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Background: Anti-freeze Glycoproteins (AFGPs) were described to preserve biological materials and protect them from different stresses., Aims: The effects of a synthetic anti-freeze glycoprotein-based compound, TFC-1326, on human skin quality and its biological actions were studied., Methods: The effects of various concentrations of TFC-1326 on the biology of human preadipocytes, differentiated in the proinflammatory microenvironment, and on human fibroblasts grown in coculture with human mature adipocytes or monocultured in stress conditions were investigated in, in vitro studies. Additionally, the efficacy of a 1% TFC-1326 topical cream was evaluated in a clinical investigation on the skin biology and appearance of 20 women aged between 40 and 65 years throughout 84 days of application., Results: The in vitro studies revealed that TFC-1326 mitigated the deleterious effects of a proinflammatory cytokine cocktail produced by human macrophages, by restoring preadipocyte adipogenic capacity and by reducing their fibroinflammatory state. TFC-1326 also stimulated the proliferative capacity of dermal fibroblasts co-cultured with mature adipocytes as well as their production of hyaluronic acid and procollagen type I, while decreasing IL6 secretion and increasing fibroblast viability. Furthermore, daily 1% TFC-1326 topical cream application, measurably improved skin radiance and laxity, as well as skin density. Finally, significant reductions of the volume and depth of the crow's feet wrinkles were also observed., Conclusions: The compound TFC-1326 significantly improved the physiological appearance and cellular functions of aging skin., (© 2024 The Author(s). Journal of Cosmetic Dermatology published by Wiley Periodicals LLC.)

Advancements in tissue engineering enable the fabrication of complex and functional tissues or organs. In particular, bioprinting enables controlled and accurate deposition of cells, biomaterials, and growth factors to create complex 3D skin constructs specific to a particular individual. Despite these advancements, challenges such as vascularization, long-term stability, and regulatory considerations hinder the clinical translation of bioprinted skin constructs. This chapter focuses on such approaches using advanced biomaterials and bioprinting techniques to overcome the current barriers in wound-healing studies. Moreover, it addresses current obstacles in wound-healing studies, highlighting the need for continued research and innovation to overcome these barriers and facilitate the practical utilization of bioprinted skin constructs in clinical settings., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Background and Aims: Internal root aeration is essential for root growth in waterlogged conditions. Aerenchyma provides a path for oxygen to diffuse to the roots. In most wetland species, including rice, a barrier to radial oxygen loss (ROL) allows more of the oxygen to diffuse to the root tip, enabling root growth into anoxic soil. Most dryland crops, including barley, do not form a root ROL barrier. We previously found that abscisic acid (ABA) signalling is involved in the induction of ROL barrier formation in rice during waterlogging. Although rice typically does not form a tight ROL barrier in roots in aerated conditions, an ROL barrier with suberized exodermis was induced by application of exogenous ABA. Therefore, we hypothesized that ABA application could also trigger root ROL barrier formation with hypodermal suberization in barley., Methods: Formation of an ROL barrier was examined in roots in different exogenous ABA concentrations and at different time points using cylindrical electrodes and Methylene Blue staining. Additionally, we evaluated root porosity and observed suberin and lignin modification. Suberin, lignin and Casparian strips in the cell walls were observed by histochemical staining. We also evaluated the permeability of the apoplast to a tracer., Key Results: Application of ABA induced suberization and ROL barrier formation in the adventitious roots of barley. The hypodermis also formed lignin-containing Casparian strips and a barrier to the infiltration of an apoplastic tracer (periodic acid). However, ABA application did not affect root porosity., Conclusions: Our results show that in artificial conditions, barley can induce the formation of ROL and apoplastic barriers in the outer part of roots if ABA is applied exogenously. The difference in ROL barrier inducibility between barley (an upland species) and rice (a wetland species) might be attributable to differences in ABA signalling in roots in response to waterlogging conditions., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Recent studies demonstrate that PDO threads, after being introduced into the facial skin, provide benefits due to collagen formation around the thread and improved vascularization., Aims: This work aims to report the ultrasound and photographic follow-up after the execution of the facelift thread technique, thus evaluating the anti-aging treatment that PDO threads will provide for human facial skin., Methods: The facelift will be performed using PDO thread 18G cannulated Sculpt i- Thread FML 100 × 185 mm, and 29G PDO thread Needled Flat i-Thread 38 × 50 mm. A total of 10 patients were selected for the study; all of them had medium-grade facial ptosis and aged 40-50 years old. In order to control tissue changes after thread, insertion ultrasound measurements will be made with Philips ultrasound 12 MHz linear probe at maximum resolution, initial evaluations and control of 30, 60, 90, and 120 days, measuring the skin layers: epidermis\dermis and hypodermis. Photographic control was performed before and after 120 days of intrusion of the PDO threads., Results: The formation of collagen around the PDO thread was evidenced through ultrasound images in all patients in this study, as well as improvement in the appearance of the skin and sagging, evaluated by photographic control., Conclusions: We can define that the PDO threads used for the facelift are effective in the formation of collagen around the thread (observed through the increase in the dermal layer), and improvement of the condition of the anti-aging factor through sagging skin, vascularization and thinning of the superficial fat layer (observed through the reduction of the subcutaneous or hypodermis layer)., (© 2023 The Author. Journal of Cosmetic Dermatology published by Wiley Periodicals LLC.)

Hypodermis is the predominant site of Staphylococcus aureus infections that cause cellulitis. Given the importance of macrophages in tissue remodeling, we examined the hypodermal macrophages (HDMs) and their impact on host susceptibility to infection. Bulk and single-cell transcriptomics uncovered HDM subsets with CCR2-dichotomy. HDM homeostasis required the fibroblast-derived growth factor CSF1, ablation of which abrogated HDMs from the hypodermal adventitia. Loss of CCR2 - HDMs resulted in accumulation of the extracellular matrix component, hyaluronic acid (HA). HDM-mediated HA clearance required sensing by the HA receptor, LYVE-1. Cell-autonomous IGF1 was required for accessibility of AP-1 transcription factor motifs that controlled LYVE-1 expression. Remarkably, loss of HDMs or IGF1 limited Staphylococcus aureus expansion via HA and conferred protection against cellulitis. Our findings reveal a function for macrophages in the regulation of HA with an impact on infection outcomes, which may be harnessed to limit the establishment of infection in the hypodermal niche., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

