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In order to study the mechanisms of sperm competition and cryptic female choice we require an understanding of the patterns of sperm storage, sperm removal and sperm digestion. Current studies infer these patterns mainly from paternity data, which only reveal the ultimate outcomes of the interactions between male and female reproductive processes. However, only with a mechanistic understanding of the fate of received sperm, and the involved patterns of postcopulatory sexual selection, can we understand the evolution of male and female reproductive morphology and physiology. The currently available approaches for tracking donor sperm in a recipient are either very time consuming, spatially imprecise or limited to model organisms for which considerable genetic knowledge and molecular know-how is available. Using the free-living flatworm Macrostomum lignano we here present a novel sperm tracking approach that uses DNA-labelling with a halogenated pyrimidine and localisation of the label using immunocytochemistry. We first outline modifications to established protocols to allow visualisation of gametic cells, in addition to somatic cells, determine the duration and patterns of spermatogenesis, and then show that labelled sperm from labelled donors can be observed in unlabelled recipients. We further show that labelled worms have a mating behaviour that is comparable to that of unlabelled worms except in one parameter. We suggest ways in which this approach can be optimised, and that it should be readily transferable to other taxa. We conclude that this approach will be a valuable tool to study postcopulatory sexual selection. [ABSTRACT FROM AUTHOR]

Neoblasts are potentially totipotent stem cells and the only proliferating cells in adult Platyhelminthes. We have examined the cellular dynamics of neoblasts during the posterior regeneration of Macrostomum lignano. Double-labeling of neoblasts with bromodeoxyuridine and the anti-phospho histone H3 mitosis marker has revealed a complex cellular response in the first 48 h after amputation; this response is different from that known to occur during regeneration in triclad platyhelminths and in starvation/feeding experiments in M. lignano. Mitotic activity is reduced during the first 8 h of regeneration but, at 48 h after amputation, reaches almost twice the value of control animals. The total number of S-phase cells significantly increases after 1 day of regeneration. A subpopulation of fast-cycling neoblasts surprisingly shows the same dynamics during regeneration as those in control animals. Wound healing and regeneration are accompanied by the formation of a distinct blastema. These results present new insights, at the cellular level, into the early regeneration of rhabditophoran Platyhelminthes. [ABSTRACT FROM AUTHOR]

The tiger flatworm Prostheceraeus crozieri (Polycladida) develops via an eight‐lobed, and three‐eyed planktonic Müller's larva. This larva has an apical organ, ultrastructural details of which remain elusive due to a scarcity of studies. The evolution and possible homology of the polyclad larva with other spiralian larvae is still controversial. Here, we provide ultrastructural data and three‐dimensional reconstructions of the apical organ of P. crozieri. The apical organ consists of an apical tuft complex and a dorso‐apical tuft complex. The apical tuft complex features a central tuft of five long cilia, which emerge from four or five individual cells that are themselves encircled by two anchor cells. The necks of six multibranched gland cells are sandwiched between ciliated tuft cell bodies and anchor cells. The proximal parts of the ciliated cell bodies are in contact with the lateral brain neuropil via gap junctions. Located dorsally of the apical tuft complex, the dorso‐apical tuft complex is characterized by several long cilia of sensory neurons, these emerge from an epidermal lumen and are closely associated with several gland cells that form a crescent apically around the dorsal anchor cell, and laterally touch the brain neuropil. Such ciliated sensory neurons emerging from a ciliated lumen are reminiscent of ampullary cells of mollusc and annelid larvae; a similar cell type can be found in the hoplonemertean decidula larva. We hypothesize that the ampullary‐like cells and the tuft‐forming sensory cells in the apical organs of these spiralian larvae could be homologous. [ABSTRACT FROM AUTHOR]

