Time-Prolonged Release of Tumor-Targeted Protein–MMAE Nanoconjugates from Implantable Hybrid Materials

The sustained release of small, tumor-targeted cytotoxic drugs is an unmet need in cancer therapies, which usually rely on punctual administration regimens of non-targeted drugs. Here, we have developed a novel concept of protein–drug nanoconjugates, which are packaged as slow-releasing chemically hybrid depots and sustain a prolonged secretion of the therapeutic agent. For this, we covalently attached hydrophobic molecules (including the antitumoral drug Monomethyl Auristatin E) to a protein targeting a tumoral cell surface marker abundant in several human neoplasias, namely the cytokine receptor CXCR4. By this, a controlled aggregation of the complex is achieved, resulting in mechanically stable protein–drug microparticles. These materials, which are mimetics of bacterial inclusion bodies and of mammalian secretory granules, allow the slow leakage of fully functional conjugates at the nanoscale, both in vitro and in vivo. Upon subcutaneous administration in a mouse model of human CXCR4+ lymphoma, the protein–drug depots release nanoconjugates for at least 10 days, which accumulate in the tumor with a potent antitumoral effect. The modification of scaffold cell-targeted proteins by hydrophobic drug conjugation is then shown as a novel transversal platform for the design of slow releasing protein–drug depots, with potential application in a broad spectrum of clinical settings.

Delta/Jagged-mediated Notch signaling induces the differentiation of agr2-positive epidermal mucous cells in zebrafish embryos

Teleosts live in aquatic habitats, where they encounter ionic and acid-base fluctuations as well as infectious pathogens. To protect from these external challenges, the teleost epidermis is composed of living cells, including keratinocytes and ionocytes that maintain body fluid ionic homeostasis, and mucous cells that secret mucus. While ionocyte progenitors are known to be specified by Delta-Notch-mediated lateral inhibition during late gastrulation and early segmentation, it remains unclear how epidermal mucous cells (EMCs) are differentiated and maintained. Here, we show that Delta/Jagged-mediated activation of Notch signaling induces the differentiation of agr2-positive (agr2+) EMCs in zebrafish embryos during segmentation. We demonstrated that agr2+ EMCs contain cytoplasmic secretory granules and express muc5.1 and muc5.2. Reductions in agr2+ EMC number were observed in mib mutants and notch3 MOs-injected notch1a mutants, while increases in agr2+ cell number were detected in notch1a- and X-Su(H)/ANK-overexpressing embryos. Treatment with γ-secretase inhibitors further revealed that Notch signaling is required during bud to 15 hpf for the differentiation of agr2+ EMCs. Increased agr2+ EMC numbers were also observed in jag1a-, jag1b-, jag2a- and dlc-overexpressing, but not jag2b-overexpressing embryos. Meanwhile, reductions in agr2+ EMC numbers were detected in jag1a morphants, jag1b mutants, jag2a mutants and dlc morphants, but not jag2b mutants. Reduced numbers of pvalb8-positive epidermal cells were also observed in mib or jag2a mutants and jag1a or jag1b morphants, while increased pvalb8-positive epidermal cell numbers were detected in notch1a-overexpressing, but not dlc-overexpressing embryos. BrdU labeling further revealed that the agr2+ EMC population is maintained by proliferation. Cell lineage experiments showed that agr2+ EMCs are derived from the same ectodermal precursors as keratinocytes or ionocytes. Together, our results indicate that specification of agr2+ EMCs in zebrafish embryos is induced by DeltaC/Jagged-dependent activation of Notch1a/3 signaling, and the cell population is maintained by proliferation.

Liquid-liquid phase separation facilitates the biogenesis of secretory storage granules

Insulin is a key regulator of human metabolism, and its dysfunction leads to diseases such as type 2 diabetes. It remains unknown how proinsulin is targeted from the trans-Golgi network (TGN) to secretory storage granules as no cargo receptor has been identified. Chromogranin proteins (CGs) are central regulators of granule biosynthesis, and it was proposed that their aggregation is critical for this process. However, the molecular mechanism by which these molecules facilitate sorting at the TGN is poorly understood. Here, we show that CGs undergo liquid-liquid phase separation (LLPS) at low pH independently of divalent cations, such as calcium. Liquid CG condensates, but not aggregates, recruit and sort proinsulin and other granule destined cargo molecules towards secretory granules. Cargo selectivity is independent of sequence or structural elements but is based on the size and concentration of the client molecules at the TGN. Finally, electrostatic interactions and the N-terminal intrinsically disordered domain of chromogranin B facilitate LLPS and are critical for granule formation. We propose that phase-separated CGs act as a cargo sponge within the TGN lumen, gathering soluble client proteins into the condensate independently of specific sequence or structural elements, facilitating receptor-independent sorting. These findings challenge the canonical TGN sorting models and provide insights into granule biosynthesis in insulin-secreting beta cells.

