Current Trends in Aerogel Use in Heritage Buildings: Case Studies from the Aerogel Architecture Award 2021.

Silica aerogels are high-performance thermal insulation materials that can be used to provide unique solutions in the envelopes of buildings when space is limited. They are most often applied in historic buildings due to thin insulation thicknesses and since they are compatible with historic structures. In 2021, the first Aerogel Architecture Award was held at Empa in Switzerland in order to collect, evaluate and award outstanding uses of this relatively new building material. From the submitted projects, three were selected for an award by an expert jury. They showcased applications in which heritage protection and the conservation of a building's character and expression were reconciled with significant improvements in the energy efficiency of the building. The submissions also showed that a broader communication of these types of solutions is important in order to provide more information and security to planners and heritage offices and to facilitate the application of these materials in the future so that they can contribute to the protection of cultural heritage and reductions in the operational and embodied emissions of our building stock by extending the life expectancy and energy efficiency of existing buildings.

p-ANCAs in autoimmune liver disorders recognise human beta-tubulin isotype 5 and cross-react with microbial protein FtsZ.

OBJECTIVE: Autoimmune hepatitis and primary sclerosing cholangitis are chronic inflammatory disorders of unknown aetiology, frequently associated with the presence of perinuclear antineutrophil cytoplasmic antibodies (p-ANCAs) directed against an unknown antigen of myeloid cells. METHODS AND RESULTS: Here, it is reported that p-ANCAs in autoimmune liver disorders react with beta-tubulin isotype 5 (TBB-5) as autoantigen as well as with its evolutionary bacterial precursor protein FtsZ. Both proteins were confirmed as antigens of p-ANCAs in autoimmune liver disorders by demonstrating reactivity of ANCA-positive sera with recombinant TBB-5 (72-88%) and FtsZ (64-82%) on immunoblots and antigen-specific abrogation of ANCA immunofluorescence when sera had been preabsorbed with tubulin and FtsZ. Using sera from interleukin 10-deficient mice (Il10(-)/(-)), an animal model of inflammatory bowel disease, it was also demonstrated that antibodies against TBB-5 are generated in response to intestinal microorganisms. However, unlike autoimmune liver disorders, human antibodies to FtsZ in the absence of TBB-5 antibodies were also a frequent finding in non-autoimmune liver diseases (up to 95%). Reactivity to TBB-5 without the presence of FtsZ antibodies was found in very few cases (<1%) in autoimmune liver disorders. CONCLUSIONS: Thus, p-ANCAs in autoimmune liver diseases are directed against human TBB-5 cross-reacting with the bacterial protein FtsZ, probably reflecting an abnormal immune response to intestinal microorganisms in susceptible, possibly genetically predisposed individuals.

Detection of endogenous lipids in chicken feathers distinct from preen gland constituents.

Bird feather lipids are usually attributed to the oily secretion product of the uropygial (preen) gland. We have observed, however, that feathers exhibit a strong reaction with osmium tetroxide (OsO4), even after treatment with detergents. This leads us to postulate the existence of endogenous feather lipids distinct from preen gland lipids. In order to substantiate our hypothesis, we investigated down feathers from a 1-day-old chicken as their uropgygial gland is not functionally active. The results confirmed the osmiophilic reaction, which was concentrated in the center of barbs and strongly reduced after lipid extraction. In these lipid extracts, we identified using thin layer chromatography, cholesterol, various ceramides, glycolipids, phospholipids, and fatty acids, which closely resembled the lipid composition of the water barrier in the chicken-cornified epidermal envelope. This composition is clearly distinct from chicken uropygeal gland secretion (UGS) known to consist of fatty alcohols as part of aliphatic monoester waxes and of free, predominantly saturated, fatty acids. A filter assay showed a strong reactivity between OsO4 and the fatty acids C18:1 and C18:2 and with feather lipid extracts, but not with UGS. These observations were confirmed by gas chromatography detecting unsaturated fatty acids including C18:1 and C18:2 as well as cholesterol exclusively in chicken feathers. Our results indicate that (1) endogenous lipids are detectable in chicken feathers and distinct from UGS and (2) in analogy to the morphogenesis of the cornified envelope of chicken feather lipids that may have derived from cellular feather-precursors, apparently enduring the specific cell death during developmental feather cornification.