The aim of this study was to examine data demonstrating that Scarpa's fascia, a superficial fascia of the anterior abdominal wall, is a vascularized tissue. Specimens of the fascia of seven volunteers undergoing abdominoplasty surgical procedures at the Plastic Surgery Unit of the University of Padova Medical Center were collected. Fractal analysis and quantitative assessment of the vascular network of the fascia was carried out, exploiting the presence of blood in the vessels. Each sample was divided and processed for histological/immunohistochemical analysis (into 5 micron-paraffin embedded sections and cryo-sectioned free-floating samples) as well as for electron microscopy study. A rich vascular pattern forming a fine, dense meshwork with an area percentage of 6.20% ± 2.10% von Willebrand factor stained vessels was noted in all the specimens of the fascia examined; the area percentage of the αSMA-stained vessels was 2.93% ± 1.80%. The diameters of the vessels fell between the 13 and 65 μm range; the network was composed of arteries, veins, capillaries and lymphatic segments. Topological results showed that the vascular network within Scarpa's fascia is well branched (segments: 6615 ± 3070 and 8.40 ± 3.40 per mm 2 ; crossing points: 3092 ± 1490 and 3.40 ± 1.90 per mm 2 ). Fractal analysis (fractal dimension = 1.063 ± 0.10; lacunarity = 0.60 ± 0.10) revealed that this particular vascular network has an optimal spatial distribution and homogeneity occupying the entire space of the superficial fascia. These findings could undoubtedly be useful to plastic surgeons as well as to pain management specialists., (© 2022 American Association of Clinical Anatomists and British Association of Clinical Anatomists.)

Recently, the superficial fascia has been recognized as a specific anatomical structure between the two adipose layers-the superficial adipose tissue (SAT) and the deep adipose tissue (DAT). The evaluation of specific characteristics of cells, fibers, blood circulation, and innervation has shown that the superficial fascia has a clear and distinct anatomical identity, but knowledge about lymphatic vessels in relation to the superficial fascia has not been described. The aim of this study was to evaluate the presence of lymphatic vessels in the hypodermis, with a specific focus on the superficial fascia and in relation to the layered subdivision of the subcutaneous tissue into SAT and DAT. Tissue specimens were harvested from three adult volunteer patients during abdominoplasty and stained with D2-40 antibody for the lymphatic endothelium. In the papillary dermis, a huge presence of lymphatic vessels was highlighted, parallel to the skin surface and embedded in the loose connective tissue. In the superficial adipose tissue, thin lymphatic vessels (mean diameter of 11.6 ± 7.71 µm) were found, close to the fibrous septa connecting the dermis to the deeper layers. The deep adipose tissue showed a comparable overall content of lymphatic vessels with respect to the superficial layer; they followed the blood vessel and had a larger diameter. In the superficial fascia, the lymphatic vessels showed higher density and a larger diameter, in both the longitudinal and transverse directions along the fibers, as well as vessels that intertwined with one another, forming a rich network of vessels. This study demonstrated a different distribution of the lymphatic vessels in the various subcutaneous layers, especially in the superficial fascia, and the demonstration of the variable gauge of the vessels leads us to believe that they play different functional roles in the collection and transport of interstitial fluid-important factors in various surgical and rehabilitation fields.

Background: Gynoid lipodystrophy is one of the most common cosmetical problems in women., Aims: The study aims to examine the pathomorphology and histology of subcutaneous tissue in women with gynoid lipodystrophy exposed to local compression/vibration therapy using a non-invasive Beautylizer Therapy Cosmospheres V medical device., Methods: The study enrolled 25 virtually healthy women aged 25-45 years with gynoid lipodystrophy grades I and II. The women included in the study completed a 10-session month-long therapeutic course with a non-invasive Beautylizer Therapy Cosmospheres V medical device. Bioptic punch-size samples were taken from the gluteal region prior to and following the 10-session therapy course with the device., Results: After completing a 10-session treatment course, a decrease in the mean adipocyte area from 123.08 ± 13.60 μm to 67.14 ± 4.20 μm was observed in punch bioptic samples of subcutaneous fat tissues of women with gynoid lipodystrophy as compared with the pre-treatment indices., Conclusion: Thus, local application of 10-session therapy with a non-invasive Beautylizer Therapy Cosmospheres V medical device in women with gynoid lipodystrophy demonstrated a positive effect on the histological structure of the hypodermis., (© 2022 Wiley Periodicals LLC.)

How serine/threonine phosphatases are spatially and temporally tuned by regulatory subunits is a fundamental question in cell biology. Ankyrin repeat, SH3 domain, proline-rich-region-containing proteins are protein phosphatase 1 catalytic subunit binding partners associated with cardiocutaneous diseases. Ankyrin repeat, SH3 domain, proline-rich-region-containing proteins localize protein phosphatase 1 catalytic subunit to cell-cell junctions, but how ankyrin repeat, SH3 domain, proline-rich-region-containing proteins localize and whether they regulate protein phosphatase 1 catalytic subunit activity in vivo is unclear. Through a Caenorhabditis elegans genetic screen, we find that loss of the ankyrin repeat, SH3 domain, proline-rich-region-containing protein homolog, APE-1, suppresses a pathology called "jowls," providing us with an in vivo assay for APE-1 activity. Using immunoprecipitations and mass spectrometry, we find that APE-1 binds the protein phosphatase 1 catalytic subunit called GSP-2. Through structure-function analysis, we discover that APE-1's N-terminal half directs the APE-1-GSP-2 complex to intercellular junctions. Additionally, we isolated mutations in highly conserved residues of APE-1's ankyrin repeats that suppress jowls yet do not preclude GSP-2 binding, implying APE-1 does more than simply localize GSP-2. Indeed, in vivo reconstitution of APE-1 suggests the ankyrin repeats modulate phosphatase output, a function we find to be conserved among vertebrate homologs., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The superficial fascia has only recently been recognized as a specific anatomical structure. Furthermore, whereas it is actually recognized that the innervation of the deep/muscular fascia plays a key role in proprioception and nociception, there are very few studies that have analyzed these characteristics in the superficial fascia. In this work, our group analyzed two different anatomical districts (abdomen and thigh), from volunteer patients, undergoing surgery procedures. Each sample was processed for histological analysis by Hematoxylin&Eosin, and by immunohistochemistry stainings (in 5-micron-paraffin embedded section and in cryosectioned free floating samples), with antibodies specific for nerve fibers: S100 antibody for myelinating and non-myelinating Schwann cells, PGP9.5 antibody as pan-neuronal marker, tyrosine hydroxylase for autonomic innervation. The results revealed a huge innervation: the nervous structures were found above all around blood vessels and close to adipocytes, but they penetrated also in the connective tissue itself and are found in the midst of fibro-adipose tissue. The tissue is pervaded by both thin (mean diameter of 4.8 ± 2.6 μm) and large nerve fiber bundles of greater diameter (21.1 ± 12.2 μm). The ratio S100/TH positivity was equal to 2.96, with a relative percentage of autonomic innervation with of 33.82%. In the light of these findings is evident that the superficial fasciae have a clear and distinct anatomical identity and a specific innervation, which should be considered to better understand their role in thermoregulation, exteroception and pain perception. The knowledge of the superficial fascia may improve grading and developing of different manual approach for treatments of fascial dysfunctions, and the understanding of how some factors like temperature or manual therapies can have an impact on sensitivity of the fascia., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © Fede, Petrelli, Pirri, Neuhuber, Tiengo, Biz, De Caro, Schleip and Stecco.)

