantity of clearing fluid for semipermanent preparations. A large quantity of Euparal in the mountant produces pronounced shrinkage. A method for permanently mounting cleared ovules with the clearing image unaltered employs a mountant which contains the ingredients of Spurr low viscosity embedding medium. Vinylcyclohexene dioxide (10 drops) is combined with diglycidyl ether of polypropylglycol (6 drops) and nonenyl succinic anhydride (26 drops). Ovules treated for 24 hr in benzyl benzoate-4 1/2 clearing fluid are passed through a graded series of clearing fluid-epoxy medium mixtures (3:1, 2:2, 1:3, and pure epoxy medium) at intervals of 15 minutes. One drop of dimethylaminoethanol, the cure accelerator, is then added to the epoxy medium and the ovules are mounted and covered immediately on a Raj slide. The preparation is cured in an oven at 60 C for 24 hr and observed with phase contrast or Nomarski interference optics.     

Replacing xylene with n-heptane for paraffin embedding

Abstract

In standard histological technique, aromatic solvents such as xylene and toluene are used as clearing agents between ethanol dehydration and paraffin embedding. In addition, these solvents are used for de-waxing paraffin sections. Unfortunately, these solvents are harmful and therefore adequate substitutes would be useful. We suggest the use of n-heptane as a convenient substitute for xylene. Paraffin sections of rat tissues processed with n-heptane and stained with hematoxylin-eosin or Masson's trichrome showed proper embedment, well preserved morphology and excellent staining.     

An analysis of methods for permanently mounting ovules cleared in four-and-a-half type clearing fluids

Ovules cleared in benzyl benzoate-4 1/2 clearing fluid can be permanently mounted in Piccolyte or Permount by replacing the cleaning fluid with absolute ethanol, upgrading the ovules in mixtures of ethanol and xylene (3:1, 2:2, 1:3, and xylene), and mounting them in either mountant under the supported coverglass of a Raj slide. Optical saggittal sections through the ovules resemble microtome sections in that the protoplasts are slightly shrunken away from the cell walls. The artifact is common in permanently mounted sections; fixation and paraffin infiltration are usually cited as the causes--its appearance in the whole-mounted ovules is caused by xylene. Although miscible with the clearing fluid, Euparal is the least satisfactory of the standard mountants for permanent preparations of cleared ovules and is best used with an equal quantity of clearing fluid for semipermanent preparations. A large quantity of Euparal in the mountant produces pronounced shrinkage. A method for permanently mounting cleared ovules with the clearing image unaltered employs a mountant which contains the ingredients of Spurr low viscosity embedding medium. Vinylcyclohexene dioxide (10 drops) is combined with diglycidyl ether of polypropylglycol (6 drops) and nonenyl succinic anhydride (26 drops). Ovules treated for 24 hr in benzyl benzoate-4 1/2 clearing fluid are passed through a graded series of clearing fluid-epoxy medium mixtures (3:1, 2:2, 1:3, and pure epoxy medium) at intervals of 14 minutes. One drop of dimethylaminoethanol, the cure accelerator, is then added to the epoxy medium and the ovules are mounted and covered immediately on a Raj slide. The preparation is cured in an oven at 60 C for 24 hr and observed with phase contrast or Nomarski interference optics.     

Opsonic Activity of Sera and Blister Fluid from Severely Burned Patients Evaluated by a Chemiluminescence Method

Burn wound sepsis is the most common and severe complication in the patients with severe burn. To know the systemic and local defect in immunity of burned patients, we measured the luminol-enhanced chemiluminescence (CL) response of normal polymorphonuclear leukocytes (PMNs) upon exposure to zymosan particles, bacteria or Candida albicans that were opsonized with any of patient's serum, blister fluid of burn wound or pooled normal serum (blood type AB). Sera from patients exhibited lower opsonic activities than those of pooled normal serum in the early postburn days. The levels of serum immunoglobulins, complement components and plasma fibronectin were found to correlate well with opsonin-index (OI), which was determined based on the CL response data obtained during the course of infusion therapy with fresh frozen plasma. Furthermore, patient's blister fluid showed much lower opsonic activity against bacteria such as Pseudomonas aeruginosa than patient's own serum. These results indicate that blister fluid is also not effective to opsonize bacteria because of the marked depression of the levels of immunoglobulins and complement components. Destruction of the skin barrier by thermal injury and impairment of systemic or local humoral immunity may predispose these patients to burn wound sepsis.     

