Search

The scarcity of age-appropriate pharmaceutical formulations is one of the major challenges impeding successful management of tuberculosis (TB) prevalence in minors. To this end, we designed and assessed the quality of a multiparticulate reconstitutable suspension powder containing fixed dose rifampicin and pyrazinamide (150 mg/300 mg per 5 mL) which was prepared employing solid−liquid direct dispersion coupled with timed dehydration, and mechanical pulverization. The optimized formulation had a high production yield (96.000 ± 3.270%), displayed noteworthy powder flow quality (9.670 ± 1.150°), upon reconstitution the suspension flow property was non-Newtonian and was easily redispersible with gentle manual agitation (1.720 ± 0.011 strokes/second). Effective drug loading was attained for both pyrazinamide (97.230 ± 2.570%w/w) and rifampicin (97.610 ± 0.020%w/w) and drug release followed a zero-order kinetic model (R2 = 0.990) for both drugs. Microscopic examinations confirmed drug encapsulation efficiency and showed that the particulates were micro-dimensional in nature (n < 700.000 µm). The formulation was physicochemically stable with no chemically irreversible drug-excipient interactions based on the results of characterization experiments performed. Findings from organoleptic evaluations generated an overall rating of 4.000 ± 0.000 for its attractive appearance and colour 5.000 ± 0.000 confirming its excellent taste and extremely pleasant smell. Preliminary cytotoxicity studies showed a cell viability above 70.000% which indicates that the FDC formulation was biocompatible. The optimized formulation was environmentally stable either as a dry powder or reconstituted suspension. Accordingly, a stable and palatable FDC antimycobacterial reconstitutable oral suspension powder, intended for flexible dosing in children and adolescents, was optimally fabricated.

The study investigated the toxicity effects of 'form specific' engineered nanomaterials (ENMs) and ions released from nano-enabled products (NEPs), namely sunscreens, sanitisers, body creams and socks on Pseudokirchneriella subcapitata , Spirodela polyrhiza , and Daphnia magna . Additionally, risk estimation emanating from the exposures was undertaken. The ENMs and the ions released from the products both contributed to the effects to varying extents, with neither being a uniform principal toxicity agent across the exposures; however, the effects were either synergistic or antagonistic. D. magna and S. polyrhiza were the most sensitive and least sensitive test organisms, respectively. The most toxic effects were from ENMs and ions released from sanitisers and sunscreens, whereas body creams and sock counterparts caused negligible effects. The internalisation of the ENMs from the sunscreens could not be established; only adsorption on the biota was evident. It was established that ENMs and ions released from products pose no imminent risk to ecosystems; instead, small to significant adverse effects are expected in the worst-case exposure scenario. The study demonstrates that while ENMs from products may not be considered to pose an imminent risk, increasing nanotechnology commercialization may increase their environmental exposure and risk potential; therefore, priority exposure cases need to be examined.

Deep eutectic solvents aid the formulation of solid pesticide dosage forms for water-insoluble actives. This was demonstrated by encapsulating Amitraz powder in a low-melting matrix based on the eutectic mixture of urea (32 wt%) and 1,3-dimethylurea. Dissolution in water of melt-cast discs, containing 20 wt% active, led to the rapid release of Amitraz in a finely dispersed form. The order of magnitude reduction in particle size, after dissolution, is ascribed to the solubilization of Amitraz in the hot deep eutectic solvent and its subsequent precipitation as a separate phase on crystallization of the matrix.

The severity of tuberculosis (TB) in children is considered a global crisis compounded by the scarcity of pharmaceutical formulations suitable for pediatric use. The purpose of this study was to optimally develop and evaluate a pyrazinamide containing edible orodispersible film formulation potentially suitable for use in pediatrics actively infected with TB. The formulation was prepared employing aqueous-particulate blending and solvent casting methods facilitated by a high performance Box Behnken experimental design template. The optimized orodispersible formulation was mechanically robust, flexible, easy to handle, exhibited rapid disintegration with initial matrix collapse occurring under 60 s (0.58 ± 0.05 min ≡ 34.98 ± 3.00 s) and pyrazinamide release was controlled by anomalous diffusion coupled with matrix disintegration and erosion mechanisms. It was microporous in nature, light weight (57.5 ± 0.5 mg) with an average diameter of 10.5 mm and uniformly distributed pyrazinamide load of 101.13 ± 2.03 % w / w . The formulation was physicochemically stable with no evidence of destructive drug-excipient interactions founded on outcomes of characterization and environmental stability investigations. Preliminary inquiries revealed that the orodispersible formulation was cytobiocompatible, palatable and remained intact under specific storage conditions. Summarily, an edible pyrazinamide containing orodispersible film formulation was optimally designed to potentially improve TB pharmacotherapy in children, particularly the under 5 year olds.