Background: Ten percent povidone-iodine (PVP-I) was initially promoted as 'tamed iodine' as the chemical activity of the active biocide, uncomplexed or free molecular iodine (I 2 ), is reduced 30- to 50-fold compared with Lugol's solution. The idea that I 2 is responsible for topical iodine staining and irritation remains widely held. However, there are no controlled studies that characterize the cytotoxicity and staining of the hydrophobic I 2 species compared with the other hydrophilic iodine species that comprise over 99.9% of the total iodine in topical iodine disinfectants., Aims: To compare the staining properties of the I 2 species with other topical iodine disinfectants; to evaluate if the concentrations of I 2 in diluted PVP-I used to reduce severe acute respiratory syndrome coronavirus-2 in the nasal cavity are potentially cytotoxic; and to determine if high concentrations of I 2 can be delivered beyond the stratum corneum into the hypodermis, which could provide a mechanistic rationale for I 2 out-gassing., Methods: Five liquid compositions that contained complexed and uncomplexed (free) I 2 in aqueous and non-aqueous carriers were used to evaluate the interaction of I 2 with mammalian cells in culture as well as human and pig skin., Findings: Concentrations of I 2 (7800 ppm) that are 1500 times higher than that found in PVP-I can be applied to skin without irritation and staining. I 2 is not cytotoxic at concentrations >100 times higher than that found in PVP-I, and does not contribute materially to staining of skin at concentrations found in Lugol's solution (approximately 170 ppm). I 2 can partition into hypodermis tissue, remain there for hours and out-gas from skin. PVP-I and Lugol's solution are highly effective topical disinfectants, but do not facilitate diffusion of I 2 through the stratum corneum., Conclusion: The maximum concentration of I 2 found in diluted PVP, approximately 25 ppm, is not cytotoxic or irritating. The potential clinical utility of I 2 has been limited by incorporating this broad-spectrum biocide into acidic aqueous formulations that contain numerous chemical species that contribute toxicity but not biocidal activity. I 2 can be delivered topically into hypodermis tissue without irritation., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Rhizophora mangle , one of the main neotropic mangrove species, has wide phenological variability associated with soil salinity. Since global warming is one of the main drivers of changes in salinity, understanding the influence of this variable at the species level would help improve the prediction of climate change in the ecological services provided by mangroves. To understand the physiological and/or anatomical responses to water stress generated by edaphic salinity and its relationship with phenological and structural diversity, we quantified the functional traits of leaf tissue subjected to a cross-seeding experiment between two forests with different ranges of natural salinity (0-18 PSU and 20 to 70 PSU). A total of 180 propagules, 90 native and 90 from the other forest, were planted in each forest. Every three months for a year, soil salinity and growth, adaptability, and survival of propagules that were transformed into seedlings were measured. The traits evaluated between the two saline regimes presented significant differences, as stated in the working hypothesis. Likewise, there were modifications in the hypodermis and the xylem vessels in the exchanged seedlings, tissues related to water storage, and conduction. These responses allowed native euhaline forest seedlings to grow in oligohaline. The opposite occurred with seedlings originating in low salinities that did not survive in high salinities. Differences in adaptability between populations of R. mangle subjected to ranges of contrasting salinity may imply changes at the structural level, zoning, and abundance of the species front to the climate change processes., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors.)

Severe injuries to skin including hypodermis require full-thickness skin replacement. Here, we bioengineered a tri-layered human skin substitute (TLSS) containing the epidermis, dermis, and hypodermis. The hypodermal layer was generated by differentiation of human adipose stem cells (ASC) in a collagen type I hydrogel and combined with a prevascularized dermis consisting of human dermal microvascular endothelial cells and fibroblasts, which arranged into a dense vascular network. Subsequently, keratinocytes were seeded on top to generate the epidermal layer of the TLSS. The differentiation of ASC into adipocytes was confirmed in vitro on the mRNA level by the presence of adiponectin, as well as by the expression of perilipin and FABP-4 proteins. Moreover, functional characteristics of the hypodermis in vitro and in vivo were evaluated by Oil Red O, BODIPY, and AdipoRed stainings visualizing intracellular lipid droplets. Further, we demonstrated that both undifferentiated ASC and mature adipocytes present in the hypodermis influenced the keratinocyte maturation and homeostasis in the skin substitutes after transplantation. In particular, an enhanced secretion of TGF-β1 by these cells affected the epidermal morphogenesis as assessed by the expression of key proteins involved in the epidermal differentiation including cytokeratin 1, 10, 19 and cornified envelope formation such as involucrin. Here, we propose a novel functional hypodermal-dermo-epidermal tri-layered skin substitute containing blood capillaries that efficiently promote regeneration of skin defects. STATEMENT OF SIGNIFICANCE: The main objective of this study was to develop and assess the usefulness of a tri-layered human prevascularized skin substitute (TLSS) containing an epidermis, dermis, and hypodermis. The bioengineered hypodermis was generated from human adipose mesenchymal stem cells (ASC) and combined with a prevascularized dermis and epidermis. The TLSS represents an exceptional model for studying the role of cell-cell and cell-matrix interactions in vitro and in vivo. In particular, we observed that enhanced secretion of TGF-β1 in the hypodermis exerted a profound impact on fibroblast and keratinocyte differentiation, as well as epidermal barrier formation and homeostasis. Therefore, improved understanding of the cell-cell interactions in such a physiological skin model is essential to gain insights into different aspects of wound healing., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The beneficial role of subcutaneous adipose tissue in skin rejuvenation derived from its capacity to fill the under-layer volumes but also from its ability to regulate the extracellular matrix production by dermis fibroblasts. Hyaluronic acid (HA), a major component of the extracellular matrix, is a commonly used injectable dermal filler showing excellent efficiencies to maintain tissue augmentation even after its biodegradation. To improve their stability, the HA molecules can also be "cross-linked" to each other. The effects of cross-linked HA-based fillers on the dermal structure are well known. For safety reasons, most of the physicians prefer to use the blunt cannula for injections. However, evidences showed that the cannula could not be located in the dermis, but it passes through immediate hypodermis and the long-lasting effect of cross-linked HA-based fillers may be related to its effects on adipose tissue. To test whether cross-linked HA has a direct effect on human adipocytes, we treated isolated adipocytes and precursors cells from human skin donors with cross-linked HA. Biochemical and cellular analysis demonstrated that treatment by cross-linked HA showed beneficial effects on differentiated cell adherence and survival as well as reduced basal and induced lipolysis in fully mature adipocytes. Taken together, these data showed that cross-linked HA promoted cell adherence and preserved the adipogenic capacity of preadipocytes during prolonged cell culture, bringing additional evidences of the beneficial role of cross-linked HA-based fillers in maintenance of the subcutaneous fat mass. This first study could defend a preventive approach to facial volume loss during natural aging., (© 2020 Laboratoires FILLMED. Journal of Cosmetic Dermatology published by Wiley Periodicals LLC.)

