Non-living Tissues Research Articles

Review on the Role of Biofilm Formation in Bacterial Pathogenesis

Biofilms are defined as a accumulation of microorganisms living in a self-assembled matrix of polymeric substances that adheres to a variety of surfaces. Depending on the interaction between the surface and constituent cells, biofilms can be monolayer or multilayer. Initial attachment, irreversible attachment, micro colony growth, biofilm maturation, and biofilm dispersal are the five steps of the biofilm formation process. The processes that contribute tobiofilm formation are a dynamic and complicated process. Biofilm Formation Causes many bacteria have the ability to transition between planktonic and biofilm forms. The biofilm state appears to be a natural and prevalent condition of bacteria. Biofilm-forming bacteria are important for animal health. The risk of infection in animal species is likely to be higher than in humans. Because of the differences in animal housing and living conditions. Microorganisms capable of generating biofilms include P. aeruginosa, S. epidermidis, E. colispp, S. aureus, E. cloacae, and K. pneumonia. Bacterial pathogenesis of biofilms occurs frequently on medical devices and bits of dead tissue, such as sequestra of dead bone; nevertheless, they canals of or nonliving tissues. Biofilms in bacterial infections can improve the pathogenicity of the organism while also protecting it from external treatment. UV radiation, pH stress, chemical exposure, phagocytosis, dehydration, and antibiotics are all threats to cells that biofilms protect them. The Role of Signaling in Biofilm Formation is the bond between environmental stimuli and the bacteria’ reciprocation of the relevant signaling is required for the formation of a biofilm. Two-component systems and Quorum sensing can both be induced by these sensing systems. Tissue culture plate, cong red agar, and bioluminescent assay are some of the laboratory tests used to diagnose biofilm. The emergence and spread of antibiotic resistance among bacteria is one of the world’s most serious health issues. One of the implications of the bacterial biofilm communities that lead to persistent illnesses is antibiotic resistance.

Plant stem tissue modeling and parameter identification using metaheuristic optimization algorithms

Bio-impedance non-invasive measurement techniques usage is rapidly increasing in the agriculture industry. These measured impedance variations reflect tacit biochemical and biophysical changes of living and non-living tissues. Bio-impedance circuit modeling is an effective solution used in biology and medicine to fit the measured impedance. This paper proposes two new fractional-order bio-impedance plant stem models. These new models are compared with three commonly used bio-impedance fractional-order circuit models in plant modeling (Cole, Double Cole, and Fractional-order Double-shell). The two proposed models represent the characterization of the biological cellular morphology of the plant stem. Experiments are conducted on two samples of three different medical plant species from the family Lamiaceae, and each sample is measured at two inter-electrode spacing distances. Bio-impedance measurements are done using an electrochemical station (SP150) in the range of 100 Hz to 100 kHz. All employed models are compared by fitting the measured data to verify the efficiency of the proposed models in modeling the plant stem tissue. The proposed models give the best results in all inter-electrode spacing distances. Four different metaheuristic optimization algorithms are used in the fitting process to extract all models parameter and find the best optimization algorithm in the bio-impedance problems.

Minimally invasive in vivo endoscopic monitoring of dextran sulfate sodium-induced murine colitis