Specific distribution of barrier-relevant ceramides in the emerging epidermis and the periderm/subperiderm during chicken embryogenesis.

During mammalian embryogenesis the emerging epidermis is temporarily covered by an epithelial monolayer, the periderm. In chicken, a second epithelial layer, the subperiderm, located underneath the periderm develops in later embryogenesis. Together the periderm and the subperiderm are referred to as the PSP unit. The cells of the PSP unit are tightly connected by tight junctions (TJ), thereby providing the embryo with an impermeable bilayered diffusion barrier. The emerging epidermis assumes its barrier function by cornification beginning at embryonic day 17 (E17) before at E18 the PSP unit undergoes desquamation. Lipid analysis of both epithelia after their mechanical separation revealed a dramatic increase to about 100-fold values of barrier-relevant ceramides, i.e. those known to essentially contribute to the diffusion barrier of the cornified envelope, in the emerging epidermis between E17 and E19. In contrast, the content of barrier-relevant ceramides in the PSP unit remained at constantly low levels throughout embryogenesis. These data strongly argue in favour of different mechanisms for the barrier function of the two epithelia. TJ in the PSP unit provide the main diffusion barrier protecting the embryo until beginning of desquamation at E18. At this developmental stage the content of cornified envelope-specific ceramides is substantially elevated, thus enabling the epidermis to fulfil its function as the major diffusion barrier after desquamation of the PSP unit. The observation that barrier-relevant ceramides are formed prior to desquamation of the PSP unit points to a precisely regulated sequence in that desquamation does not occur until the lipid-based barrier of the cornified envelope is completed and suggests in addition that these lipids might be essential regulators of the interaction between the PSP unit and the emerging epidermis.

Thyroid functions of mouse cathepsins B, K, and L.

Thyroid function depends on processing of the prohormone thyroglobulin by sequential proteolytic events. From in vitro analysis it is known that cysteine proteinases mediate proteolytic processing of thyroglobulin. Here, we have analyzed mice with deficiencies in cathepsins B, K, L, B and K, or K and L in order to investigate which of the cysteine proteinases is most important for proteolytic processing of thyroglobulin in vivo. Immunolabeling demonstrated a rearrangement of the endocytic system and a redistribution of extracellularly located enzymes in thyroids of cathepsin-deficient mice. Cathepsin L was upregulated in thyroids of cathepsin K(-/-) or B(-/-)/K(-/-) mice, suggesting a compensation of cathepsin L for cathepsin K deficiency. Impaired proteolysis resulted in the persistence of thyroglobulin in the thyroids of mice with deficiencies in cathepsin B or L. The typical multilayered appearance of extracellularly stored thyroglobulin was retained in cathepsin K(-/-) mice only. These results suggest that cathepsins B and L are involved in the solubilization of thyroglobulin from its covalently cross-linked storage form. Cathepsin K(-/-)/L(-/-) mice had significantly reduced levels of free thyroxine, indicating that utilization of luminal thyroglobulin for thyroxine liberation is mediated by a combinatory action of cathepsins K and L.

Defective epidermal barrier in neonatal mice lacking the C-terminal region of connexin43.