Collagen-enriched cuticle forms the outermost layer of skin in nematode Caenorhabditis elegans. The nematode's genome encodes 177 collagens, but little is known about their role in maintaining the structure or barrier function of the cuticle. In this study, we found six permeability determining (PD) collagens. Loss of any of these PD collagens-DPY-2, DPY-3, DPY-7, DPY-8, DPY-9, and DPY-10-led to enhanced susceptibility of nematodes to paraquat (PQ) and antihelminthic drugs- levamisole and ivermectin. Upon exposure to PQ, PD collagen mutants accumulated more PQ and incurred more damage and death despite the robust activation of antioxidant machinery. We find that BLMP-1, a zinc finger transcription factor, maintains the barrier function of the cuticle by regulating the expression of PD collagens. We show that the permeability barrier maintained by PD collagens acts in parallel to FOXO transcription factor DAF-16 to enhance survival of insulin-like receptor mutant, daf-2. In all, this study shows that PD collagens regulate cuticle permeability by maintaining the structure of C. elegans cuticle and thus provide protection against exogenous toxins., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

An intact skin is essential in high-quality apples. Ongoing deposition of cuticular material during fruit development may decrease microcracking. Our objective was to establish a system for quantifying cutin and wax deposition in developing apple fruit. Oleic acid ( 13 C and 14 C labelled) and palmitic acid ( 14 C labelled) were fed to developing apples and the amounts incorporated in the cutin and wax fractions were quantified. The incorporation of 14 C oleic acid (C18) was significantly higher than that of 14 C palmitic acid (C16) and the incorporation in the cutin fraction exceeded that in the wax fraction. The amount of precursor incorporated in the cutin increased asymptotically with time, but the amount in the wax fraction remained about constant. Increasing the concentration of the precursor applied generally increased incorporation. Incorporation in the cutin fraction was high during early development (43 days after full bloom) and decreased towards maturity. Incorporation was higher from a dilute donor solution (infinite dose feeding) than from a donor solution subjected to drying (finite dose feeding) or from perfusion of the precursor by injection. Feeding the skin of a developing apple with oleic acid resulted in significant incorporation in the cutin fraction under both laboratory and field conditions.

Understanding how animals detect and respond to pathogen threats is central to dissecting mechanisms of host immunity. The oomycetes represent a diverse eukaryotic group infecting various hosts from nematodes to humans. We have previously shown that Caenorhabditis elegans mounts a defense response consisting of the induction of chitinase-like (chil) genes in the epidermis to combat infection by its natural oomycete pathogen Myzocytiopsis humicola. We provide here evidence that C. elegans can sense the oomycete by detecting an innocuous extract derived from animals infected with M. humicola. The oomycete recognition response (ORR) leads to changes in the cuticle and reduction in pathogen attachment, thereby increasing animal survival. We also show that TAX-2/TAX-4 function in chemosensory neurons is required for the induction of chil-27 in the epidermis in response to extract exposure. Our findings highlight that neuron-to-epidermis communication may shape responses to oomycete recognition in animal hosts., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Calcium is one of the most poorly reutilized nutrients. Its deficiencies cause various physiological disturbances and, consequently, reduce the quantity and quality of yields. Reduced content of Ca 2+ ions in cells leads to development of, e.g., bitter pit in apples. Efficient and instantaneous mitigation of Ca 2+ deficiencies is provided by foliar feeding. There are no detailed data on the effect of foliar feeding with various calcium forms on the cell structure or on the microanalysis and mapping of this element in apple fruit cells. Therefore, we carried out comparative studies of the ultrastructure of epidermis and hypodermis cells, to assess the content and distribution of calcium in the cell wall, cytoplasmic membrane, cytoplasm, and precipitates of Malus domestica Borkh. 'Szampion' fruit exposed to four Ca treatments, including the control with no additional Ca supplementation (I) and foliar applications of Ca(NO 3 ) 2 (II), CaCl 2 (III), and Ca chelated with EDTA (IV). Light and transmission electron microscopy and an X-ray microanalyzer were used and showed a beneficial effect of calcium preparations on the ultrastructure of fruit epidermis and hypodermis cells, manifested in the presence of a normally developed cell wall with a regular middle lamella, preserved continuity of cytoplasmic membranes, and stabilized cell structure. In the selected elements of apical epidermis cells, the highest level of Ca 2+ ions was detected in the middle lamella, cell wall, plasmalemma, and cytoplasm. The highest increase in the Ca 2+ content in these cell constituents was recorded in treatment IV, whereas the lowest value of the parameters was noted in variant III.

Russeting is an economically important surface disorder in apple ( Malus × domestica Borkh.). Indirect evidence suggests an irregular skin structure may be the cause of the phenomenon. The objective of this study was to characterize epidermal and hypodermal cell morphology and the mechanical properties of the skins of apple cultivars of differing russet susceptibility. Dimensions of epidermal and hypodermal cells were determined using microscopy. Stiffness ( S ), maximum force (Fmax), and maximum strain (ɛmax) at failure were quantified using uniaxial tensile tests of skin strips. Particularly during early fruit development, epidermal cells (EC) and hypodermal cells (HC) in russet non-susceptible cultivars occurred in greater numbers per unit area than in russet-susceptible ones. The EC and HC were lower in height, shorter in length, and of reduced tangential surface area. There were little differences in S or Fmax between non-susceptible and susceptible cultivars. However, the ɛmaxwere higher for the skins of non-susceptible cultivars, than for those of susceptible ones. This difference was larger for the young than for the later growth stages. It is concluded that russet-susceptible cultivars generally have larger cells and a wider distribution of cell sizes for both EC and HC. These result in decreased ɛmax for the skin during early fruit development when russet susceptibility is high. This increases the chances of skin failures which is known to trigger russeting., Competing Interests: There are no known competing interests that could influence the work reported in this paper.