Ulcerative colitis (UC) is a gastrointestinal, autoimmune disease that causes ulceration and inflammation of the colon with an incidence of 10 out of every 100,000 people in North America and Western Europe. Though the specific cause is unknown, several studies have demonstrated that inflammatory cells as well as environmental variables, genetics, and lifestyle behaviors can play a role in the long-term inflammatory response. Researchers have commonly used immunohistochemistry, western blotting and gene sequencing to establish the cellular processes behind UC relapse and remission. However, because these destructive methods necessitate the removal of a sample, they can only be used on non-living tissues. The use of minimally invasive approaches to evaluate the in vivo, longitudinal effects of UC on the mucosa in the colon is gaining popularity among clinicians and researchers. We have created a dextran sulfate sodium-induced model of UC in C57 mice based on the work of Wirtz et al., and a minimally invasive imaging modality to explore the changes in mucosal tissue during “active” and “in remission” UC. Briefly, C57 mice were given dextran sulfate sodium (DSS) dissolved in water in 5-day cycles with a remission/recovery period of 10 days. After 7 days post-DSS treatment and 7 days post-recovery, mice were anesthetized and exploratory endoscopies were performed to assess the mucosal changes that occur during the “active” and “remission” periods of UC. Value of protocol:•Minimally invasive induction of ulcerative colitis in a murine mouse model.•Minimally invasive longitudinal monitoring of “active” and “in remission” ulcerative colitis.•Our endoscopic based imaging modality can be used to validate the induction of ulcerative colitis and the potential treatment response for pre-clinical trials.

A study on health care monitoring of femoral shaft fracture healing by using implanted antenna for wireless in-to-out body channel communication

This work proposes a health care monitoring method of femoral fractured bone healing as a novel application, based on the oblique incidence case and transmission of electromagnetic waves. The difference between the transmitted average power densities of normal and fractured bone cases is used to monitor the bone healing status. The study demonstrates the in-to-out body channel characteristics regarding the electromagnetic wave propagation and angle of incidence on a multi-layered human tissue in the range of frequencies from 100 MHz to 10 GHz. The monitoring method is presented by the analysis of electromagnetic wave propagation through a multilayer model and simulated by the CST Microwave studio. Fully insulated planar and meander half-wave dipole antennas are designed and used to simulate both types of bone fracture healing. Results show good monitoring of common types of fractures. Verification is carried out by experimental measurements on non-living animal tissues.

Fractional-Order Bio-Impedance Modeling for Interdisciplinary Applications: A Review

Bio-impedance circuit modeling is a popular and effective non-invasive technique used in medicine and biology to fit the measured spectral impedance data of living or non-living tissues. The variations in impedance magnitude and/or phase at different frequencies reflect implicit biophysical and biochemical changes. Bio-impedance is also used for sensing environmental changes and its use in the agriculture industry is rapidly increasing. In this paper, we review and compare among the fractional-order circuit models that best fit bio-impedance data and the different methods for identifying the parameters of these circuits. Four different vegetables species (Carrot, Tomato, Zucchini, Cucumber) in three different conditions (fresh, frozen, and heated) are used to demonstrate the differences among these models.

Isolation, Purification, and Characterization of Serratiopeptidase Enzyme from Serratia marcescens

Serratiopeptidase is a proteolytic enzyme that is derived from a member of Enterobacteriaceae. Serratia marcescens is a gram-negative bacteria identified characteristically, which produces a red pigment called prodigiosin. Serratiopeptidase is a multifunctional proteolytic enzyme that dissolves non-living tissues such as fibrin, blood clots, inflammation in all forms without harming living tissues. In this study, the organism was isolated from the diseased silkworm's pupa by using Luria- Bertani (LB) agar media. The enzyme production can be enhanced by applying different physical and chemical parameters. Serratia marcescens was subjected to production such that in order to obtain the maximum level of cell-free supernatant Serratiopeptidase enzyme with all the optimized conditions. The enzyme was subjected to purification by four methods such as salt precipitation, dialysis, ion-exchange chromatography and gel filtration. When subjected to enzyme kinetics, Serratiopeptidase was active at temperature 350C, pH-9 with 8 minutes of the incubation period. The molecular weight of serratiopep