More than 97% of mice in which the C-terminal region of connexin43 (Cx43) was removed (designated as Cx43K258stop) die shortly after birth due to a defect of the epidermal barrier. The abnormal expression of Cx43K258stop protein in the uppermost layers of the epidermis seems to perturb terminal differentiation of keratinocytes. In contrast to Cx43-deficient mice, neonatal Cx43K258stop hearts show no lethal obstruction of the right ventricular outflow tract, but signs of dilatation. Electrocardiographies of neonatal hearts reveal repolarization abnormalities in 20% of homozygous Cx43K258stop animals. The very rare adult Cx43K258stop mice show a compensation of the epidermal barrier defect but persisting impairment of cardiac function in echocardiography. Female Cx43K258stop mice are infertile due to impaired folliculogenesis. Our results indicate that the C-terminally truncated Cx43K258stop mice lack essential functions of Cx43, although the truncated Cx43 protein can form open gap junctional channels.

Misfolded BiP is degraded by a proteasome-independent endoplasmic-reticulum-associated degradation pathway.

Misfolded proteins are removed from the ER (endoplasmic reticulum) by retrotranslocation to the cytosol and degradation by the ubiquitin-proteasome system in a process designated ERAD (ER-associated degradation). Analysing the turnover of a misfolded form of the ER-resident chaperone BiP (heavy-chain binding protein) (BiPDeltaA), we found that the degradation of BiPDeltaA did not follow this general ERAD pathway. In transfected cells, BiPDeltaA was degraded, although proteasome-dependent ERAD was inactivated either by proteasome inhibitors or by ATP depletion. In semi-permeabilized cells, which did not support the degradation of the proteasomal substrate alpha1-antitrypsin, the degradation of BiPDeltaA was still functional, excluding the Golgi apparatus or lysosomes as the degradative compartment. The degradation of BiPDeltaA was recapitulated in biosynthetically loaded brain microsomes and in an extract of luminal ER proteins. In contrast with proteasome-dependent ERAD, degradation fragments were detectable inside the microsomes and in the extract, and the degradation was prevented by a serine protease inhibitor. These results show that the degradation of BiPDeltaA was initiated in the ER lumen by a serine protease, and support the view that proteasome-independent ERAD pathways exist.

Increased expression and processing of the Alzheimer amyloid precursor protein in pancreatic cancer may influence cellular proliferation.

Abnormal cleavage of amyloid precursor protein (APP) in the central nervous system has been linked to the development of Alzheimer's disease. Recent work has identified additional roles for APP in peripheral tissue, such as cellular proliferation and motility. APP undergoes proteolytic processing to release a soluble NH(2)-terminal ectodomain fragment (sAPP), an Abeta or p3 peptide, and cytosolic COOH-terminal fragments. We have identified the up-regulation of APP expression in pancreatic cancer cells both in vitro and in vivo. APP undergoes high levels of proteolytic processing in pancreatic cancer cells, and sAPP can be detected in collected medium in vitro. Inhibition of sAPP signaling reduces pancreatic cancer cell number via a reduction in cellular proliferation. We propose that APP may function to promote growth in pancreatic cancer cells via signaling through sAPP and may therefore represent a novel therapeutic target in pancreatic cancer.

sAPP as a regulator of dendrite motility and melanin release in epidermal melanocytes and melanoma cells.

Numerous factors including ultraviolet (UV) radiation and growth factors regulate the specific function of epidermal melanocytes. A recently discovered epidermal growth factor is sAPP, the soluble N-terminal ectodomain of the beta-amyloid precursor protein (APP). Using whole mount preparations of isolated human epidermis, we detected a small population of basal cells, which expressed exceptionally high levels of APP. These cells were identified as melanocytes, which, similar to keratinocytes and neuronal cells, expressed the three APP isoforms 695, 751, and 770. They differed in their expression pattern from that of neuronal cells by expressing only low levels of APP 695. Melanocytes and melanoma cells in vitro released, in addition to keratinocytes, large quantities of sAPP. Because of its growth factor function, we studied possible effects of sAPP on melanocytes. Recombinant sAPP strongly increased lamellipodia activity at dendritic tips, an effect that coincided with increased release of melanin particles. Our observations point to the possible use of APP as an immunocytochemical marker for melanocytes. They suggest that sAPP derived from keratinocytes and/or melanocytes belongs to a family of factors operating in the paracrine and/or autocrine regulation of melanocyte function.