Objective: High frequency ultrasound allows visualization of epidermis, dermis and hypodermis, precise measurement of skin thickness, as well as assessment of skin oedema, fibrosis and atrophy. The aim of this pilot cross-sectional observational study was to assess the performance and multiobserver variability of ultra-high-frequency (UHF) (50 MHz) ultrasound (US) in measuring skin thickness as well as the capacity of UHF-derived skin features to differentiate SSc patients from healthy controls., Methods: Twenty-one SSc patients (16 limited and five diffuse SSc) and six healthy controls were enrolled. All subjects underwent US evaluation by three experts at three anatomical sites (forearm, hand and finger). Dermal thickness was measured and two rectangular regions of interest, one in dermis and one in hypodermis, were established for texture feature analysis., Results: UHF-US allowed a precise identification and measurement of the thickness of the dermis. The dermal thickness in the finger was significantly higher in patients than in controls (P < 0.05), while in the forearm it was significantly lower in patients than in controls (P < 0.001). Interobserver variability for dermal thickness was good to excellent [forearm intraclass correlation coefficient (ICC) = 0.754; finger ICC = 0.699; hand ICC = 0.602]. Texture computed analysis of dermis and hypodermis was able to discriminate between SSc and healthy subjects (area under the curve >0.7)., Conclusion: These preliminary data show that skin UHF-US allows a very detailed imaging of skin layers, a reliable measurement of dermal thickness, and a discriminative capacity between dermis and hypodermis texture features in SSc and healthy subjects., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Adrenoleukodystrophy is a neurometabolic disorder caused by a defective peroxisomal ABCD1 transporter of very long-chain fatty acids (VLCFAs). Its pathogenesis is incompletely understood. Here we characterize a nematode model of X-ALD with loss of the pmp-4 gene, the worm orthologue of ABCD1. These mutants recapitulate the hallmarks of X-ALD: i) VLCFAs accumulation and impaired mitochondrial redox homeostasis and ii) axonal damage coupled to locomotor dysfunction. Furthermore, we identify a novel role for PMP-4 in modulating lipid droplet dynamics. Importantly, we show that the mitochondria targeted antioxidant MitoQ normalizes lipid droplets size, and prevents axonal degeneration and locomotor disability, highlighting its therapeutic potential. Moreover, PMP-4 acting solely in the hypodermis rescues axonal and locomotion abnormalities, suggesting a myelin-like role for the hypodermis in providing essential peroxisomal functions for the nematode nervous system., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Fascia in the veterinary sciences is drawing attention, such that physiotherapists and animal practitioners are now applying techniques based on the concept of fascia studies in humans. A comprehensive study of fascia is therefore needed in animals to understand the arrangement of the fascial layers in an unguligrade horse and a digitigrade dog. This study has examined the difference between the horse and the dog fascia at specific regions, in terms of histology, and has compared it with the human model. Histological examinations show that in general the fascia tissue of the horse exhibits a tight and dense composition, while in the dog it is looser and has non-dense structure. Indeed, equine fascia appears to be different from both canine fascia and the human fascia model, whilst canine fascia is very comparable to the human model. Although regional variations were observed, the superficial fascia (fascia superficialis) in the horse was found to be trilaminar in the trunk, yet multilayered in the dog. Moreover, crimping of collagen fibers was more visible in the horse than the dog. Blood vessels and nerves were present in the loose areolar tissue of the superficial and the profound compartment of hypodermis. The deep fascia (fascia profunda) in the horse was thick and tightly attached to the underlying muscle, while in the dog the deep fascia was thin and loosely attached to underlying structures. Superficial and deep fascia fused in the extremities. In conclusion, gross dissection and histology have revealed species variations that are related to the absence or presence of the superficial adipose tissue, the retinacula cutis superficialis, the localization and amount of elastic fibers, as well as the ability to slide and glide between the different layers. Further research is now needed to understand in more detail whether these differences have an influence on the biomechanics, movements and proprioception of these animals., (© 2019 Anatomical Society.)

In apple (Malus×domestica) fruit, the different layers of the exocarp (cuticle, epidermis, and hypodermis) protect and maintain fruit integrity, and resist the turgor-driven expansion of the underlying thin-walled cortical cells during growth. Using in situ immunolocalization and size exclusion epitope detection chromatography, distinct cell type differences in cell wall composition in the exocarp were revealed during apple fruit development. Epidermal cell walls lacked pectic (1→4)-β-d-galactan (associated with rigidity), whereas linear (1→5)-α-l-arabinan (associated with flexibility) was exclusively present in the epidermal cell walls in expanding fruit and then appeared in all cell types during ripening. Branched (1→5)-α-l-arabinan was uniformly distributed between cell types. Laser capture microdissection and RNA sequencing (RNA-seq) were used to explore transcriptomic differences controlling cell type-specific wall modification. The RNA-seq data indicate that the control of cell wall composition is achieved through cell-specific gene expression of hydrolases. In epidermal cells, this results in the degradation of galactan side chains by possibly five β-galactosidases (BGAL2, BGAL7, BGAL10, BGAL11, and BGAL103) and debranching of arabinans by α-arabinofuranosidases AF1 and AF2. Together, these results demonstrate that flexibility and rigidity of the different cell layers in apple fruit during development and ripening are determined, at least in part, by the control of cell wall pectin remodelling., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Aim: To investigate the structural and functional characteristics of palmar hypodermal tissue vascularization in Dupuytren's contracture patients of different age groups., Methods: Eighty-seven Dupuytren's contracture patients underwent partial fasciectomy. Twenty-two of them were less than 55 years old (Y-group, n = 22); the others were 55 and older (O-group, n = 65). In surgically excised representative tissue samples, a histomorphometric analysis of the perforating arteries of the palmar aponeurosis and stereologic analysis of hypodermis vascularity were performed. The method of laser flowmetry estimated the microcirculation of the skin of the palm., Results: Frequency of cases with rapid development of contracture (less than 5 years) was 13.6% in the Y-group and 40% in the O-group, P < 0.05. The external and luminal diameters of perforating arteries in palmar fascia were decreased more severely in Y. The thickness of intima increased three times compared with healthy control, and the intima/media relation also increased, especially in O. Increased numerical and volumetric micro-vessel densities in hypodermis, percentage of large vessels (more than 12 μm in diameter), and percentage of vessels with signs of periadventitial inflammatory infiltration were noted in Y. The percentage of vessels with adventitial fibrosis was greater in O than in Y. Base capillary flow in Y was increased compared to healthy control subjects and to O, and peak capillary flow was increased in comparison with control., Conclusion: Compared to the O-group, Y-group patients exhibited more severe constrictive remodeling of palmar fascia perforating arteries supplying hypodermis but more expressed compensatory changes of its capillarization., Competing Interests: Conflict-of-interest statement: The authors declare no conflict of interest.