In the present work, because of the importance of the lowering sulfur level in fuels, the new materials have been designed for adsorptive desulfurization. In this line, carbon spheres and sulfur-doped carbon spheres have been prepared from the acetylene gas and sulfur powder using chemical vapor deposition method. To increase the level of desulfurization, the prepared spheres were decorated with gold nanoparticles with different percentages (5, 10, 15 and 20%). The structures and surface properties of the products were fully characterized using common methods. The study of thermal behaviors showed the sulfur-doped spheres has higher thermal stabilities than the carbon spheres. The gold-decorated products were employed to adsorb dibenzothiophene from n-octane solution as a model fuel. The 95% adsorption efficiency and 364 (mg/g) of dibenzothiophene was achieved using 10% gold-decorated sulfur-doped spheres at 25 °C. The results showed that the sulfur-doped product is more appropriate that the simple carbon spheres and modification of this product with gold increased its adsorption capacities. Image 1 • Gold-decorated carbon spheres were employed for desulfurization. • The sulfur-doped products showed and higher adsorption potencies and thermal stabilities. • The 95% removal and 364 mg/g maximum adsorption capacity was achieved. • The adsorption obeyed from the Temkin and pseudo-second order kinetic models. [ABSTRACT FROM AUTHOR]

Flatworms can very rapidly attach to and detach from many substrates. In the presented work, we analysed the adhesive system of the marine proseriate flatworm Minona ileanae. We used light-, scanning- and transmission electron microscopy to analyse the morphology of the adhesive organs, which are located at the ventral side of the tail-plate. We performed transcriptome sequencing and differential RNA-seq for the identification of tail-specific transcripts. Using in situ hybridization expression screening, we identified nine transcripts that were expressed in the cells of the adhesive organs. Knock-down of five of these transcripts by RNA interference led to a reduction of the animal's attachment capacity. Adhesive proteins in footprints were confirmed using mass spectrometry and antibody staining. Additionally, lectin labelling of footprints revealed the presence of several sugar moieties. Furthermore, we determined a genome size of about 560 Mb for M. ileanae. We demonstrated the potential of Oxford Nanopore sequencing of genomic DNA as a cost-effective tool for identifying the number of repeats within an adhesive protein and for combining transcripts that were fragments of larger genes. A better understanding of the molecules involved in flatworm bioadhesion can pave the way towards developing innovative glues with reversible adhesive properties. This article is part of the theme issue 'Transdisciplinary approaches to the study of adhesion and adhesives in biological systems'. [ABSTRACT FROM AUTHOR]

The flatworm Macrostomum lignano features a duo-gland adhesive system that allows it to repeatedly attach to and release from substrates in seawater within a minute. However, little is known about the molecules involved in this temporary adhesion. In this study, we show that the attachment of M. lignano relies on the secretion of two large adhesive proteins, M. lignano adhesion protein 1 (Mlig-ap1) and Mlig-ap2. We revealed that both proteins are expressed in the adhesive gland cells and that their distribution within the adhesive footprints was spatially restricted. RNA interference knockdown experiments demonstrated the essential function of these two proteins in flatworm adhesion. Negatively charged modified sugars in the surrounding water inhibited flatworm attachment, while positively charged molecules impeded detachment. In addition, we found that M. lignano could not adhere to strongly hydrated surfaces. We propose an attachment-release model where Mlig-ap2 attaches to the substrate and Mlig-ap1 exhibits a cohesive function. A small negatively charged molecule is secreted that interferes with Mlig-ap1, inducing detachment. These findings are of relevance for fundamental adhesion science and efforts to mitigate biofouling. Further, this model of flatworm temporary adhesion may serve as the starting point for the development of synthetic reversible adhesion systems for medicinal and industrial applications. [ABSTRACT FROM AUTHOR]

Auditory sensitivity and frequency resolution depend on the physical properties of the basilar membrane in combination with outer hair cell-based amplification in the cochlea. The physiological role of the tectorial membrane (TM) in hair cell transduction has been controversial for decades. New insights into the TM structure and function have been gained from studies of targeted gene disruption. Several missense mutations in genes regulating the human TM structure have been described with phenotypic expressions. Here, we portray the remarkable gradient structure and molecular organization of the human TM. Ultrastructural analysis and confocal immunohistochemistry were performed in freshly fixed human cochleae obtained during surgery. Based on these findings and recent literature, we discuss the role of human TMs in hair cell activation. Moreover, the outcome proposes that the α-tectorin-positive amorphous layer of the human TM is replenished and partly undergoes regeneration during life. [ABSTRACT FROM AUTHOR]