Membrane and raft association of reggie-1/flotillin-2: role of myristoylation, palmitoylation and oligomerization and induction of filopodia by overexpression.

The reggie protein family consists of two proteins, reggie-1 and -2, also called flotillins, which are highly ubiquitous and evolutionarily conserved. Both reggies have been shown to be associated with membrane rafts and are involved in various cellular processes such as T-cell activation, phagocytosis and insulin signalling. However, the exact molecular function of these proteins remains to be determined. In addition, the mechanism of membrane association of reggie-1, which does not contain any transmembrane domain, is not known. In this study, we have produced a fusion protein of reggie-1 with enhanced green fluorescent protein and generated targeted substitutions for the inactivation of putative palmitoylation and myristoylation sites. We were able to show that reggie-1 is myristoylated and multiply palmitoylated and that lipid modifications are necessary for membrane association of reggie-1. Overexpression of reggie-1 resulted in the induction of numerous filopodia-like protrusions in various cell lines, suggesting a role for reggie-1 as a signalling protein in actin-dependent processes.

Endoplasmic reticulum-associated degradation: exceptions to the rule.

Quality control mechanisms in the endoplasmic reticulum (ER) ensure that misfolded proteins are recognized and targeted for degradation. According to the current view of ER-associated degradation (ERAD), the degradation does not occur in the ER itself but requires the retrotranslocation of the proteins to the cytosol where they are degraded by proteasomes. Although this model appears to be valid for many different proteins a number of exceptions from this rule suggest that additional proteasome-independent ERAD pathways may exist. In this review, we will summarize what is known about these alternative ERAD pathways.

The secretory beta-amyloid precursor protein is a motogen for human epidermal keratinocytes.

Cell migration is known to be triggered by constituents of the extracellular matrix such as fibronectin and by soluble mediators commonly summarized as motogens. Many growth factors such as the epidermal growth factor (EGF) have been shown to act as motogens. Recently, the secretory N-terminal portion of the beta-amyloid precursor protein (sAPP) has been identified as a keratinocyte growth factor. Hence, in this study we analysed whether sAPP stimulates also keratinocyte migration employing the stroboscopic cell motility assay. The migration velocity as well as the frequency of lamellipodia protrusion and ruffle formation were increased about two-fold thus corresponding to the effect of EGF. Using a newly developed beta1-integrin migration track assay we observed that sAPP increased the proportion of migrating keratinocytes and their directional persistence. sAPP appeared to operate synergistically with fibronectin with respect to its motogenic effect. Using a modified Boyden chamber assay we showed that sAPP besides its chemokinetic effect functions as a chemoattractant. Like EGF, sAPP exerted its motogenic effect through the activation of Rac kinase but the receptor for sAPP appears to be distinct. The results suggest that sAPP operates as a motogen in the human epidermis, where it may participate in the regulation of reepithelialization during wound healing.

Biological roles of APP in the epidermis.

The amyloid precursor protein (APP) was initially detected in cells of the central nervous system where it is considered to be involved in the pathogenesis of Alzheimer's disease. However, APP is also found in peripheral organs with exceptionally strong expression in the mammalian epidermis where it fulfils a variety of distinct biological roles. Full length APP appears to facilitate keratinocyte adhesion due to its ability to interact with the extracellular matrix. The C-terminus of APP also serves as adapter protein for binding the motor protein kinesin thereby mediating the centripetal transport of melanosomes in epidermal melanocytes. By the action of alpha-secretase sAPPalpha, the soluble N-terminal portion of APP, is released. sAPPalpha has been shown to be a potent epidermal growth factor thus stimulating proliferation and migration of keratinocytes as well as the exocytic release of melanin by melanocytes. The release of sAPPalpha can be almost completely blocked by inhibiting alpha-secretase with hydroxamic acid-based zinc metalloproteinase inhibitors. In hyperproliferative keratinocytes from psoriatic skin this inhibition results in normalized growth.

