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Marine mussels fabricate tough collagenous fibers known as byssal threads to anchor themselves. Threads are produced individually in minutes via secretion of liquid crystalline (LC) collagenous precursors (preCols); yet the physical and chemical parameters influencing thread formation remain unclear. Here, we characterized the structural anisotropy of native and artificially induced threads using quantitative polarized light microscopy and transmission electron microscopy to elucidate spontaneous vs regulated aspects of thread assembly, discovering that preCol LC phases form aligned domains of several hundred microns, but not the cm-level alignment of native threads. We then explored the hypothesized roles of mechanical shear, pH, and metal ions on thread formation through in vitro assembly studies employing a microfluidic flow focusing device using purified preCol secretory vesicles. Our results provide clear evidence for the role of all three parameters in modulating the structure and properties of the final product with relevance for fabrication of collagenous scaffolds for tissue engineering applications.

The mussel byssus stem provides a strong and compact mechanically mismatched biointerface between living tissue and a nonliving biopolymer. Yet, in a poorly understood process, mussels can simply jettison their entire byssus, rebuilding a new one in just hours. We characterized the structure and composition of the byssus biointerface using histology, confocal Raman mapping, phase contrast-enhanced microcomputed tomography, and advanced electron microscopy, revealing a sophisticated junction consisting of abiotic biopolymer sheets interdigitated between living extracellular matrix. The sheet surfaces are in intimate adhesive contact with billions of motile epithelial cilia that control biointerface strength and stem release through their collective movement, which is regulated neurochemically. We posit that this may involve a complex sensory pathway by which sessile mussels respond to environmental stresses to release and relocate.

Functionally graded interfaces are prominent in biological tissues and are used to mitigate stress concentrations at junctions between mechanically dissimilar components. Biological mechanical gradients serve as important role models for bioinspired design in technically and biomedically relevant applications. However, this necessitates elucidating exactly how natural gradients mitigate mechanical mismatch and how such gradients are fabricated. Here, we applied a cross-disciplinary experimental approach to understand structure, function, and formation of mechanical gradients in byssal threads─collagen-based fibers used by marine mussels to anchor on hard surfaces. The proximal end of threads is approximately 50-fold less stiff and twice as extensible as the distal end. However, the hierarchical structure of the distal-proximal junction is still not fully elucidated, and it is unclear how it is formed. Using tensile testing coupled with video extensometry, confocal Raman spectroscopy, and transmission electron microscopy on native threads, we identified a continuous graded transition in mechanics, composition, and nanofibrillar morphology, which extends several hundreds of microns and which can vary significantly between individual threads. Furthermore, we performed in vitro fiber assembly experiments using purified secretory vesicles from the proximal and distal regions of the secretory glands (which contain different precursor proteins), revealing spontaneous self-assembly of distinctive distal- and proximal-like fiber morphologies. Aside from providing fundamental insights into the byssus structure, function, and fabrication, our findings reveal key design principles for bioinspired design of functionally graded polymeric materials.

Mussels ( Mytilus edulis ) adhere to hard surfaces in intertidal marine habitats with a porous underwater glue called the byssus plaque. The plaque is an established role model for bioinspired underwater glues and comprises at least six proteins, most of which are highly cationic and enriched in the post-translationally modified amino acid 3,4-dihydroxyphenylalanine (DOPA). While much is known about the chemistry of plaque adhesion, less is understood about the natural plaque formation process. Here, we investigated plaque structure and formation using 3D electron microscopic imaging, revealing that micro- and nanopores form spontaneously during secretion of protein-filled secretory vesicles. To better understand this process, we developed a method to purify intact secretory vesicles for in vitro assembly studies. We discovered that each vesicle contains a sulfate-associated fluid condensate consisting of ∼9 histidine- and/or DOPA-rich proteins, which are presumably the required ingredients for building a plaque. Rupturing vesicles under specific buffering conditions relevant for natural assembly led to controlled multiphase liquid-liquid phase separation (LLPS) of different proteins, resulting in formation of a continuous phase with coexisting droplets. Rapid coarsening of the droplet phase was arrested through pH-dependent cross-linking of the continuous phase, producing native-like solid porous "microplaques" with droplet proteins remaining as fluid condensates within the pores. Results indicate that histidine deprotonation and sulfates figure prominently in condensate cross-linking. Distilled concepts suggest that combining phase separation with tunable cross-linking kinetics could be effective for microfabricating hierarchically porous materials via self-assembly.