Hyperacidification-induced, clear appearance of venules caused by the swelling of erythrocytes in microcirculation

In the mesenterium of rats it was found that an overacidity of the blood flowing in the area of microcirculation and caused by irrigation with acid media may be objectively represented in the clearing of venols measurable by means of video technique. The cause of this clearing process is the swelling of erythrocytes setting in at lower pH-values. According to in-vitro findings this swelling of erythrocytes will lead to an increase of the apparent viscosity of the blood fluid or blood cell suspension respectively in conformity with the increase of hematocrit connected with it.     

Profiles of Extracellular miRNA in Cerebrospinal Fluid and Serum from Patients with Alzheimer's and Parkinson's Diseases Correlate with Disease Status and Features of Pathology

The discovery and reliable detection of markers for neurodegenerative diseases have been complicated by the inaccessibility of the diseased tissue- such as the inability to biopsy or test tissue from the central nervous system directly. RNAs originating from hard to access tissues, such as neurons within the brain and spinal cord, have the potential to get to the periphery where they can be detected non-invasively. The formation and extracellular release of microvesicles and RNA binding proteins have been found to carry RNA from cells of the central nervous system to the periphery and protect the RNA from degradation. Extracellular miRNAs detectable in peripheral circulation can provide information about cellular changes associated with human health and disease. In order to associate miRNA signals present in cell-free peripheral biofluids with neurodegenerative disease status of patients with Alzheimer''s and Parkinson''s diseases, we assessed the miRNA content in cerebrospinal fluid and serum from postmortem subjects with full neuropathology evaluations. We profiled the miRNA content from 69 patients with Alzheimer''s disease, 67 with Parkinson''s disease and 78 neurologically normal controls using next generation small RNA sequencing (NGS). We report the average abundance of each detected miRNA in cerebrospinal fluid and in serum and describe 13 novel miRNAs that were identified. We correlated changes in miRNA expression with aspects of disease severity such as Braak stage, dementia status, plaque and tangle densities, and the presence and severity of Lewy body pathology. Many of the differentially expressed miRNAs detected in peripheral cell-free cerebrospinal fluid and serum were previously reported in the literature to be deregulated in brain tissue from patients with neurodegenerative disease. These data indicate that extracellular miRNAs detectable in the cerebrospinal fluid and serum are reflective of cell-based changes in pathology and can be used to assess disease progression and therapeutic efficacy.     

Carbon stock loss from deforestation through 2013 in Brazilian Amazonia

The largest carbon stock in tropical vegetation is in Brazilian Amazonia. In this ~5 million km² area, over 750 000 km² of forest and ~240 000 km² of nonforest vegetation types had been cleared through 2013. We estimate current carbon stocks and cumulative gross carbon loss from clearing of premodern vegetation in Brazil's ‘Legal Amazonia’ and ‘Amazonia biome’ regions. Biomass of ‘premodern’ vegetation (prior to major increases in disturbance beginning in the 1970s) was estimated by matching vegetation classes mapped at a scale of 1 : 250 000 and 29 biomass means from 41 published studies for vegetation types classified as forest (2317 1‐ha plots) and as either nonforest or contact zones (1830 plots and subplots of varied size). Total biomass (above and below‐ground, dry weight) underwent a gross reduction of 18.3% in Legal Amazonia (13.1 Pg C) and 16.7% in the Amazonia biome (11.2 Pg C) through 2013, excluding carbon loss from the effects of fragmentation, selective logging, fires, mortality induced by recent droughts and clearing of forest regrowth. In spite of the loss of carbon from clearing, large amounts of carbon were stored in stands of remaining vegetation in 2013, equivalent to 149 Mg C ha^?1 when weighted by the total area covered by each vegetation type in Legal Amazonia. Native vegetation in Legal Amazonia in 2013 originally contained 58.6 Pg C, while that in the Amazonia biome contained 56 Pg C. Emissions per unit area from clearing could potentially be larger in the future because previously cleared areas were mainly covered by vegetation with lower mean biomass than the remaining vegetation. Estimates of original biomass are essential for estimating losses to forest degradation. This study offers estimates of cumulative biomass loss, as well as estimates of premodern carbon stocks that have not been represented in recent estimates of deforestation impacts.     