The GI mucus layer represents a significant block to drug carriers absorption. Taking an example from nature, virus-mimicking nanoparticles (NPs) with highly densely charged surface were designed with the aim to improve their mucus permeation ability. NPs were formulated by combining chitosan with chondroitin sulfate and were characterized by particle size, ζ-potential and hydrophobicity. The interaction occurring between NPs and diluted porcine intestinal mucus was investigated by a new method. Furthermore, the rotating tube technique was exploited to evaluate the NPs permeation ability in fresh undiluted porcine intestinal mucus. NPs (400–500 nm) presenting a slightly positive (4.02 mV) and slightly negative (−3.55 mV) ζ-potential resulted to be hydrophobic and hydrophilic, respectively. On the one hand the hydrophobic NPs undergo physico-chemical changes when incubated with mucus, namely the size increased and the ζ-potential decreased. On the other hand, the hydrophilic NPs did not significantly change size and net charge during incubation with mucus. Both types of NPs showed a 3-fold higher diffusion ability compared to the reference 50/50 dl -lactide/glycolide copolymer NPs (136 nm, −23 mV, hydrophilic). Based on these results, this work gives valuable information for the further design of mucus-penetrating NPs. [ABSTRACT FROM AUTHOR]

The objective of this study was to generate nanoparticles with a slightly negative zeta potential which switches to positive values under the influence of intestinal alkaline phosphatase in order to address two major physiological barriers (mucus and membrane barrier). Carboxymethyl cellulose and chitosan were modified with phosphotyrosine by means of a water-soluble carbodiimide and polyelectrolyte complexes were formed by mixing two polymer solutions in an appropriate ratio. Due to this modification, phosphate ions could potentially be released which would lead to a change in zeta potential. Their sizes were found to be between 200 and 300 nm while their zeta potentials ranged from −8 mV to −5 mV prior to incubation with the enzyme. It could be shown that phosphate ions are released from the modified polymers and nanoparticles by isolated phosphatase and in a Caco-2 cell model. Incubation with phosphatase led to a change in zeta potential of the nanoparticles up to +8 mV. As neither polymers nor particles display toxic properties within the resazurin assay, these nanoparticles appear to be useful tools in future drug delivery systems as they have appropriate properties regarding particle size and surface charge in order to overcome the mucus and the membrane barrier. [ABSTRACT FROM AUTHOR]

Abstract: A key feature of the aged human immune system is the accumulation of highly differentiated CD8+CD28− T cells, a phenomenon that negatively influences immune function in the elderly. However, the mechanisms that regulate survival or death of CD8+CD28− T cells remain incompletely understood. Macroautophagy has been shown to protect cells from unfavorable environmental conditions and extend lifespan of various cells and organisms. In this study, we investigated autophagy in CD8+CD28+ and CD8+CD28− T cells following T cell receptor (TCR) engagement. We demonstrate that TCR-mediated activation led to a potent induction of autophagy in CD8+CD28+ T cells which was accompanied by an increased activity of the mammalian target of rapamycin complex 1 (mTORC1). This was surprising, as mTORC1 is generally perceived as an inhibitor of autophagy. Inhibition of mTORC1 by rapamycin could still enhance activation-induced autophagy. In contrast, CD8+CD28− T cells induced autophagy to a significantly lower extent in response to TCR engagement compared to CD8+CD28+ T cells and failed to increase autophagy upon mTORC1 inhibition. In conclusion, we describe for the first time the induction of autophagy in human CD8+ T cells following TCR engagement and the decreased ability of CD8+CD28− T cells to induce autophagy, suggesting that they cannot meet the metabolic needs of antigen receptor-mediated activation and are therefore unlikely to survive when confronted by their specific antigens. [Copyright &y& Elsevier]