Safe application of water-insoluble acaricides requires fast release from solid dosage systems into aquatic environments. Dextrin is a water-soluble form of partially hydrolyzed starch, which may be used as matrix material for these systems if retrogradation can be inhibited by the inclusion of nanofillers. Several glycerol-plasticized thermoplastic dextrin-based nanocomposites were prepared with a twin-screw extrusion-compounding process. The nanofillers included a layered double hydroxide (LDH), cellulose nanofibers (CNF), and stearic acid. The time-dependent retrogradation of the compounds was monitored by X-ray diffraction (XRD) and dynamic mechanical thermal analysis (DMA). XRD showed that composite samples that included stearic acid in the formulation led to the formation of an amylose-lipid complex and a stable crystallinity during aging. The most promising nanocomposite included both stearic acid and CNF. It was selected as the carrier material for the water-insoluble acaricide Amitraz. Fast release rates were observed for composites containing 5, 10, and 20% (w/w) of the pesticide. A significant reduction in the particle size of the released Amitraz powder was observed, which is ascribed to the high-temperature compounding procedure.

To improve traditional cancer therapies, we synthesized polylactide (PLA) spheres coencapsulating magnetic nanoparticles (MNPs, Fe3O4) and an anticancer drug (doxorubicin, DOX). The synthesis process involves the preparation of Fe3O4 NPs by a coprecipitation method and then PLA/DOX/Fe3O4 spheres using the solvent evaporation (oil-in-water) technique. The Fe3O4 NPs were coated with oleic acid to improve their hydrophobicity and biocompatibility for medical applications. The structure, morphology and properties of the MNPs and PLA/DOX/Fe3O4 spheres were studied using various techniques, such as FTIR, SEM, TEM, TGA, VSM, UV-vis spectroscopy, and zeta potential measurements. The in vitro DOX release from the spheres was prolonged, sustained, and pH-dependent and fit a zero-order kinetics model and an anomalous mechanism. Interestingly, the spheres did not show a DOX burst effect, ensuring the minimal exposure of the healthy cells and an increased drug payload at the tumor site. The pronounced biocompatibility of the PLA/DOX/Fe3O4 spheres with HeLa cells was proven by a WST assay. In summary, the synthesized PLA/DOX/Fe3O4 spheres have the potential for magnetic targeting of tumor cells to transform conventional methods.

As a step toward functional annotation of genes required for floral initiation and development within the Eucalyptus genome, we used short read sequencing to analyze transcriptomes of floral buds from early and late developmental stages, and compared these with transcriptomes of diverse vegetative tissues, including leaves, roots, and stems. A subset of 4807 genes (13% of protein-coding genes) were differentially expressed between floral buds of either stage and vegetative tissues. A similar proportion of genes were differentially expressed among all tissues. A total of 479 genes were differentially expressed between early and late stages of floral development. Gene function enrichment identified 158 gene ontology classes that were overrepresented in floral tissues, including 'pollen development' and 'aromatic compound biosynthetic process'. At least 40 floral-dominant genes lacked functional annotations and thus may be novel floral transcripts. We analyzed several genes and gene families in depth, including 49 putative biomarkers of floral development, the MADS-box transcription factors, 'S-domain'-receptor-like kinases, and selected gene family members with phosphatidylethanolamine-binding protein domains. Expanded MADS-box gene subfamilies in Eucalyptus grandis included SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), SEPALLATA (SEP) and SHORT VEGETATIVE PHASE (SVP) Arabidopsis thaliana homologs. These data provide a rich resource for functional and evolutionary analysis of genes controlling eucalypt floral development, and new tools for breeding and biotechnology., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)