Fluid intelligence and psychosocial outcome: from logical problem solving to social adaptation

Background

While fluid intelligence has proved to be central to executive functioning, logical reasoning and other frontal functions, the role of this ability in psychosocial adaptation has not been well characterized.

Methodology/Principal Findings

A random-probabilistic sample of 2370 secondary school students completed measures of fluid intelligence (Raven''s Progressive Matrices, RPM) and several measures of psychological adaptation: bullying (Delaware Bullying Questionnaire), domestic abuse of adolescents (Conflict Tactic Scale), drug intake (ONUDD), self-esteem (Rosenberg''s Self Esteem Scale) and the Perceived Mental Health Scale (Spanish adaptation).Lower fluid intelligence scores were associated with physical violence, both in the role of victim and victimizer. Drug intake, especially cannabis, cocaine and inhalants and lower self-esteem were also associated with lower fluid intelligence. Finally, scores on the perceived mental health assessment were better when fluid intelligence scores were higher.

Conclusions/Significance

Our results show evidence of a strong association between psychosocial adaptation and fluid intelligence, suggesting that the latter is not only central to executive functioning but also forms part of a more general capacity for adaptation to social contexts.     

The Human Frontier Science Program: its aims and a proposal for its implementation

Reactions to the Human Frontier Science Program have been mixed. It has been welcomed as an important initiative in basic biological research yet regarded with suspicion by some outside Japan who see it as facilitating access to European and North American fundamental research. However, suspicion is clearing away and the governments concerned are looking forward to the Program's implementation. In this article, the Program's aims and the mechanisms for its execution are outlined as proposed in the light of a recent feasibility study.     

THE DOUBLE REFRACTION OF CHITIN

The double refraction of the chitinous hair of the crayfish is positive with respect to the axis of the hair, and is largely caused by the arrangement of submicroscopic, elongated chitin particles parallel to this axis (form birefringence). Using a series of relatively unreactive liquids and fluid mixtures which permeate the chitin framework, the type of curve relating double refraction and refractive index of the imbibed fluid is found to depend greatly on the chemical nature of the fluid. Either a positive or a negative residual birefringence may be found, depending on the choice of imbibing liquid. Separation of form and crystalline elements in double refraction by means of Ambronn''s imbibition technique is therefore unsafe in a system like chitin, where some type of oriented association of the imbibed molecules with the chitin framework is prevalent.     

Reduction of Photo Bleaching and Long Term Archiving of Chemically Cleared GFP-Expressing Mouse Brains

Tissue clearing allows microscopy of large specimens as whole mouse brains or embryos. However, lipophilic tissue clearing agents as dibenzyl ether limit storage time of GFP-expressing samples to several days and do not prevent them from photobleaching during microscopy. To preserve GFP fluorescence, we developed a transparent solid resin formulation, which maintains the specimens'' transparency and provides a constant signal to noise ratio even after hours of continuous laser irradiation. If required, high-power illumination or long exposure times can be applied with virtually no loss in signal quality and samples can be archived for years.     

An Overview of Botanical Clearing Technique

Clearing techniques are outlined with reference to their action on the chemical cunstituents of plant tissue. The most general technique would include pretreatment with solvents, dissolution of protoplasm, dissolution of other substances, bleaching, infiltration with a dense fluid, and staining. Extensive chemical changes go on during these steps and may prevent satisfactory clearing, an important example King the discoloration of phenolic compounds. Rational design of clearing methods for the chemically distinct cell types and tissues seems a likely future development.     