Background: Cryolipolysis is a non-surgical procedure for subcutaneous fat layer reduction by controlled cooling. During the past few years, the use of cryolipolysis for non-invasive body contouring has increased significantly., Objectives: This retrospective study examines patient satisfaction, recommendation rate and patient comfort with the use of an FDA-cleared system (CoolSculpting Elite, Allergan Aesthetics, AbbVie Company, Irvine CA) for cryolipolysis at a single clinic and reports on the results., Methods: Between December 2020 and January 2022, 91 patients were treated with an FDA-cleared cryolipolysis system. To assess patient satisfaction, patients were asked to complete clinical questionnaires three months after their last treatment session. The following questions were asked: painfulness of the treatment, complications, satisfaction with the treatment, consideration of further treatment sessions, and willingness to further recommend the treatment., Results: Eighty-four percent of the 91 patients were female, and 16% were male. The average age was 45.5 years and the mean BMI was 26 kg/m 2 . Patients rated the treatment in terms of pain and discomfort experienced during the procedures on a scale of 1 to 5, with the value 1 representing not painful and 5 as extremely painful. 40% of the patients evaluated the procedure with 1, 38% with 2, 19% with 3, 1% with 4 and 2% with 5. With respect to satisfaction rates, 66% rated the treatment on a scale from 1 to 5 with 1, 18% with 2, 7% with 3, 7% with 4 and 3% with 5, with 1 indicating very satisfied and 5 indicating very dissatisfied. Thus, the overall level of satisfaction (the sum of scale values 1 and 2) amounts to 84%. Of 91 patients, 88% would agree to further treatment and 92% would recommend the therapy to others. All patients reported temporary tissue reactions such as swelling and redness which did not require any further treatment and were self-limiting. Serious or permanent complications did not occur., Conclusions: The results of our study show that cryolipolysis is a safe and effective method for non-surgical body contouring, providing a high degree of patient satisfaction and recommendation rate., (© 2022 The Aesthetic Society.)

To anchor in seashore habitats, mussels fabricate adhesive byssus fibers that are mechanically reinforced by protein-metal coordination mediated by 3,4-dihydroxyphenylalanine (DOPA). The mechanism by which metal ions are integrated during byssus formation remains unknown. In this study, we investigated the byssus formation process in the blue mussel, Mytilus edulis , combining traditional and advanced methods to identify how and when metals are incorporated. Mussels store iron and vanadium ions in intracellular metal storage particles (MSPs) complexed with previously unknown catechol-based biomolecules. During adhesive formation, stockpiled secretory vesicles containing concentrated fluid proteins are mixed with MSPs within a microfluidic-like network of interconnected channels where they coalesce, forming protein-metal bonds within the nascent byssus. These findings advance our understanding of metal use in biological materials with implications for next-generation metallopolymers and adhesives.

Protein-based biological materials are important role models for the design and fabrication of next generation advanced polymers. Marine mussels ( Mytilus spp.) fabricate hierarchically structured collagenous fibers known as byssal threads via bottom-up supramolecular assembly of fluid protein precursors. The high degree of structural organization in byssal threads is intimately linked to their exceptional toughness and self-healing capacity. Here, we investigated the hypothesis that multidomain collagen precursor proteins, known as preCols, are stored in secretory vesicles as a colloidal liquid crystal (LC) phase prior to thread self-assembly. Using advanced electron microscopy methods, including scanning TEM and FIB-SEM, we visualized the detailed smectic preCol LC nanostructure in 3D, including various LC defects, confirming this hypothesis and providing quantitative insights into the mesophase structure. In light of these findings, we performed an in-depth comparative analysis of preCol protein sequences from multiple Mytilid species revealing that the smectic organization arises from an evolutionarily conserved ABCBA pentablock copolymer-like primary structure based on demarcations in hydropathy and charge distribution as well as terminal pH-responsive domains that trigger fiber formation. These distilled supramolecular assembly principles provide inspiration and strategies for sustainable assembly of nanostructured polymeric materials for potential applications in engineering and biomedical applications.