Background and Aims: Conservation of the genetic diversity afforded by recalcitrant seeds is achieved by cryopreservation, in which excised embryonic axes (or, where possible, embryos) are treated and stored at temperatures lower than -180 °C using liquid nitrogen. It has previously been shown that intracellular ice forms in rapidly cooled embryonic axes of Acer saccharinum (silver maple) but this is not necessarily lethal when ice crystals are small. This study seeks to understand the nature and extent of damage from intracellular ice, and the course of recovery and regrowth in surviving tissues., Methods: Embryonic axes of A. saccharinum, not subjected to dehydration or cryoprotection treatments (water content was 1·9 g H2O g(-1) dry mass), were cooled to liquid nitrogen temperatures using two methods: plunging into nitrogen slush to achieve a cooling rate of 97 °C s(-1) or programmed cooling at 3·3 °C s(-1). Samples were thawed rapidly (177 °C s(-1)) and cell structure was examined microscopically immediately, and at intervals up to 72 h in vitro. Survival was assessed after 4 weeks in vitro. Axes were processed conventionally for optical microscopy and ultrastructural examination., Key Results: Immediately following thaw after cryogenic exposure, cells from axes did not show signs of damage at an ultrastructural level. Signs that cells had been damaged were apparent after several hours of in vitro culture and appeared as autophagic decomposition. In surviving tissues, dead cells were sloughed off and pockets of living cells were the origin of regrowth. In roots, regrowth occurred from the ground meristem and procambium, not the distal meristem, which became lethally damaged. Regrowth of shoots occurred from isolated pockets of surviving cells of peripheral and pith meristems. The size of these pockets may determine the possibility for, the extent of and the vigour of regrowth., Conclusions: Autophagic degradation and ultimately autolysis of cells following cryo-exposure and formation of small (0·2-0·4 µm) intracellular ice crystals challenges current ideas that ice causes immediate physical damage to cells. Instead, freezing stress may induce a signal for programmed cell death (PCD). Cells that form more ice crystals during cooling have faster PCD responses., (Published by Oxford University Press on behalf of the Annals of Botany Company 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Background and Aims: Cryopreservation is the only long-term conservation strategy available for germplasm of recalcitrant-seeded species. Efforts to cryopreserve this form of germplasm are hampered by potentially lethal intracellular freezing events; thus, it is important to understand the relationships among cryo-exposure techniques, water content, structure and survival., Methods: Undried embryonic axes of Acer saccharinum and those rapidly dried to two different water contents were cooled at three rates and re-warmed at two rates. Ultrastructural observations were carried out on radicle and shoot tips prepared by freeze-fracture and freeze-substitution to assess immediate (i.e. pre-thaw) responses to cooling treatments. Survival of axes was assessed in vitro., Key Results: Intracellular ice formation was not necessarily lethal. Embryo cells survived when crystal diameter was between 0·2 and 0·4 µm and fewer than 20 crystals were distributed per μm(2) in the cytoplasm. Ice was not uniformly distributed within the cells. In fully hydrated axes cooled at an intermediate rate, the interiors of many organelles were apparently ice-free; this may have prevented the disruption of vital intracellular machinery. Intracytoplasmic ice formation did not apparently impact the integrity of the plasmalemma. The maximum number of ice crystals was far greater in shoot apices, which were more sensitive than radicles to cryo-exposure., Conclusions: The findings challenge the accepted paradigm that intracellular ice formation is always lethal, as the results show that cells can survive intracellular ice if crystals are small and localized in the cytoplasm. Further understanding of the interactions among water content, cooling rate, cell structure and ice structure is required to optimize cryopreservation treatments without undue reliance on empirical approaches.

The buccal mucosal route offers several advantages but the delivery of certain drugs can be limited by low membrane permeability. This study investigated the buccal permeability properties of didanosine (ddI) and assessed the potential of Aloe vera gel (AVgel) as a novel buccal permeation enhancer. Permeation studies were performed using Franz diffusion cells, and the drug was quantified by UV spectroscopy. Histomorphological evaluations were undertaken using light and transmission electron microscopy. The permeability of ddI was concentration-dependent, and it did not have any adverse effects on the buccal mucosae. A linear relationship (R² = 0.9557) between the concentrations and flux indicated passive diffusion as the mechanism of drug transport. AVgel at concentrations of 0.25 to 2 %w/v enhanced ddI permeability with enhancement ratios from 5.09 (0.25 %w/v) to 11.78 (2 %w/v) but decreased permeability at 4 and 6 %w/v. Ultrastructural analysis of the buccal mucosae treated with phosphate buffer saline pH 7.4 (PBS), ddI/PBS, and ddI/PBS/AVgel 0.5 %w/v showed cells with normal plasmalemma, well-developed cristae, and nuclei with regular nuclear envelopes. However, cells from 1, 2, and 6 %w/v AVgel-treated mucosae showed irregular nuclear outlines, increased intercellular spacing, and plasmalemma crenulations. This study demonstrates the potential of AVgel as a buccal permeation enhancer for ddI to improve anti-HIV and AIDS therapy., (© Georg Thieme Verlag KG Stuttgart · New York.)