The aim of this study was to design thiolated surface stabilized superparamagnetic iron oxide nanoparticles (TSS-SPIONs) for efficient internalization with high MRI sensitivity. TSS-SPIONs were developed by chelation between thiolated chitosan–thioglycolic acid (chitosan–TGA) hydrogel and iron ions (Fe2+/Fe3+). Likely, unmodified chitosan hydrogel SPIONs (UC-SPIONs) and uncoated SPIONs were used as control. Moreover, TSS-SPIONs were investigated regarding to their iron core size, hydrodynamic diameter, zeta potential, iron contents, molar relaxivities (r 1 and r 2), and cellular internalization. TSS-SPIONs demonstrated an iron oxide core diameter (crystallite size by XRD) of 3.1±0.02nm, a hydrodynamic diameter of 94±20nm, a zeta potential of +21±5mV, and an iron content of 3.6±0.9mg/mL. In addition, internalization of TSS-SPIONs into human endothelial progenitor cells (EPC) from umbilical cord blood was more than threefold and 17-fold higher in contrast to UC-SPIONs and SPIONs, respectively. With twofold lower incubation iron concentration of TSS-SPIONs, more than threefold higher internalization was achieved as compared to Resovist®. Also, cell viability of more than 90% was observed in the presence of TSS-SPIONs after 24h. The molar MR relaxivities (r 2) value at 1.5T was threefold higher than that of Resovist® and demonstrated that TSS-SPIONs have the potential as very effective T 2 contrast-enhancement agent. According to these findings, TSS-SPIONs with efficient internalization, lower cytotoxicity, and high MRI sensitivity seem to be promising for cell tracking. [ABSTRACT FROM AUTHOR]

Sodium glucose transporter (SGLT)-2 inhibitors have consistently shown cardioprotective effects independent of the glycemic status of treated patients. In this study we aimed to investigate underlying mechanisms of short-term empagliflozin treatment in a mouse model of type II diabetes. Male db/db mice were fed a western type diet with or without enrichment with empagliflozin for 7 days. While glucose tolerance was significantly improved in empagliflozin treated mice, body weight and fasting insulin levels were comparable in both groups. Cardiac insulin signaling activity indicated by reduced proteinkinase B (AKT) phosphorylation was significantly decreased in the empagliflozin treated group. Remarkably, mitochondrial mass estimated by citrate synthase activity was significantly elevated in empagliflozin treated mice. Accordingly, mitochondrial morphology was significantly altered upon treatment with empagliflozin as analysed by transmission electron microscopy. Additionally, short-term empagliflozin therapy was associated with a changed cardiac tissue cytokine expression in favor of an anti-inflammatory pattern. Our data suggest that early cardioprotection in empagliflozin treated mice is independent of a reduction in body weight or hyperinsulinemia. Ameliorated mitochondrial ultrastructure, attenuated cardiac insulin signaling and diminished cardiac inflammation might contribute to the cardioprotective effects of empagliflozin. [ABSTRACT FROM AUTHOR]

Polycladida are the only free-living flatworms with a planktonic larval stage in some species. Currently, it is not clear if a larval stage is ancestral in polyclads, and which type of larva that would be. Known polyclad larvae are Müller's larva, Kato's larva and Goette's larva, differing by body shape and the number of lobes and eyes. A valuable character for the comparison and characterisation of polyclad larval types is the ultrastructural composition of the apical organ. This organ is situated at the anterior pole of the larva and is associated with at least one ciliary tuft. The larval apical organ of Theama mediterranea features two multiciliated apical tuft sensory cells. Six unfurcated apical tuft gland cell necks are sandwiched between the apical tuft sensory cells and two anchor cells and have their cell bodies located lateral to the brain. Another type of apical gland cell necks is embedded in the anchor cells. Ventral to the apical tuft, ciliated sensory neurons are present, which are neighbouring the cell necks of two furcated apical tuft gland cells. Based on the ultrastructural organisation of the apical organ and other morphological features, like a laterally flattened wedge-shaped body and three very small lobes, we recognise the larva of T. mediterranea as a new larval type, which we name Curini-Galletti's larva after its first discoverer. The ultrastructural similarities of the apical organ in different polyclad larvae support their possible homology, that is, all polyclad larvae have likely evolved from a common larva., (© 2024 The Authors. Cell Biology International published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.)