Pus solvents as new drugs with unique physical and chemical properti

The literature review shows that standards for the treatment of purulent diseases to date include antiseptic and disinfectants, but do not include agents that dilute and/or dissolve thick pus. It is shown that the pharmacodynamics of antiseptic and disinfectants consists in the local disinfecting effect of these agents. With local interaction with the surface of living and non-living tissues, these agents are able to sterilize it. It was found that for disinfection of the selected surfaces, these agents are used in solutions that contain these agents in concentrations that provide a denaturing effect. It is shown that denaturing action of antiseptic and disinfectants is a special case of dependence of local action of solutions of all medicines and chemical compounds on their concentration. The fact is that increasing the concentration of chemical compounds in solutions sooner or later turns that solutions into a liquid medium that is not compatible with life. Therefore, this liquid kills all cells of the micro-and macroworld. That is why the use of solutions with denaturing action provides a detrimental effect on the cells of all microorganisms and cells of the macroorganism. With local interaction with the tissues of the macroorganism, the drugs cause the following pharmacological effects: local sterilizing, irritating (local inflammatory) and cauterizing (necrotic) action. Thus solutions with denaturing concentration of one means increase the hardness, other means do not change the hardness, and the third means decrease the hardness of biological tissues, including purulent masses. From this it is concluded that today in the treatment of purulent diseases are used means, detrimental effect on all forms of life, and not means, unidirectionally acting on purulent masses. At the same time, it is shown that some of the modern antiseptic and disinfectants can change the properties of purulent masses in the right direction and optimize their removal by diluting and dissolving the thick pus. It is established that the leaders in the solvent action on thick and sticky pus are sodium bicarbonate solutions in combination with hydrogen peroxide. It is shown that solutions that effectively dissolve thick pus have special physical and chemical properties: all of them have moderate alkaline, temperature, boiling and osmotic activity. Preparations that most effectively dissolve thick pus and most quickly and completely remove it out of the purulent cavities, additionally contain carbon dioxide gas or oxygen gas under excessive pressure. In chronological order are recipes invented hygiene products with special physical and chemical properties, providing them with the ability to quickly and effectively liquefy, dissolve and remove the thick pus out of the purulent cavities. It is shown that a new group of hygiene products was discovered in Russia and was called Solvents of pus. The most effective and safe solvents of pus are warm (heated to a temperature of 42 C) solutions containing 24% sodium bicarbonate and 0.53% hydrogen peroxide.

Persistence of Fusarium oxysporum f. sp. fragariae in Soil Through Asymptomatic Colonization of Rotation Crops.

Asymptomatic plant colonization is hypothesized to enhance persistence of pathogenic forms of Fusarium oxysporum. However, a correlation between pathogen populations on living, asymptomatic plant tissues and soilborne populations after tillage has not been demonstrated. Living and dead tissues of broccoli, lettuce, spinach, wheat, cilantro, raspberry, and strawberry plants grown in soil infested with F. oxysporum f. sp. fragariae (the cause of Fusarium wilt of strawberry) were assayed to quantify the incidence of infection and extent of colonization by this pathogen. All crops could be infected by F. oxysporum f. sp. fragariae but the extent of colonization varied between plant species. Pathogen population densities on nonliving crown tissues incorporated into the soil matrix were typically greater than those observed on living tissues. Crop-dependent differences in the inoculum density of F. oxysporum f. sp. fragariae in soil were only observed after decomposition of crop residue. Forty-four weeks after plants were incorporated into the soil, F. oxysporum f. sp. fragariae soil population densities were positively correlated with population densities on plant tissue fragments recovered at the same time point. Results indicate that asymptomatic colonization can have a significant, long-term impact on soilborne populations of Fusarium wilt pathogens. Cultural practices such as crop rotation should be leveraged to favor pathogen population decline by planting hosts that do not support extensive population growth on living or decomposing tissues.

Bioinspired Temperature-Responsive Multilayer Films and Their Performance under Thermal Fatigue.