Effects of sequential procedures required for cryopreservation of embryos excised from the recalcitrant seeds of Haemanthus montanus were assessed ultrastructurally and in conjunction with respiratory activity and the rate of protein synthesis. Fresh material (water content, 5.05 ± 0.92 g g(-1) dry mass) afforded ultrastructural evidence of considerable metabolic activity, borne out by respiratory rates. Neither exposure to glycerol nor sucrose as penetrating and non-penetrating cryoprotectants, respectively, brought about degradative changes, although increased vacuolation and autophagy accompanied both, while respiratory and protein synthetic activity were not adversely affected. Glycerol-cryoprotected embryos flash dried to water contents >0.4 g g(-1) showed organised ultrastructural features and considerable autophagy consistent with metabolic activity, and although respiratory activity was lower, protein synthesis rate was enhanced relative to fresh material. However, at water contents <0.4 g g(-1), embryo tissue presented a mosaic of cells of variable density and ultrastructural status, but trends in rates of respiration and protein synthesis remained similar. Flash drying after sucrose exposure was accompanied by considerable ultrastructural abnormality particularly at water contents <0.4 g g(-1), lysis of individual and groups of cells and considerable depression of respiration, but not of protein synthesis. Success, assessed as ≥50% axes forming seedlings after cryogen exposure, was obtained only when glycerol-cryoprotected embryos at water contents >0.4 g g(-1)-in which the degree of vacuolation remained moderate-were rapidly cooled. The outcomes of this study are considered particularly in terms of the stresses imposed by prolonged, relatively slow dehydration and ultimate water contents, on embryos showing considerable metabolic activity.

Cryostorage (usually in, or above liquid nitrogen) is presently the only option for long-term germplasm conservation of species producing recalcitrant (desiccation-sensitive) seeds. The present study investigated the ultrastructural responses of zygotic embryos excised from recalcitrant Amaryllis belladonna seeds to the sequential steps involved in cryopreservation. Flash-dried embryos, with and without prior sucrose (non-penetrating) or glycerol (penetrating) cryoprotection, were cooled rapidly or slowly, recovered in vitro and then assessed for ultrastructural and viability responses. Untreated embryos were 100% viable, the ultrastructure being indicative of their actively metabolic condition. Although nuclear morphology changed, viability was unaffected after exposure to either glycerol or sucrose, but mitochondrial ultrastructure suggested enhancement of metabolic activity particularly after sucrose treatment. When flash dried after sucrose cryoprotection, a significant increase in the degree of vacuolation, abnormal plastid ultrastructure and some wall abnormality accompanied a decline in survival to 70% and 60% at water contents > and <0.4 g g(-1), respectively. In contrast, glycerol cryoprotection, which promoted retention of generally normal ultrastructure and also counteracted any increase in the degree of vacuolation, was associated with 100% and 90% survival of embryos at the higher and lower water contents. After exposure to liquid nitrogen (LN), ultrastructural irregularities were minimal in rapidly cooled glycerol-cryoprotected embryos, at water content <0.4 g g(-1), which showed 70% survival after retrieval from cryogenic conditions. At the other extreme, no embryos survived LN exposure when sucrose cryoprotected. The study relates the cumulative effects of subcellular abnormality and declining viability, in relation to experimental parameters for cryopreservation.

Flight necessitates that the feather rachis is extremely tough and light. Yet, the crucial filamentous hierarchy of the rachis is unknown-study hindered by the tight chemical bonding between the filaments and matrix. We used novel microbial biodegradation to delineate the fibres of the rachidial cortex in situ. It revealed the thickest keratin filaments known to date (factor >10), approximately 6 microm thick, extending predominantly axially but with a small outer circumferential component. Near-periodic thickened nodes of the fibres are staggered with those in adjacent fibres in two- and three-dimensional planes, creating a fibre-matrix texture with high attributes for crack stopping and resistance to transverse cutting. Close association of the fibre layer with the underlying 'spongy' medulloid pith indicates the potential for higher buckling loads and greater elastic recoil. Strikingly, the fibres are similar in dimensions and form to the free filaments of the feather vane and plumulaceous and embryonic down, the syncitial barbules, but, identified for the first time in 140+ years of study in a new location-as a major structural component of the rachis. Early in feather evolution, syncitial barbules were consolidated in a robust central rachis, definitively characterizing the avian lineage of keratin.