The Hydra nervous system is the paradigm of a 'simple nerve net'. Nerve cells in Hydra , as in many cnidarian polyps, are organized in a nerve net extending throughout the body column. This nerve net is required for control of spontaneous behavior: elimination of nerve cells leads to polyps that do not move and are incapable of capturing and ingesting prey (Campbell, 1976). We have re-examined the structure of the Hydra nerve net by immunostaining fixed polyps with a novel antibody that stains all nerve cells in Hydra . Confocal imaging shows that there are two distinct nerve nets, one in the ectoderm and one in the endoderm, with the unexpected absence of nerve cells in the endoderm of the tentacles. The nerve nets in the ectoderm and endoderm do not contact each other. High-resolution TEM (transmission electron microscopy) and serial block face SEM (scanning electron microscopy) show that the nerve nets consist of bundles of parallel overlapping neurites. Results from transgenic lines show that neurite bundles include different neural circuits and hence that neurites in bundles require circuit-specific recognition. Nerve cell-specific innexins indicate that gap junctions can provide this specificity. The occurrence of bundles of neurites supports a model for continuous growth and differentiation of the nerve net by lateral addition of new nerve cells to the existing net. This model was confirmed by tracking newly differentiated nerve cells., Competing Interests: AK, SS, CB, CC, BB, WS, MH, OA, KG, RS, BH, TH, CD No competing interests declared, (© 2023, Keramidioti, Schneid, Busse et al.)

Aims/hypothesis: Sodium-glucose cotransporter 2 (SGLT2) inhibitors are widely used in the treatment of type 2 diabetes, heart failure and chronic kidney disease. Their role in the prevention of diet-induced metabolic deteriorations, such as obesity, insulin resistance and fatty liver disease, has not been defined yet. In this study we set out to test whether empagliflozin prevents weight gain and metabolic dysfunction in a mouse model of diet-induced obesity and insulin resistance., Methods: C57Bl/6 mice were fed a western-type diet supplemented with empagliflozin (WDE) or without empagliflozin (WD) for 10 weeks. A standard control diet (CD) without or with empagliflozin (CDE) was used to control for diet-specific effects. Metabolic phenotyping included assessment of body weight, food and water intake, body composition, hepatic energy metabolism, skeletal muscle mitochondria and measurement of insulin sensitivity using hyperinsulinaemic-euglycaemic clamps., Results: Mice fed the WD were overweight, hyperglycaemic, hyperinsulinaemic and insulin resistant after 10 weeks. Supplementation of the WD with empagliflozin prevented these metabolic alterations. While water intake was significantly increased by empagliflozin supplementation, food intake was similar in WDE- and WD-fed mice. Adipose tissue depots measured by MRI were significantly smaller in WDE-fed mice than in WD-fed mice. Additionally, empagliflozin supplementation prevented significant steatosis found in WD-fed mice. Accordingly, hepatic insulin signalling was deteriorated in WD-fed mice but not in WDE-fed mice. Empagliflozin supplementation positively affected size and morphology of mitochondria in skeletal muscle in both CD- and WD-fed mice., Conclusions/interpretation: Empagliflozin protects mice from diet-induced weight gain, insulin resistance and hepatic steatosis in a preventative setting and improves muscle mitochondrial morphology independent of the type of diet., (© 2022. The Author(s).)

How did cells of early metazoan organisms first organize themselves to form a body axis? The canonical Wnt pathway has been shown to be sufficient for induction of axis in Cnidaria, a sister group to Bilateria, and is important in bilaterian axis formation. Here, we provide experimental evidence that in cnidarian Hydra the Hippo pathway regulates the formation of a new axis during budding upstream of the Wnt pathway. The transcriptional target of the Hippo pathway, the transcriptional coactivator YAP, inhibits the initiation of budding in Hydra and is regulated by Hydra LATS. In addition, we show functions of the Hippo pathway in regulation of actin organization and cell proliferation in Hydra . We hypothesize that the Hippo pathway served as a link between continuous cell division, cell density, and axis formation early in metazoan evolution.

Hedgehog (Hh) ligands act as morphogens to direct patterning and proliferation during embryonic development. Protein kinase A (PKA) is a central negative regulator of Hh signalling, and in the absence of Hh ligands, PKA activity prevents inappropriate expression of Hh target genes. The orphan G-protein-coupled receptor Gpr161 contributes to the basal Hh repression machinery by activating PKA. Gpr161 acts as an A-kinase-anchoring protein, and is itself phosphorylated by PKA, but the functional significance of PKA phosphorylation of Gpr161 in the context of Hh signalling remains unknown. Here, we show that loss of Gpr161 in zebrafish leads to constitutive activation of medium and low, but not maximal, levels of Hh target gene expression. Furthermore, we find that PKA phosphorylation-deficient forms of Gpr161, which we show directly couple to G αs , display an increased sensitivity to Shh, resulting in excess high-level Hh signalling. Our results suggest that PKA feedback-mediated phosphorylation of Gpr161 may provide a mechanism for fine-tuning Gpr161 ciliary localisation and PKA activity., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