In its natural habitat, the nematode Caenorhabditis elegans encounters a plethora of other organisms, including many that are pathogenic [1, 2]. The study of interactions between C. elegans and various pathogens has contributed to characterizing key mechanisms of innate immunity [2-4]. However, how C. elegans recognizes different pathogens to mount pathogen-specific immune responses remains still largely unknown [3, 5-8]. Expanding the range of known C. elegans-infecting pathogens and characterizing novel pathogen-specific immune responses are key steps toward answering this question. We report here that the oomycete Myzocytiopsis humicola is a natural pathogen of C. elegans, and we describe its infection strategy. We identify a new host immune response to pathogen exposure that involves induction of members of a previously uncharacterized gene family encoding chitinase-like (CHIL) proteins. We demonstrate that this response is highly specific against M. humicola and antagonizes the infection. We propose that CHIL proteins may diminish the ability of the oomycete to infect by hindering pathogen attachment to the host cuticle. This work expands our knowledge of natural eukaryotic pathogens of C. elegans and introduces a new pathosystem to address how animal hosts recognize and respond to oomycete infections., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The disproportionate length of an axon makes its structural and functional maintenance a major task for a neuron. The heterochronic gene lin-14 has previously been implicated in regulating the timing of key developmental events in the nematode C. elegans. Here, we report that LIN-14 is critical for maintaining neuronal integrity. Animals lacking lin-14 display axonal degeneration and guidance errors in both sensory and motor neurons. We demonstrate that LIN-14 functions both cell autonomously within the neuron and non-cell autonomously in the surrounding tissue, and we show that interaction between the axon and its surrounding tissue is essential for the preservation of axonal structure. Furthermore, we demonstrate that lin-14 expression is only required during a short period early in development in order to promote axonal maintenance throughout the animal's life. Our results identify a crucial role for LIN-14 in preventing axonal degeneration and in maintaining correct interaction between an axon and its surrounding tissue., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The nematode Caenorhabditis elegans is a simple metazoan animal that is widely used as a model to understand the genetic control of development. The completely sequenced C. elegans genome contains 22 T-box genes, and they encode factors that show remarkable diversity in sequence, DNA-binding specificity, and function. Only three of the C. elegans T-box factors can be grouped into the conserved subfamilies found in other organisms, while the remaining factors are significantly diverged and unlike those in most other animals. While some of the C. elegans factors can bind canonical T-box binding elements, others bind and regulate target gene expression through distinct sequences. The nine genetically characterized T-box factors have varied functions in development and morphogenesis of muscle, hypodermal tissues, and neurons, as well as in early blastomere fate specification, cell migration, apoptosis, and sex determination, but the functions of most of the C. elegans T-box factors have not yet been extensively characterized. Like T-box factors in other animals, interaction with a Groucho-family corepressor and posttranslational SUMOylation have been shown to affect C. elegans T-box factor activity, and it is likely that additional mechanisms affecting T-box factor activity will be discovered using the effective genetic approaches in this organism., (© 2017 Elsevier Inc. All rights reserved.)

Administration into the subcutaneous (SC) tissue is a typical route of delivery for therapeutic proteins, especially for frequent treatments, long-term regimens, or self-administration. It is currently believed that the maximum volume for SC injections is approximately 1.5 mL. Larger SC injection volumes are considered to be associated with injection pain and adverse events at the injection site. However, no controlled clinical studies and actual evidence exist to support this assumption. In this review, we discuss current and publically available data related to SC administration volumes. We conclude that injection volumes higher than 3.5 mL are worth exploring if required for the development of efficacious drug treatments. Studying tissue back pressure, injection site leakage, local tolerability, and injection-related adverse events, such as injection pain, should be considered for the development of higher SC injection volumes., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

A gene encoding fatty acid- and retinoid-binding protein was isolated from the cereal cyst nematode Heterodera avenae and the biochemical function of the protein that it encodes was analysed. The full-length cDNA of the Ha-far-1 gene is 827 bp long and includes a 22- nucleotide trans-spliced leader sequence (SL1) at its 5-end. The genomic clone of Ha-far-1 consists of eight exons separated by seven introns, which range in size from 48 to 186 bp. The Ha-far-1 cDNA contains an open reading frame encoding a 191 amino acid protein, with a predicted secretory signal peptide. Sequence analysis showed that Ha-FAR-1 has highest similarity to the Gp-FAR-1 protein from the potato cyst nematode, Globodera pallida and that the protein was grouped with all homologues from other plant-parasitic nematodes in a phylogenetic analysis. Fluorescence-based ligand binding analysis confirmed that the recombinant Ha-FAR-1 protein was able to bind fatty acids and retinol. Spatial and temporal expression assays showed that the transcripts of Ha-far-1 accumulated mainly in the hypodermis and that the gene is most highly expressed in third-stage juveniles of H. avenae. Fluorescence immunolocalization showed that the Ha-FAR-1 protein was present on the surface of the infective second-stage juveniles of H. avenae. Nematodes treated with dsRNA corresponding to Ha-far-1 showed significantly reduced reproduction compared to nematodes exposed to dsRNA from a non-endogenous gene, suggesting that Ha-far-1 may be an effective target gene for control of H. avenae using an RNAi strategy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

L-Rhamnose is a common component of cell-wall polysaccharides, glycoproteins and some natural products in bacteria and plants, but is rare in fungi and animals. In the present study, we identify and characterize a biosynthetic pathway for dTDP-rhamnose in Caenorhabditis elegans that is highly conserved across nematode species. We show that RML-1 activates glucose 1-phosphate (Glc-1-P) in the presence of either dTTP or UTP to yield dTDP-glucose or UDP-glucose, respectively. RML-2 is a dTDP-glucose 4,6-dehydratase, converting dTDP-glucose into dTDP-4-keto-6-deoxyglucose. Using mass spectrometry and NMR spectroscopy, we demonstrate that coincubation of dTDP-4-keto-6-deoxyglucose with RML-3 (3,5-epimerase) and RML-4 (4-keto-reductase) produces dTDP-rhamnose. RML-4 could only be expressed and purified in an active form through co-expression with a co-regulated protein, RML-5, which forms a complex with RML-4. Analysis of the sugar nucleotide pool in C. elegans established the presence of dTDP-rhamnose in vivo Targeting the expression of the rhamnose biosynthetic genes by RNAi resulted in significant reductions in dTDP-rhamnose, but had no effect on the biosynthesis of a closely related sugar, ascarylose, found in the ascaroside pheromones. Therefore, the rhamnose and ascarylose biosynthetic pathways are distinct. We also show that transcriptional reporters for the rhamnose biosynthetic genes are expressed highly in the embryo, in the hypodermis during molting cycles and in the hypodermal seam cells specifically before the molt to the stress-resistant dauer larval stage. These expression patterns suggest that rhamnose biosynthesis may play an important role in hypodermal development or the production of the cuticle or surface coat during molting., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