Conservation of arboreal herbivores: The Australian scene

Abstract A common global issue in biological conservation, that of land use allocation and subsequent management practice, is also at the core of the problem of conserving arboreal herbivores in Australia. The problem is caused by a number of biological and cultural factors. Many populations of Australia's terrestrial vertebrate fauna species of forest and woodland environments, including notably the arboreal herbivores, are concentrated in habitats on soils relatively rich in nutrients. Such habitats are limited due to (i) Australia's general aridity and its naturally poor endowment of soils of reasonable quality, and (ii) the extent to which habitats on the better soils have been cleared to service Australia's social and economic development. Land clearing has focused on these better soils because of cultural traditions in agriculture, and society's recognition of the importance of ‘good’ soils to a nation's development. As a result, Australia has legislation and numerous government policies and programmes that aim to identify and make maximum use of the productive potential of Australia's land. For example, the Crown Lands Alienation Act of 1861 was enacted expressly to transfer land from public to private or leasehold tenure and to develop those lands best suited for agriculture and pastoralism. As a result, from 1861 to the end of the nineteenth century, large areas were extensively cleared and potentially commercial forests were destroyed. The remaining uncleared land in private tenure today carries a major part of the woodland and forest resource on the better soils. Woodland and forest associations on these soils are generally poorly represented in reserves. Furthermore, the Australian constitution, and laws concerning the rights of the private landholder and the use of forest resources on leasehold land, indirectly or implicitly encourage or otherwise act to minimize the constraints on continued land clearing. From the above facts and because human populations are expected to increase, the prognosis for Australia's woodland and forest fauna, including the arboreal herbivores, is one of ongoing loss of important habitats. This paper suggests the following measures for ameliorating this loss: identify strategically important habitats, and encourage private philanthropic purchase or government purchase of timber rights, or subsidize the management of these habitats; examine possible trade-offs in public expenditures on forest administration and management in return for public purchase of privately held forests; establish educational programmes that encourage a conservation ethic and a better community understanding of the realities and the extent of the difficulties to be addressed.     

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure^1,2, alterations in brain vasculature of a Alzheimer''s disease mouse model³, portal vein abnormalities in portal hypertensive and cirrhotic mice⁴, developmental steps in rat lymphatic maturation between immature and adult lungs⁵, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury⁶.Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.     

不同经营模式对川西亚高山天然次生林林地水文效应的影响

天然次生林是川西亚高山林区经历大规模砍伐后形成的主要森林类型之一,是我国西南林区水源涵养林的重要组成部分。以不同经营模式(抚育经营、清林+补植经营以及封育经营)的川西亚高山次生桦木林和桦木、岷江冷杉混交林为研究对象,通过样方取样法获取和分析了林地苔藓、枯落物和土壤的水文指标。结果表明,与封山育林经营相比,抚育经营下的两种林型的苔藓最大持水率均显著升高(F=8.147,P=0.010;F=15.525,P=0.006)、桦木林的蓄积量显著降低(F=4.979,P=0.022),而苔藓最大持水量变化不显著;混交林则均无显著变化。在清林+补植经营下,混交林苔藓水文效应变化不显著(F=2.280,P=0.183),而桦木林虽然苔藓最大持水率无显著变化(F=4.072,P=0.098),但蓄积量的显著降低(F=3.536,P=0.044)导致了其最大持水量的降低(F=3.782,P=0.042)。两种经营方式基本上促进了天然林的枯落物最大持水率、降低了林下枯落物蓄积量;其中抚育经营效果更显著,但两种经营方式下枯落物最大持水量变化不显著。两种经营方式下,桦木林和混交林的林下土壤容重均降低(F=10.715,P0.01;F=5.148,P0.05),同时桦木林土壤最大持水量增加(F=4.499,P0.05),其中抚育经营的影响程度都更显著。从4年来的短期效应来看,两种经营方式均对天然林的林地持水能力具有促进作用,抚育经营较清林+补植经营更显著,但这仅是短期的结果,两种经营方式对于退化天然林水文以及其他生态功能恢复的长期影响还有待于进一步的观测研究。