We isolated a filamentous, thermophilic, and first anaerobic representative of the genus Thermoactinomyces, designated strain AMNI-1 T , from a biogas plant in Tyrol, Austria and report the results of a phenotypic, genetic, and phylogenetic investigation. Strain AMNI-1 T was observed to form a white branching mycelium that aggregates into pellets when grown in liquid medium. Cells could primarily utilize lactose, glucose, and mannose as carbon and energy sources, with acetate accelerating and yeast extract being mandatory for growth. The optimum growth temperature and pH turned out to be 55 °C and pH 7.0, respectively, with an optimum NaCl concentration of 0-2% (w/v). 16S rRNA gene sequence comparison indicated that the genetic relatedness between strain AMNI-1 T and Thermoactinomyces intermedius, Thermoactinomyces khenchelensis, and Thermoactinomyces vulgaris was less than 97%. The G + C content of the genomic DNA was 44.7 mol%. The data obtained suggest that the isolate represents a novel and first anaerobic species of the genus Thermoactinomyces, for which the name Thermoactinomyces mirandus is proposed. The type strain is AMNI-1 T (= DSM 110094 T  = LMG 31503 T ). The description of the genus Thermoactinomyces is emended accordingly.

Tunicates populate a great variety of marine underwater substrates worldwide and represent a significant concern in marine shipping and aquaculture. Adhesives are secreted from the anterior papillae of their swimming larvae, which attach and metamorphose into permanently adhering, filter-feeding adults. We recently described the cellular composition of the sensory adhesive organ of the model tunicate Ciona intestinalis in great detail. Notably, the adhesive secretions of collocytes accumulate at the tip of the organ and contain glycoproteins. Here, we further explore the components of adhesive secretions and have screened for additional specificities that may influence adhesion or cohesion of the Ciona glue, including other carbohydrate moieties, catechols and substrate properties. We found a distinct set of sugar residues in the glue recognized by specific lectins with little overlap to other known marine adhesives. Surprisingly, we also detect catechol residues that likely originate from an adjacent cellular reservoir, the test cells. Furthermore, we provide information on substrate preferences where hydrophobicity outperforms charge in the attachment. Finally, we can influence the settlement process by the addition of hydrophilic heparin. The further analysis of tunicate adhesive strategies should provide a valuable knowledge source in designing physiological adhesives or green antifoulants. This article is part of the theme issue 'Transdisciplinary approaches to the study of adhesion and adhesives in biological systems'.

Electron microscopy (EM) has long been indispensable for flatworm research, as most of these worms are microscopic in dimension and provide only a handful of characters recognizable by eye or light microscopy. Therefore, major progress in understanding the histology, systematics, and evolution of this animal group relied on methods capable of visualizing ultrastructure. The rise of molecular and cellular biology renewed interest in such ultrastructural research. In the light of recent developments, we offer a best-practice guide for users of transmission EM and provide a comparison of well-established chemical fixation protocols with cryo-processing methods (high-pressure freezing/freeze-substitution, HPF/FS). The organisms used in this study include the rhabditophorans Macrostomum lignano, Polycelis nigra and Dugesia gonocephala, as well as the acoel species Isodiametra pulchra., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Hydra is a classical model organism in developmental and cell biology with a simple body plan reminiscent of a gastrula with one body axis and a limited number of cell types. This rather simple organism exhibits a regeneration capacity that is unique among all eumetazoans and is largely dependent on the stem cell properties of its epithelial stem cell population. Molecular work in the past few years has revealed an unexpected genetic complexity of these simple animals, making them an interesting model for studying the generation of animal form and regeneration. In addition, Hydra has an interstitial stem cell system with a unique population of nematocytes, neuronal cells that are characterized by an explosive exocytotic discharge. Here, we compare classical and modern transmission electron microscopy (TEM) fixation protocols including protocols for TEM immunocytochemistry (post-embedding immunogold labeling). We presume that TEM studies will become an important tool to analyze cell-cell interactions as well as cell matrix interrelationships in Hydra in the future., (Copyright © 2010 Elsevier Inc. All rights reserved.)