Complex hierarchical structure governs emergent properties in biopolymeric materials; yet, the material processing involved remains poorly understood. Here, we investigated the multi-scale structure and composition of the mussel byssus cuticle before, during and after formation to gain insight into the processing of this hard, yet extensible metal cross-linked protein composite. Our findings reveal that the granular substructure crucial to the cuticle's function as a wear-resistant coating of an extensible polymer fiber is pre-organized in condensed liquid phase secretory vesicles. These are phase-separated into DOPA-rich proto-granules enveloped in a sulfur-rich proto-matrix which fuses during secretion, forming the sub-structure of the cuticle. Metal ions are added subsequently in a site-specific way, with iron contained in the sulfur-rich matrix and vanadium coordinated by DOPA-catechol in the granule. We posit that this hierarchical structure self-organizes via phase separation of specific amphiphilic proteins within secretory vesicles, resulting in a meso-scale structuring that governs cuticle function.

Biotechnology offers an exciting avenue toward the sustainable production of high performance proteinaceous polymeric materials. In particular, the mussel byssus-a high performance adhesive bio-fiber used by mussels to cling on hard surfaces-has become a veritable archetype for bio-inspired self-healing fibers, tough coatings, and versatile wet adhesives. However, successful translation of mussel-inspired design principles into man-made materials hinges upon elucidating structure-function relationships and biological fabrication processes. This review provides a detailed survey of the state-of-the-art understanding of the biochemical structure-function relationships defining byssus performance with a particular focus on structural hierarchy and metal coordination-based cross-linking. The efforts to mimic the byssus in man-made materials are then discussed. While there has been a strong push to mimic the byssus via synthetic chemistry taking a reductionist approach, herein the focus is specifically on recent progress of biotechnology-based strategies that more closely approximate the biochemical complexity of the natural material. As an outlook, an overview of recent research toward understanding the natural byssus assembly process is provided, as processing remains a critical factor in achieving native-like properties., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bottom-up control over structural hierarchy from the nanoscale through the macroscale is a critical aspect of biological materials fabrication and function, which can inspire production of advanced materials. Mussel byssal threads are a prime example of protein-based biofibers in which hierarchical organization of protein building blocks coupled via metal complexation leads to notable mechanical behaviors, such as high toughness and self-healing. Using a natural amino acid sequence from byssal thread proteins, which functions as a pH-triggered self-assembly point, we created free-standing peptide films with complex hierarchical organization across multiple length scales that can be controlled by inclusion of metal ions (Zn 2+ and Cu 2+ ) during the assembly process. Additionally, analysis of film mechanical performance indicates that metal coordination bestows up to an order of magnitude increase in material stiffness, providing a paradigm for creating tunable polymeric materials with multiscale organizational structure.

Cell accumulation is a prerequisite for adipose tissue inflammation. The leukocyte integrin Mac-1 (CD11b/CD18, α M β 2 ) is a classic adhesion receptor critically regulating inflammatory cell recruitment. Here, we tested the hypothesis that a genetic deficiency and a therapeutic modulation of Mac-1 regulate adipose tissue inflammation in a mouse model of diet-induced obesity (DIO). C57Bl6/J mice genetically deficient (Mac-1 -/- ) or competent for Mac-1 (WT) consumed a high fat diet for 20 weeks. Surprisingly, Mac-1 -/- mice presented with increased diet-induced weight gain, decreased insulin sensitivity in skeletal muscle and in the liver in insulin-clamps, insulin secretion deficiency and elevated glucose levels in fasting animals, and dyslipidaemia. Unexpectedly, accumulation of adipose tissue macrophages (ATMs) was unaffected, while gene expression indicated less inflamed adipose tissue and macrophages in Mac-1 -/- mice. In contrast, inflammatory gene expression at distant locations, such as in skeletal muscle, was not changed. Treatment of ATMs with an agonistic anti-Mac-1 antibody, M1/70, induced pro-inflammatory genes in cell culture. In vivo, treatment with M1/70 induced a hyper-inflammatory phenotype with increased expression of IL-6 and MCP-1, whereas accumulation of ATMs did not change. Finally, inhibition of Mac-1's adhesive interaction to CD40L by the peptide inhibitor cM7 did not affect myeloid cell accumulation in adipose tissue. We present the surprising finding that adhesive properties of the leukocyte integrin Mac-1 are not required for macrophage accumulation in adipose tissue. Instead, Mac-1 modulates inflammatory gene expression in macrophages. These findings question the net effect of integrin blockade in cardio-metabolic disease.