Cytoprotective function of sAppalpha in human keratinocytes.

sAPPalpha, the soluble form of the beta-amyloid precursor protein, has been shown to act as a potent epidermal growth factor by stimulating keratinocyte proliferation and migration. In this report we provide evidence for a cytoprotective role of sAPPalpha. As a model we used HaCaT cells and normal human keratinocytes (NHK) cultured in the absence of fetal calf serum and bovine pituitary extract. Under these conditions keratinocytes began to undergo apoptosis at increasing rates after 96 h of culture. Surprisingly, keratinocytes were protected from apoptosis by the addition of 50 nM recombinant sAPPalpha. Subsequent experiments were performed to elucidate the regulatory basis of the cytoprotective role of sAPPalpha. We found that recombinant sAPPalpha facilitated the substrate adhesion of keratinocytes in the first 30 minutes after seeding. The basis for this adhesion-promoting function was shown by the ability of recombinant sAPPalpha to continuously coat the culture dish thereby promoting the ability to bind keratinocytes. A second mechanism explaining the cytoprotective role was found in the significant inhibition of apoptosis by recombinant sAPPalpha. In HaCaT cells moderate UV-B irradiation was sufficient to induce apoptosis. In contrast, induction of apoptosis in NHK required additionally the depletion of endogenous sAPPalpha suggesting that sAPPalpha mediates protection against UV-B irradiation. Staurosporine-induced apoptosis rates were significantly reduced by about 59% after addition of recombinant sAPPalpha. These results show that sAPPalpha exerts a pronounced cytoprotective effect and that this effect is mediated by facilitated cell adhesion and by the antiapoptotic function of sAPPalpha.

Cell migration: mechanisms of rear detachment and the formation of migration tracks.

Cell migration is central to many biological and pathological processes, including embryogenesis, tissue repair and regeneration as well as cancer and the inflammatory response. In general, cell migration can be usefully conceptualized as a cyclic process. The initial response of a cell to a migration-promoting agent is to polarize and extend protrusions in the direction of migration. These protrusions can be large, broad lamellipodia or spike-like filopodia, are usually driven by actin polymerization, and are stabilized by adhering to the extracellular matrix (ECM) via transmembrane receptors of the integrin family linked to the actin cytoskeleton. These adhesions serve as traction sites for migration as the cell moves forward over them, and they must be disassembled at the cell rear, allowing it to detach. The mechanisms of rear detachment and the regulatory processes involved are not well understood. The disassembly of adhesions that is required for detachment depends on a coordinated interaction of actin and actin-binding proteins, signaling molecules and effector enzymes including proteases, kinases and phosphatases. Originally, the biochemically regulated processes leading to rear detachment of migrating cells were thought not to be necessarily accompanied by any loss of cell material. However, it has been shown that during rear detachment long tubular extensions, the retracting fibers, are formed and that "membrane ripping" occurs at the cell rear. By this process, a major fraction of integrin-containing cellular material is left behind forming characteristic migration tracks that exactly mark the way a cell has taken.

Release of integrin macroaggregates as a mechanism of rear detachment during keratinocyte migration.