The structure of certain nonliving tissues determines their self-shaping and self-folding capabilities in response to a stimulus. Predetermined movements are realized according to changes in the environmental conditions due to the generated stresses of the multilayer anisotropic structure. In this study, we present bioinspired responsive anisotropic multilayer films and their fabrication process which comprises low-cost techniques. The anisotropic multilayer materials are capable of deforming their geometry caused by small temperature changes (<40 °C). The mismatch in the thermo-mechanical properties between three or more anisotropic thin layers creates responsive materials that alter their shape owing to the developed internal stresses. The movements of the material can be controlled by forming anisotropic homogenous metallic strips over an anisotropic thermoplastic layer. As a result, responsive multilayer films made of common materials can be developed to passively react to a temperature stimulus. We demonstrate the ability of the anisotropic materials to transform their geometry and we present a promising fabrication process and the thermal fatigue resistance of the developed materials. The thermal fatigue performance is strongly related to the fabrication method and the thickness of the strips. We studied the thermal fatigue performance of the materials and how the thermal cycling affects their sensitivity, as well as their failure modes and crack formation.

Isolation of tissues and preservation of RNA from intact, germinated barley grain.

Isolated barley (Hordeum vulgare L.) aleurone layers have been widely used as a model system for studying gene expression and hormonal regulation in germinating cereal grains. A serious technological limitation of this approach has been the inability to confidently extrapolate conclusions obtained from isolated tissues back to the whole grain, where the co-location of several living and non-living tissues results in complex tissue-tissue interactions and regulatory pathways coordinated across the multiple tissues. Here we have developed methods for isolating fragments of aleurone, starchy endosperm, embryo, scutellum, pericarp-testa, husk and crushed cell layers from germinated grain. An important step in the procedure involves the rapid fixation of the intact grain to freeze the transcriptional activity of individual tissues while dissection is effected for subsequent transcriptomic analyses. The developmental profiles of 19611 gene transcripts were precisely defined in the purified tissues and in whole grain during the first 24h of germination by RNA sequencing. Spatial and temporal patterns of transcription were validated against well-defined data on enzyme activities in both whole grain and isolated tissues. Transcript profiles of genes involved in mitochondrial assembly and function were used to validate the very early stages of germination, while the profiles of genes involved in starch and cell wall mobilisation matched existing data on activities of corresponding enzymes. The data will be broadly applicable for the interrogation of co-expression and differential expression patterns and for the identification of transcription factors that are important in the early stages of grain and seed germination.

Adsorption-induced deformation of nanoporous materials—A review

When a solid surface accommodates guest molecules, they induce noticeable stresses to the surface and cause its strain. Nanoporous materials have high surface area and, therefore, are very sensitive to this effect called adsorption-induced deformation. In recent years, there has been significant progress in both experimental and theoretical studies of this phenomenon, driven by the development of new materials as well as advanced experimental and modeling techniques. Also, adsorption-induced deformation has been found to manifest in numerous natural and engineering processes, e.g., drying of concrete, water-actuated movement of non-living plant tissues, change of permeation of zeolite membranes, swelling of coal and shale, etc. In this review, we summarize the most recent experimental and theoretical findings on adsorption-induced deformation and present the state-of-the-art picture of thermodynamic and mechanical aspects of this phenomenon. We also reflect on the existing challenges related both to the fundamental understanding of this phenomenon and to selected applications, e.g., in sensing and actuation, and in natural gas recovery and geological CO2 sequestration.

Contributions of North American endophytes to the phylogeny, ecology, and taxonomy of Xylariaceae (Sordariomycetes, Ascomycota)