The ultrastructure of dermal fibres of a 200Myr thunniform ichthyosaur, Ichthyosaurus, specifically the 67nm axial repeat D-banding of the fibrils, which characterizes collagen, is presented for the first time by means of scanning electron microscopy (SEM) analysis. The fragment of material investigated is part of previously described fossilized skin comprising an architecture of layers of oppositely oriented fibre bundles. The wider implication, as indicated by the extraordinary quality of preservation, is the robustness of the collagen molecule at the ultrastructural level, which presumably contributed to its survival during the initial processes of decomposition prior to mineralization. Investigation of the elemental composition of the sample by SEM-energy dispersive X-ray spectroscopy indicates that calcite and phosphate played important roles in the rapid mineralization and fine replication of the collagen fibres and fibrils. The exceedingly small sample used in the investigation and high level of information achieved indicate the potential for minimal damage to prized museum specimens; for example, ultrastructural investigations by SEM may be used to help resolve highly contentious questions, for example, 'protofeathers' in the Chinese dinosaurs.

A study on cryopreservation of excised embryonic axes of fifteen species of the amaryllidaceae is reported. Embryonic axes that after flash-drying had a water content in the range 0.4 to 0.1 g/g and survival greater than 60% were selected for cryopreservation procedures. The highest post-thaw viabilities (roots and shoots produced) across all species were recorded for embryonic axes subjected to rapid rather than slow cooling. With rapid cooling and no cryoprotection, the highest post-thaw viabilities for the fifteen species investigated was 0% in one species; ranged between 10 and 35% for nine species; and between 45 and 55% for five species. With cryoprotection and rapid cooling the highest post-thaw viabilities for these fifteen species was 0% for one species; ranged between 15 and 35% for six species; and between 40 and 75% for eight species. The highest post-thaw survival in ten out of fifteen species was obtained for axes dried to between 0.24 +/- 0.06 and 0.14 +/- 0.08 g/g(-1) (and rapidly cooled). With only one exception (Strumaria discifera; 45%), post-thaw survival after slow cooling ranged between 10 and 30%. Survival after vitrification plus slow cooling was achieved for seven species but was never higher than post-thaw survival in non-cryoprotected, rapidly cooled axes. The results suggest that species within the same family can exhibit commonalities in terms of amenability to cryopreservation techniques but for maximum success, axis water content and cooling rate particularly, must be optimised for each species in the family independently.

The present study investigated the relative contributions of water content and non-equilibrium cooling and warming rates to the survival of cryopreserved axes of recalcitrant P. trifoliata seeds. Reducing water contents from 1.7 and 0.26 g water per g dry mass is believed to increase cytoplasmic viscosity. Cooling to -196 degree C was done at rates averaging between 0.17 and 1300 degree C per second, and warming at 600 or 1.35 degree C per second. Survival was assessed after 4 weeks in vitro. Rapid warming resulted in higher survival and normal development of axes at all water contents. The effects of cooling rate were dependent on the water content of axes. Cooling rates resulting in >70 percent normal development ranged between 0.17 and about 1300 degree C per second for axes at a water content of 0.26 g water per g dry mass narrowing with increasing hydration to an apparent optimum at about 686 degree C per second in axes at 0.8 g water per g dry mass At 1.7 g water per g dry mass, axes cooled at 0.17 degree C per second yielded nearly 40 percent normal development, whereas faster cooling was deleterious. Results are interpreted in the context of the effect of water content on cytoplasmic viscosity and the rate of intracellular ice formation. At low water contents, the high intracellular viscosity slows ice crystallization making survival independent of cooling rate. At higher water contents, the reduced viscosity requires faster cooling to prevent ice crystal damage. The ability to cool rapidly with increasing hydration is balanced with an increasing limitation to dissipate heat fast enough to prevent severe damage.