Background: Costimulatory cascades such as the CD40L-CD40 dyad enhance immune cell activation and inflammation during atherosclerosis. Here, we tested the hypothesis that CD40 directly modulates traits of the metabolic syndrome in diet-induced obesity in mice., Methods and Results: To induce the metabolic syndrome, wild-type or CD40(-/-) mice consumed a high-fat diet for 20 weeks. Unexpectedly, CD40(-/-) mice exhibited increased weight gain, impaired insulin secretion, augmented accumulation of inflammatory cells in adipose tissue, and enhanced proinflammatory gene expression. This proinflammatory and adverse metabolic phenotype could be transplanted into wild-type mice by reconstitution with CD40-deficient lymphocytes, indicating a major role for CD40 in T or B cells in this context. Conversely, therapeutic activation of CD40 signaling by the stimulating antibody FGK45 abolished further weight gain during the study, lowered glucose levels, improved insulin sensitivity, and suppressed adipose tissue inflammation. Mechanistically, CD40 activation decreased the expression of proinflammatory cytokines in T cells but not in B cells or macrophages. Finally, repopulation of lymphocyte-free Rag1(-/-) mice with CD40(-/-) T cells provoked dysmetabolism and inflammation, corroborating a protective role of CD40 on T cells in the metabolic syndrome. Finally, levels of soluble CD40 showed a positive association with obesity in humans, suggesting clinical relevance of our findings., Conclusions: We present the surprising finding that CD40 deficiency on T cells aggravates whereas activation of CD40 signaling improves adipose tissue inflammation and its metabolic complications. Therefore, positive modulation of the CD40 pathway might describe a novel therapeutic concept against cardiometabolic disease., (© 2014 American Heart Association, Inc.)

Background: Adipose tissue inflammation fuels the metabolic syndrome. We recently reported that CD40L--an established marker and mediator of cardiovascular disease--induces inflammatory cytokine production in adipose cells in vitro. Here, we tested the hypothesis that CD40L deficiency modulates adipose tissue inflammation in vivo., Methodology/principal Findings: WT or CD40L(-/-) mice consumed a high fat diet (HFD) for 20 weeks. Inflammatory cell recruitment was impaired in mice lacking CD40L as shown by a decrease of adipose tissue macrophages, B-cells, and an increase in protective T-regulatory cells. Mechanistically, CD40L-deficient mice expressed significantly lower levels of the pro-inflammatory chemokine MCP-1 both, locally in adipose tissue and systemically in plasma. Moreover, levels of pro-inflammatory IgG-antibodies against oxidized lipids were reduced in CD40L(-/-) mice. Also, circulating low-density lipoproteins and insulin levels were lower in CD40L(-/-) mice. However, CD40L(-/-) mice consuming HFD were not protected from the onset of diet-induced obesity (DIO), insulin resistance, and hepatic steatosis, suggesting that CD40L selectively limits the inflammatory features of diet-induced obesity rather than its metabolic phenotype. Interestingly, CD40L(-/-) mice consuming a low fat diet (LFD) showed both, a favorable inflammatory and metabolic phenotype characterized by diminished weight gain, improved insulin tolerance, and attenuated plasma adipokine levels., Conclusion: We present the novel finding that CD40L deficiency limits adipose tissue inflammation in vivo. These findings identify CD40L as a potential mediator at the interface of cardiovascular and metabolic disease.

In genome-wide screens we studied CA/C1 peptidases of malaria-causing plasmodia and their hosts (man and mouse). For Plasmodium falciparum and P. berghei, several new CA/C1 peptidase genes encoding proteases of the L- and B-family with specific promoter modules were identified. In addition, two new human CA/C1 peptidase loci and one new mouse gene locus were found; otherwise, the sets of CA/C1 peptidase genes in man and mouse seem to be complete now. In each species studied there is a multitude of CA/C1 peptidases with lysosomal localization signals and partial functional overlap according to similar but subfamily-specific structures. Individual target structures in plasmodia include residues specifically different in CA/C1 peptidase subsite 2. This is of medical interest considering CA/C1 peptidase inhibition for chemotherapy in malaria, malignancies and other diseases. Promoter structures and mRNA regulation differ widely among CA/C1 peptidase subfamilies and between mammals and plasmodia. We characterized promoter modules conserved in mouse and man for the CA/C1 peptidase families B and L (with the L-like subfamily, F-like subfamily and mouse-specific J-like subfamily). RNA motif searches revealed conserved regulatory elements such as GAIT elements; plasmodial CA/C1 peptidase mRNA elements include ARE elements and mammalian mRNAs contain 15-lox DICE elements.