The Xylariaceae (Sordariomycetes) comprise one of the largest and most diverse families of Ascomycota, with at least 85 accepted genera and ca. 1343 accepted species. In addition to their frequent occurrence as saprotrophs, members of the family often are found as endophytes in living tissues of phylogenetically diverse plants and lichens. Many of these endophytes remain sterile in culture, precluding identification based on morphological characters. Previous studies indicate that endophytes are highly diverse and represent many xylariaceous genera; however, phylogenetic analyses at the family level generally have not included endophytes, such that their contributions to understanding phylogenetic relationships of Xylariaceae are not well known. Here we use a multi-locus, cumulative supermatrix approach to integrate 92 putative species of fungi isolated from plants and lichens into a phylogenetic framework for Xylariaceae. Our collection spans 1933 isolates from living and senescent tissues in five biomes across the continental United States, and here is analyzed in the context of previously published sequence data from described species and additional taxon sampling of type specimens from culture collections. We found that the majority of strains obtained in our surveys can be classified in the hypoxyloid and xylaroid subfamilies, although many also were found outside of these lineages (as currently circumscribed). Many endophytes were placed in lineages previously not known for endophytism. Most endophytes appear to represent novel species, but inferences are limited by potential gaps in public databases. By linking our data, publicly available sequence data, and records of ascomata, we identify many geographically widespread, host-generalist clades capable of symbiotic associations with diverse photosynthetic partners. Concomitant with such cosmopolitan host use and distributions, many xylariaceous endophytes appear to inhabit both living and non-living plant tissues, with potentially important roles as saprotrophs. Overall, our study reveals major gaps in the availability of multi-locus datasets and metadata for this iconic family, and provides new hypotheses regarding the ecology and evolution of endophytism and other trophic modes across the family Xylariaceae.

Stability of Ostreid herpesvirus-1 (OsHV-1) and assessment of disinfection of seawater and oyster tissues using a bioassay

Microvariant genotypes of Ostried herpesvirus-1 (OsHV-1 μVar) have recently emerged as a cause of epizootic mortality in Pacific oysters (Crassostrea gigas) in many countries. Measures that can reduce the spread of the virus are required to decrease the incidence and distribution of the disease at the local and global scales. Disease management strategies and biosecurity plans require data describing the stability of OsHV-1 in the environment and the methods required for effective disinfection of the virus. A bioassay using intramuscular injection of 10month old oyster spat had a limit of detection of 3.6×103 copies of OsHV-1 genome per oyster. This was 10-fold more sensitive compared to immersion challenge of 5month old spat, even though the oysters were exposed to a greater volume of inoculum over a period of 2h. OsHV-1 remained infectious in seawater for 2days at 20°C and in wet or dry, non-living oyster tissues for at least 7days at 20°C. OsHV-1 was inactivated by: commercial multipurpose disinfectants used according to label directions (Virkon-S, Dupont; quaternary ammonium preparation, Livingston); sodium hydroxide (20g/L 10min), iodine (0.1% 5min) and formalin (10% v/v 30min); and physical measures including heating to 50°C for 5min and exposure to a high dose of ultraviolet light. Ineffective disinfectant treatments were: heating to 42°C for 5min, and alkaline detergent (2000ppm, 10min) (Pyroneg, Johnson Diversey). Sodium hypochlorite (50ppm available chlorine, 15min) inactivated OsHV-1 in relatively clean seawater, but this treatment was not effective after addition of protein, 10% v/v foetal bovine serum. A concentration of 200ppm available chlorine for 15min did not inactivate OsHV-1 in oyster tissue. This study provides information that will assist in modelling the disease risk posed by OsHV-1 and data that are necessary to devise effective biosecurity strategies to control the spread of OsHV-1 in a variety of situations. The potential role of fomites in the spread of the virus, and recurrence of disease in endemically infected areas were highlighted by the persistence of infectivity in non-living oyster tissues. Statement of relevanceData on the duration of infectivity and effective disinfection of OsHV-1 in filed relevant preparations is needed for improved disease control.

Polyphenol oxidase as a biochemical seed defense mechanism.

Seed dormancy and resistance to decay are fundamental survival strategies, which allow a population of seeds to germinate over long periods of time. Seeds have physical, chemical, and biological defense mechanisms that protect their food reserves from decay-inducing organisms and herbivores. Here, we hypothesize that seeds also possess enzyme-based biochemical defenses, based on induction of the plant defense enzyme, polyphenol oxidase (PPO), when wild oat (Avena fatua L.) caryopses and seeds were challenged with seed-decaying Fusarium fungi. These studies suggest that dormant seeds are capable of mounting a defense response to pathogens. The pathogen-induced PPO activity from wild oat was attributed to a soluble isoform of the enzyme that appeared to result, at least in part, from proteolytic activation of a latent PPO isoform. PPO activity was also induced in wild oat hulls (lemma and palea), non-living tissues that cover and protect the caryopsis. These results are consistent with the hypothesis that seeds possess inducible enzyme-based biochemical defenses arrayed on the exterior of seeds and these defenses represent a fundamental mechanism of seed survival and longevity in the soil. Enzyme-based biochemical defenses may have broader implications since they may apply to other defense enzymes as well as to a diversity of plant species and ecosystems.