The present study investigated the rate of temperature change within axes of Poncirus trifoliata during cooling and warming by various methods. Cooling rates ranged between 0.17 and 170 degree (C per second, and warming rates of 1.25 and 600 degree C per second were measured when axes were warmed at room temperature or in water at 40 degree C, respectively. Partial drying increased the cooling rate within axes in direct contact with the cryogen, but did not affect the cooling or warming rates within axes enclosed in a double layer of lightweight aluminium foil. The procedures described illustrate the orders of magnitude that separate extremes of the range of cooling or warming rates attained using methods commonly employed in cryopreservation studies. Quantifying these rates allows the relationship between cooling rate, water content and survival of hydrated embryonic axes to be explored.

Seeds of the endangered species Zizania texana are recalcitrant, making it difficult to preserve the remaining genetic diversity of this species in genebanks. Excised embryos can be cryopreserved using solution-based cryoprotection protocols. Survival following cryoexposure increased from less than 5% to about 75% by preculturing embryos in high concentrations of sugars, bathing them in cryoprotectant solutions, and partially drying them to water contents of about 0.6 g H2O/g dry mass.

This study investigated the interactions among water content, rapid (nonequilibrium) cooling to -196 degrees C using isopentane or subcooled nitrogen, and survival of embryonic axes of Aesculus hippocastanum. Average cooling rates in either cryogen did not exceed 60 degrees C s(-1) for axes containing more than 1.0 g H(2)O g(-1)dw (g g(-1)). Partial dehydration below 0.5 g gg(-1) facilitated faster cooling, averaging about 200 and 580 degrees C s(-1) in subcooled nitrogen and isopentane, respectively. The combination of partial drying and rapid cooling led to increased survival and reduced cellular damage in axes. Electrolyte leakage was 10-fold higher from fully hydrated axes cooled in either cryogen than from control axes that were not cooled. Drying of axes to 0.5 g g(-1), reduced electrolyte leakage of cryopreserved axes to levels similar to those of control material. Axis survival was assayed by germination in vitro. Axes with water contents greater than 1.0 g g(-1), did not survive cryogenic cooling. Between 1.0 and 0.75 g g(-1), axes survived cryogenic exposure but developed abnormally. The proportion of axes developing normally after being cooled in isopentane increased with increasing dehydration below 0.75 g g(-1), reaching a maximum between 0.5 and 0.25 g g(-1) after being cooled at > or =300 degrees C s(-1). Cooling rates attained in subcooled nitrogen did not exceed 250 degrees C s(-1), and normal development of axes was observed only at < or =0.4 g g(-1). These results support the hypothesis that rapid cooling enhances the feasibility of cryopreservation of desiccation-sensitive embryonic axes by increasing the upper limit of allowable water contents and overall survival., (Copyright 2001 Academic Press.)

This investigation assessed the extent of rehydration of dehydrated plant tissues during aqueous fixation in comparison with the fine structure revealed by freeze-substitution. Radicles from desiccation-tolerant pea (Pisum sativum L.), desiccation-sensitive jackfruit seeds (Artocarpus heterophyllus Lamk.), and leaves of the resurrection plant Eragrostis nindensis Ficalho & Hiern. were selected for their developmentally diverse characteristics. Following freeze-substitution, electron microscopy of dehydrated cells revealed variable wall infolding. Plasmalemmas had a trilaminar appearance and were continuous and closely appressed to cell walls, while the cytoplasm was compacted but ordered. Following aqueous fixation, separation of the plasmalemma and the cell wall, membrane vesiculation and distortion of cellular substructure were evident in all material studied. The sectional area enclosed by the cell wall in cortical cells of dehydrated pea and jackfruit radicles and mesophyll of E. nindensis increased after aqueous fixation by 55, 20, and 30%, respectively. Separation of the plasmalemma and the cell wall was attributed to the characteristics of aqueous fixatives, which limited the expansion of the plasmalemma and cellular contents but not that of the cell wall. It is proposed that severed plasmodesmatal connections, plasmalemma discontinuities, and membrane vesiculation that frequently accompany separation of walls and protoplasm are artefacts of aqueous fixation and should not be interpreted as evidence of desiccation damage or membrane recycling. Evidence suggests that, unlike aqueous fixation, freeze-substitution facilitates reliable preservation of tissues in the dehydrated state and is therefore essential for ultrastructural studies of desiccation.