The heterochronic pathway controls temporal patterning during Caenorhabditis elegans larval development. The highly conserved let-7 microRNA (miRNA) plays a key role in this pathway, directing the larval-to-adult (L/A) transition. Hence, knowledge of the genetic interactome of let-7 has the potential to provide insight into both control of temporal cell fates and mechanisms of regulation and function of miRNAs. Here, we report the results of a genome-wide, RNAi-based screen for suppressors of let-7 mutant vulval bursting. The 201 genetic interaction partners of let-7 thus identified include genes that promote target silencing activity of let-7, seam cell differentiation, or both. We illustrate the suitability of our approach by uncovering the mitotic cyclin-dependent kinase CDK-1 as a downstream effector of let-7 that affects both seam cell proliferation and differentiation, and by identifying a core set of candidate modulators of let-7 activity, which includes all subunits of the condensin II complex. We propose that the genes identified in our screen thus constitute a valuable resource for studies of the heterochronic pathway and miRNAs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background and Objective: Radiofrequency currents are commonly used in dermatology to treat cutaneous and subcutaneous tissues by heating. The subcutaneous morphology of tissue consists of a fine, collagenous and fibrous septa network enveloping clusters of adipocyte cells. The architecture of this network, namely density and orientation of septa, varies among patients and, furthermore, it correlates with cellulite grading. In this work we study the effect of two clinically relevant fibrous septa architectures on the thermal and elastic response of subcutaneous tissue to the same RF treatment; in particular, we evaluate the thermal damage and thermal stress induced to an intermediate- and a high-density fibrous septa network architecture that correspond to clinical morphologies of 2.5 and 0 cellulite grading, respectively., Study Design/materials and Methods: We used the finite element method to assess the electric, thermal and elastic response of a two-dimensional model of skin, subcutaneous tissue and muscle subjected to a relatively long, constant, low-power RF treatment. The subcutaneous tissue is constituted by an interconnected architecture of fibrous septa and fat lobules obtained by processing micro-MRI sagittal images of hypodermis. As comparison criteria for the RF treatment of the two septa architectures, we calculated the accumulated thermal damage that corresponds to 63% loss in cell viability., Results: Electric currents preferentially circulated through the fibrous septa in the subcutaneous tissue. However, the intensity of the electric field was higher within the fat because it is a poor electric conductor. The power absorption in the fibrous septa relative to that in the fat varied with septum orientation: it was higher in septa with vertical orientation and lower in septa with horizontal orientation. Overall, maximum values of electric field intensity, power absorption and temperature were similar for both fibrous septa architectures. However, the high-density septa architecture (cellulite grade 0) had a more uniform and broader spatial distribution of power absorption, resulting in a larger cross-sectional area of thermal damage (≈1.5 times more). Volumetric strains (expansion and contraction) were small and similar for both network architectures. During the first seconds of RF exposure, the fibrous septa were subjected to thermal expansion regardless of orientation. In the long term, the fibrous septa contracted due to the thermal expansion of fat. Skin and muscle were subjected to significantly higher Von Mises stresses (measure of yield) or distortion energy than the subcutaneous tissue., Conclusion: The distribution of electric currents within subcutaneous tissues depends on tissue morphology. The electric field is more intense in septum oriented along the skin to muscle (top to bottom) direction, creating lines or planes of preferential heating. It follows that the more septum available for preferential heating, the larger the extent of volumetric RF-heating and thermal damage to the subcutaneous tissue. Thermal load alone, imposed by long-exposure to heating up to 50 °C, results in small volumetric expansion and contraction in the subcutaneous tissue. The subcutaneous tissue is significantly less prone to non-reversible deformation by a thermal load than the skin and muscle., (© 2015 Wiley Periodicals, Inc.)

The rigid crustacean exoskeleton, the cuticle, is composed of the polysaccharide chitin, structural proteins and mineral deposits. It is periodically replaced to enable growth and its construction is an energy-demanding process. Ecdysis, the shedding event of the old cuticle, is preceded by a preparatory phase, termed premolt, in which the present cuticle is partially degraded and a new one is formed underneath it. Procambarus clarkii (Girard 1852), an astacid crustacean, was used here to comprehensively examine the changing patterns of gene expression in the hypodermis underlying the cuticle of the carapace at seven time points along ~14 premolt days. Next generation sequencing was used to construct a multi-tissue P. clarkii transcript sequence assembly for general use in a variety of transcriptomic studies. A reference transcriptome was created here in order to perform digital transcript expression analysis, determining the gene expression profiles in each of the examined premolt stages. The analysis revealed a cascade of sequential expression events of molt-related genes involved in chitin degradation, synthesis and modification, as well as synthesis of collagen and four groups of cuticular structural genes. The new description of major transcriptional events during premolt and the determination of their timing provide temporal markers for future studies of molt progress and regulation. The peaks of the expression of the molt-related genes were preceded by expression peaks of cytoskeletal genes that are hypothesized to be essential for premolt progress through regulating protein synthesis and/or transport, probably by remodeling the cytoskeletal structure., (© 2014. Published by The Company of Biologists Ltd.)

Although Caenorhabditis elegans is commonly used as a model organism for studies of cell biology, development and physiology, the small size of the worm has impeded measurements of ion transport by the excretory cell and hypodermis. Here, we use the scanning ion-selective microelectrode technique to measure efflux and influx of K(+), H(+), Na(+) and Ca(2+) in intact worms. Transport of ions into, or out of, immobilized worms produces small gradients in ion concentration in the unstirred layer near the surface of the worm. These gradients are readily detectable with ion-selective microelectrodes and the corresponding ion fluxes can be estimated using the Fick equation. Our data show that effluxes of K(+), H(+), Na(+) and Ca(2+) are localized to the region of the excretory pore, consistent with release of these ions from the excretory cell, and that effluxes increase after experimental preloading with Na(+), K(+) or Ca(2+). In addition, the hypodermis is a site of Na(+) influx., (© 2014. Published by The Company of Biologists Ltd.)

Plants that are adapted to waterlogged conditions develop aerenchyma in roots for ventilation. Some wetland plant species also form an apoplastic barrier at the outer cell layers of roots that reduces radial oxygen loss (ROL) from the aerenchyma and prevents toxic compounds from entering the root. The composition of the apoplastic barrier is not well understood. One potential component is suberin, which accumulates at the hypodermal/exodermal cell layers of the roots under waterlogged soil conditions or in response to other environmental stimuli. However, differences in suberin content and composition between plant species make it difficult to evaluate whether suberin has a role in preventing ROL. In this article, we summarize recent advances in understanding apoplastic barrier formation in roots and, between various plant species, compare the chemical compositions of the apoplastic barriers in relation to their permeability to oxygen. Moreover, the relationship between suberin accumulation and the barrier to ROL is discussed.

Among citrus rootstocks, higher specific root length (SRL, root length/d. wt) has been linked to several specific morphological and physiological traits, including smaller average root diameter, higher root hydraulic conductivity and higher rates of root proliferation. In this study, thickness of the outer tangential exodermal (hypodermal) wall and its suberin layer, number of passage cells, presence of epidermis, and stelar anatomy were examined and related to variation in root diameter of field roots of known maximum age. We also compared root morphology and anatomy of young roots in the field with those of potted rootstock seedlings in the glasshouse. Fibrous roots were measured separately from pioneer (framework) roots. Among the fibrous roots, only the first-order (root links having a root tip) and second-order (root links bearing first-order roots) laterals were examined. Among first-order field roots, larger root diameter was caused by larger rather than more numerous cells in the cortex. Root diameter of first-order roots was positively correlated with both number of passage cells in the exodermis and thickness of the secondary walls of the exodermis in both field and potted plants. Exodermal walls were about 80% thicker in field- than pot-grown roots. In the field, in more than 50% of the first-order roots examined less than 30% of the root surface was still covered by epidermis, with few differences among rootstocks. In contrast, in roots of 19-wk-old glasshouse plants generally 70-100% of the epidermis was still intact. There was no evidence of secondary xylem development in second-order fibrous roots in the field; in seedling, pot-grown rootsystems, 75-97% of second-order roots had formed secondary xylem despite their small diameter (<0.8 mm). It is argued that there can be suites of physiological, morphological and anatomical traits in roots that co-vary with specific root length. Investigations of how root morphology and anatomy are linked to root function, moreover, need to recognize trait variability and the potentially important differences between field- and pot- grown (seedling) roots.