Volcanic mercury in Pinus canariensis

Mercury (Hg) is a toxic element that is emitted to the atmosphere by both human activities and natural processes. Volcanic emissions are considered a natural source of mercury in the environment. In some cases, tree ring records taken close to volcanoes and their relation to volcanic activity over time are contradictory. In 1949, the Hoyo Negro volcano (La Palma-Canary Islands) produced significant pyroclastic flows that damaged the nearby stand of Pinus canariensis. Recently, 60 years after the eruption, we assessed mercury concentrations in the stem of a pine which survived volcano formation, located at a distance of 50 m from the crater. We show that Hg content in a wound caused by pyroclastic impacts (22.3 μg kg(-1)) is an order of magnitude higher than the Hg concentrations measured in the xylem before and after the eruption (2.3 μg kg(-1)). Thus, mercury emissions originating from the eruption remained only as a mark-in pyroclastic wounds-and can be considered a sporadic and very high mercury input that did not affect the overall Hg input in the xylem. In addition, mercury contents recorded in the phloem (9.5 μg kg(-1)) and bark (6.0 μg kg(-1)) suggest that mercury shifts towards non-living tissues of the pine, an aspect that can be related to detoxification in volcanism-adapted species.

Sorologia e sobrevivência do vírus do amarelo letal do mamoeiro

O vírus do amarelo letal do mamoeiro (Papaya lethal yellowing virus, PLYV) é responsável por uma das principais doenças do mamoeiro (Carica papaya) no Nordeste brasileiro. O PLYV pode ser transmitido através do solo, água, instrumento de corte e mãos contaminadas. A presente pesquisa teve como objetivo estudar as características biológicas, sorológicas e físicas de um isolado do vírus e avaliar sua sobrevivência em tecido seco infetado. O PLYV foi detectado por "Enzyme linked immunosorbent assay" (ELISA) indireto e isolado em mudas de mamoeiro, através da inoculação mecânica. A sobrevivência do PLYV em folhas e raízes secas de mamoeiro infetado foi avaliada por sorologia e inoculação em plantas sadias. A presença do vírus foi detectada em folhas e raízes de mamoeiro secas, indicando que o vírus pode permanecer ativo em restos de cultura por até 120 dias. A purificação do PLYV permitiu a obtenção de 309.5 mg de vírus/kg de folha e o anti-soro obtido mostrou-se altamente específico, com títulos de 1:128 em dupla difusão em Agar e 1:1.024.000 em Elisa indireto. Estudos das propriedades físicas do PLYV em mamoeiro revelaram um ponto de inativação térmica (PIT) em torno de 80 ºC, um ponto máximo de diluição (PMD) de ac. 10-6 e uma longevidade in vitro (LIV) acima de 50 dias.

Midface Reconstruction

In the midface, two polyhedron-shaped maxillary units are separated by the central midportion, which includes the nasal area. The midface includes such facial features as the nose, cheek, and upper lip, and posteriorly it extends to the anterior skull base. In the superoinferior direction, the midface includes the soft and bony tissue from the orbital cavity to the oral cavity. Laterally, the midface extends to the temporal bone. Although most superficial skin defects of the midface can be covered by various standard reconstructive modalities, because of the need to evaluate the nature of the tissues involved and because of structural and also functional considerations, the management of large, full-thickness defects is a challenge for reconstructive surgeons. Advances in microsurgical techniques have permitted reliable wound closure and a substantial decrease in patient morbidity with low complication rates while allowing a variety of reconstructive flap options in a single stage. To create a reconstructive algorithm, several classification systems have been proposed, mostly relating to the extension, location, and tissue involvement of the defect. Defects can be classified as simple soft tissue defects and complex defects. The complex three-dimensional defect is classified under four types: types I to IV. Although maxillary prostheses are nonliving tissues and may cause discomfort for the patient, in special situations they can be reconstructive options requiring special experience. Essentially, the method of reconstruction should be selected on an individual basis, bearing in mind the medical situation; the age and prognosis of the patient; the size, extension, and composition of the defect; and the availability of local or distant tissues.