Cell-substrate adhesion can be mediated by the relatively short-lived focal complexes and focal adhesions and by the more stable hemidesmosomes. During cell migration both types of cell-substrate adhesions must be disrupted allowing the cell rear to detach. Rear detachment has been described to be accompanied by membrane ripping and the loss of cellular material in a variety of cell types including fibroblasts and chondrocytes, but also in fast moving cells such as keratinocytes. Here we show that migrating keratinocytes leave behind "migration tracks" of cellular remnants that can be classified due to their size, distribution and molecular composition. Type I macroaggregates appeared as spherical and tubular structures with a diameter of about 50-100 nm that were arranged like "pearls on a string". These structures apparently derived from fragmentation of long tubular extensions, the retracting fibers, at the cell rear and contained high amounts of beta1 integrin and different alpha integrins that are components of fibronectin and laminin receptors in migrating keratinocytes usually found in focal adhesions. Type II macroaggregates were recognized as spherical structures with a diameter of about 30 - 50 nm that were arranged in clusters scattered over the gaps between type I, macroaggregates. In contrast to type I type II macroaggregates contained high amounts of beta4 integrin and seemed to derive from former hemidesmosomes. Both types of macroaggregates were completely membrane covered, impermeable compartments devoid of cytosolic proteins. Our observations strongly support the concept that the release of macroaggregates represents a distinct cellular mechanism of rear detachment based on the loss of adhesive receptors embedded in membrane-covered cellular remnants.

Normalized proliferation of normal and psoriatic keratinocytes by suppression of sAPPalpha-release.

The soluble form of the beta-amyloid precursor protein (sAPPalpha) is known to function in the autocrine regulation of epidermal growth and repair. Here we show that its proteolytic release by alpha-secretase in normal human keratinocytes is susceptible to hydroxamic-acid-based zinc metalloproteinase inhibitors and suppressed by these inhibitors by 80%-90%. As various other growth factors participate in regulating epidermal growth we investigated whether the inhibitor-induced sAPPalpha-deficiency would affect keratinocyte proliferation. At optimal inhibitor concentrations the suppression of sAPPalpha-release was followed by a decline in proliferation by 50%-60%, indicating that sAPPalpha is a major growth factor that cannot be compensated for by other growth factors. This finding was the basis for the treatment of human lesional psoriatic keratinocytes with these inhibitors, which resulted in the normalization of their increased proliferation rates. The reversibility of these effects and the lack of toxicity underline the value of these inhibitors and suggest their therapeutic application in psoriatic skin diseases.

BiP-dependent export of cholera toxin from endoplasmic reticulum-derived microsomes.

Cholera toxin (CT) is transported from the cell surface to the endoplasmic reticulum (ER) from where it is translocated to the cytosol in a process depending on ATP and luminal ER proteins. To test whether the molecular chaperone BiP (heavy chain binding protein), which is an ER-luminal ATPase, was one of the required proteins the export of CT was analyzed using ER-derived CT-loaded microsomes. The resubstitution of extracted export-incompetent microsomes with purified BiP was sufficient to restore the export of CT. As BiP protected CT from aggregation it is proposed that BiP maintains CT in a soluble, export-competent state.

Improved Technique for Electron Microscope Visualization of Yeast Membrane Structure.

Yeast cells represent a powerful model system in cell biology mainly due to their amenability to genetic manipulations. Increasingly, studies focus on mutant genes resulting in alterations of cellular structures and organelles. To ascertain the phenotypic changes involved, it is often desirable to use the resolving power of electron microscopy. In contrast to higher eukaryotic cells, yeast cells are particularly difficult to preserve mainly due to the presence of a thick cell wall that acts as a barrier against diffusion of fixatives. Although several procedures are targeted to overcome these difficulties, none of them have become established as a standard procedure. As a consequence, electron microscopy is still not used routinely as a tool in yeast cell biology. This prompted us to develop an easy-to-follow protocol for yeast transmission electron microscopy that should be useful in all cases where membrane integrity and organelle morphology is emphasized. One means of making the yeast cytoplasm more attainable to fixation and staining solutions is by enzymatic digestion of the cell wall. Following this approach, we were able to reliably preserve yeast cells and their cellular organelles. Enzymatic treatment with zymolyase 20T to partially remove the yeast cell wall allowed the fixation, preservation, and visualization of the yeast cytoplasm revealing detailed ultrastructure. The advancement of this technique is demonstrated with mitochondria as a model organelle. Our studies on various yeast mutants clearly show the power of the enzymatic digestion technique in visualizing subtle changes of membrane structure and organelle morphology.