Following our publication of a new method of calculating rates of water uptake by roots from measurements of the rate of accumulation on the roots of a marker solute, this paper describes the sites of accumulation of the solute, which indicate the sites where the water entered the symplast. Sulphorhodamine G (SR) was supplied in aeroponic mist culture to large maize plants with fully developed root systems. Root samples were collected after 4 to 8 h of transpiration in the dye-mist from both axes and branches of the main roots, and from non-transpiring (detopped) controls, frozen rapidly, freeze-substituted, and embedded and sectioned by an anhydrous procedure that preserves the SR in place. Whole mounts and sections were examined by bright-field, polarizing and epifluorescence microscopy. Major accumulations of SR were all at the outer surface of the roots, on Epidermal or root hair cell walls, or, in older roots where the epidermal cells were separating or dead, on the outer wall of the hypodermis. On some branch roots, though not on any main axes, the accumulations of SR were conspicuously aligned in the grooves over anticlinal cell walls of the epidermis. Non-transpiring plants showed very slight accumulations. Diffusion of SR into the cell wall apoplast was limited by the suberized lamellae and Casparian bands of the hypodermis, except in some branch roots, where SR diffused throughout the cortical cell walls. In parts of roots where the epidermis and hypodermis had been damaged, SR diffused through cell walls of the cortex from the wound site. These patterns of accumulation show that water enters the symplast of roots at the outermost cell membranes of the root, whether they are epidermal or hypodermal cells. Water enters roots with fully developed hypodermises at high rates. The rote of the hypodermal suberization is to limit solute movement in the wall apoplast. A symplastic path for water throughout the cortex, endodermis and living cells of the stele is suggested.

Ultrastructural study of the body wall of preparasitic, parasitic, and postparasitic stages of Romanomermis culicivorax showed that the cuticle of all three stages was permeable to lanthanum. The cuticle of the parasitic stage was the thinnest and showed the greatest permeability. Lanthanum accumulated on the apical surfaces of the hypodermal cells but was not found intracellularly. The negative staining characteristics of lanthanum enhanced the detection of numerous smooth septate junctions in the hypodermis of the parasitic stage.

The body wall fine structure including the cuticle, hypodermis, and somatic muscles is similar in males of Meloidogyne incognita and Heterodera glycines. The cuticle can be regarded as basically three-layered in both species, but is much thicker in M. incognita than in H. glycines, and differences occur in surface markings. The chordal and interchordal hypodermis is syncytial. Hypodermal tissue pervades the lip region, and lines the stomatal cavity and stylet shaft. Various organelles and structures, some previously undescribed, are concentrated in the chords. Their possible role in lipid metabolism is considered, as well as the probable function of the hypodermis in fornlation of the cephalic framework and stylet. The interchordal hypodermis which encloses peripheral nerves, is periodically transversed by bundles of fibrils which are homologous with the subcuticular striation previously observed in the light microscope. The somatic musculature is meromyarian, and the muscle cells are of the platymyarian type with I, A, and H bands, but without Z bands or T tubules. Thin dense bands are present in the H bands, and appear to be associated with sarcoplasmic reticulum.

Iontophoretic cobalt staining of the nematode Phocanema decipiens results in the deposition of cobalt between the contractile bases of adjacent muscle cells and in a hexagonal lattice pattern in the hypodermis. The possibility of staining sarcolemmal invaginations between muscle cells which are proprioceptive or coordinate the activity of adjacent muscle cells is suggested.

The development of suberin lamellae in the hypodermis of Zea mays cv. LG 11 was observed by electron microscopy and the presence of suberin inferred from autoliuorescence and by Sudan black B staining in nodal (adventitious) and primary (seminal) root axes. Suberin lamellae were evident at a distance of 30-50 mm from the tip of roots growing at 20°C and became more prominent with distance from the tip. Both oxygen deficiency and growth at 13°C produced shorter roots in which the hypodermis was suberized closer to the root tip. There were no suberin lamellae in epidermal cells or cortical collenchyma adjacent to the hypodermis. Plasmodesmata were not occluded by the suberin lamellae: there were twice as many of them in the inner tangential hypodermal wall (1,14 μn -2 ) as in the junction between the epidermis and hypodermis (0.54 μm -2 ). Water uptake by seminal axes (measured by micropotometry) was greater at distances more than 100 mm from the root lip than in the apical zone where the hypodermis was unsuberized. In the more mature zones of roots grown at 13°C rates of water uptake were greater than in roots grown at 20°C even though hypodermal suberization was more marked. Sleeves of epidermal/hypodermal cells (plus some accessory collenchyma) were isolated from the basal 60 mm of nodal axes by enzymatic digestion (drisclase). The roots were either kept totally immersed in culture solution or had the basal 50 mm exposed to moist air above the solution surface. In both treatments the permeabilities to tritiated water and 86 Rb were low (circa 10 -5 mms -1 ) in sleeves isolated from the extreme base. In roots grown totally immersed, however, the permeability of sleeves increased 10 to 50-fold over a distance of 40 mm. In roots exposed to moist air the permeability remained at a low level until the point where the root entered the culture solution and then increased rapidly (> 50-fold in a distance of 8 mm). Growth of roots in oxygen depleted (5% O 2 ) solutions promoted the development of extensive cortical aerenchymas. These developments were not associated with any reduction in permeability of sleeves isolated from the basal 40 mm of the axis. It was concluded that the presence of suberin lamellae in hypodermal walls does not necessarily indicate low permeability of cells or tissues to water or solutes. The properties of the walls (lamellae?) can be greatly changed by exposure to moist air, perhaps due to increased oxygen availability.

The association of neurons and hypodermal cells in longitudinal nerve chords and in interchordal positions (perhaps dorso-ventral commissures) in the body wall of Trichuris myocastoris Enigk has been examined with the electron microscope. Nerve chords are composed of several groups of neurons separated by processes of hypodermal cells; such groups may be functionally separate. Nerve cell bodies occur within the chords, and appear to be at least bipolar. Neurons emerge from the median chords and enter the interchordal regions, showing progressive invagination in hypodermal tissue with increased distance from the median line. Such invagination results in formation of mesaxons and may be significant in the nutrition of the neuron. Mesaxons may branch and may enclose single or groups of neurons; no spiralling of the mesaxon was seen. Occasionally more than one mesaxon was traced to a single neuron.