DEVELOPMENT, TAXONOMIC SIGNIFICANCE AND ECOLOGICAL ROLE OF THE CUTICULAR EPITHELIUM IN THE SANTALALES

All genera in the mistletoe family Viscaceae develop a secondary protective covering, the cuticular epithelium, that replaces the epidermis. The cuticular epithelium also occurs in the Eremolepidaceae and some genera within the related family Santalaceae. This secondary covering, unlike the periderm, lacks lenticels or their functional equivalent. We suggest that the cuticular epithelium provides a greater degree of control over transpirational water loss in older plant parts. The cuticular epithelium may arise in the epidermis, subepidermal layer, or in deeper tissues of the stem. Bark strands of the endophytic system, where they are in contact with either nonliving host tissues or the external environment, also form a cuticular epithelium. The epidermal feature, stomatal orientation, was also studied. All genera in the Viscaceae and Eremolepidaceae have stomata with a transverse orientation. The presence or absence of a cuticular epithelium and stomatal orientation are vegetative characters with potential taxonomic value.

Osteopontin is associated with bioprosthetic heart valve calcification in humans

Calcification of non-osseous tissues such as heart valves or vessels is a major concern in clinical practice. The exact mechanism is still unknown. Numerous studies have shown that mineral deposits of crystalline hydroxyapatite within these tissues were associated with increased non-collagenous protein content. More recently osteopontin was found to be associated with calcification in living tissues such as vessels and native human aortic valves. The aim of this study was to determine whether or not non-collagenous proteins can also be found in non-living tissues such as glutaraldehyde-pretreatedporcine valves after implantation in humans. Thirty-eight glutaraldéhyde pretreated porcine bioprostheses were studied: 16 not implanted and 22 after 11 years of implantation in the aortic and mitral valve position in humans. In areas of calcification vizualized by Von Kossa staining and microradiography, immunostaining using polyclonal antibodies against calcium-bindingproteins showed osteopontin positive staining and no staining for osteocalcin, bone sialoprotein or osteonectin. In uncalcified areas and in non-implanted valves, staining for osteopontin or other calcium-binding proteins was negative. Western blot analysis of macroscopically calcified and uncalcified areas showed that several proteins were adsorbed in implanted valves and confirmed the presence of osteopontin in the calcified areas, while no immunolabelling was found in non-calcified areas, in uncalcified valves and in non-implanted valves. Thus the presence of osteopontin in the calcified areas of bioprosthetic heart valves implanted in human indicates that this protein is associated with bioprosthetic valvular calcification. Since these valves are made of non-living connective tissue, and no cell immunostained for osteopontin was found around the calcified area, this study suggests that a non-cellular mediated mechanism involving protein adsorption may play a role in bioprosthetic valvular calcification.

THE FEEDING BEHAVIOR OFPARANOPHRYS CARNIVORA(CILIATA, PHILASTERIDAE)

The marine ciliate Paranophrys carnivora, isolated from the Mediterranean coast of Israel, was found to feed on a varied diet of bacteria, algae, and living and non-living tissues. Chlorococcum sp. and Dunaliella parva, the algal species on which P. carnivora grew best, did not elicit a chemosensory response; tissues and bacteria did. In experiments on stationary phase ciliates, betaine, choline, L-histidine, and trimethylamine oxide elicited a positive chemosensory response at concentrations as low as l0-6 M to l0-3 M.