Interspecific Analysis of Sea Urchin Adhesive Composition Emphasizes Variability of Glycans Conjugated With Putative Adhesive Proteins

Sea urchins possess specialized adhesive organs, tube feet. Although initially believed to function as suckers, it is currently accepted that they rely on adhesive and de-adhesive secretions to attach and detach repeatedly from the substrate. Given the biotechnological potential of their strong reversible adhesive, sea urchins are under investigation to identify the protein and glycan molecules responsible for its surface coupling, cohesion and polymerization properties. However, this characterization has only focused on a single species, Paracentrotus lividus. To provide a broader insight into sea urchins adhesion, a comparative study was performed using four species belonging to different taxa and habitats: Diadema africanum, Arbacia lixula, Paracentrotus lividus and Sphaerechinus granularis. Their tube feet external morphology and histology was studied, together with the ultrastructure of their adhesive secretory granules. In addition, one antibody and five lectins were used on tube foot histological sections and extracts, and on adhesive footprints to detect the presence of adhesion-related (glyco)proteins like those present in P. lividus in other species. Results confirmed that the antibody raised against P. lividus Nectin labels the adhesive organs and footprints in all species. This result was further confirmed by a bioinformatic analysis of Nectin-like sequences in ten additional species, increasing the comparison to seven families and three orders. The five tested lectins (GSL II, WGA, STL, LEL, and SBA) demonstrated that there is high interspecific variability of the glycans involved in sea urchin adhesion. However, there seems to be more conservation among taxonomically closer species, like P. lividus and S. granularis. In these species, lectin histochemistry and lectin blots indicated the presence of high molecular weight putative adhesive glycoproteins bearing N-acetylglucosamine residues in the form of chitobiose in the adhesive epidermis and footprints. Our results emphasize a high selective pressure for conservation of functional domains in large putative cohesive proteins and highlight the importance of glycosylation in sea urchin adhesion with indications of taxonomy-related conservation of the conjugated glycans.

Weibel Palade Bodies: Unique Secretory Organelles of Endothelial Cells that Control Blood Vessel Homeostasis

Vascular endothelial cells produce and release compounds regulating vascular tone, blood vessel growth and differentiation, plasma composition, coagulation and fibrinolysis, and also engage in interactions with blood cells thereby controlling hemostasis and acute inflammatory reactions. These interactions have to be tightly regulated to guarantee smooth blood flow in normal physiology, but also allow specific and often local responses to blood vessel injury and infectious or inflammatory insults. To cope with these challenges, endothelial cells have the remarkable capability of rapidly changing their surface properties from non-adhesive (supporting unrestricted blood flow) to adhesive (capturing circulating blood cells). This is brought about by the evoked secretion of major adhesion receptors for platelets (von-Willebrand factor, VWF) and leukocytes (P-selectin) which are stored in a ready-to-be-used form in specialized secretory granules, the Weibel-Palade bodies (WPB). WPB are unique, lysosome related organelles that form at the trans-Golgi network and further mature by receiving material from the endolysosomal system. Failure to produce correctly matured VWF and release it through regulated WPB exocytosis results in pathologies, most importantly von-Willebrand disease, the most common inherited blood clotting disorder. The biogenesis of WPB, their intracellular motility and their fusion with the plasma membrane are regulated by a complex interplay of proteins and lipids, involving Rab proteins and their effectors, cytoskeletal components as well as membrane tethering and fusion machineries. This review will discuss aspects of WPB biogenesis, trafficking and exocytosis focussing on recent findings describing factors contributing to WPB maturation, WPB-actin interactions and WPB-plasma membrane tethering and fusion.

V-ATPase V0a1 promotes Weibel–Palade body biogenesis through the regulation of membrane fission

Membrane fission, the division of a membrane-bound structure into two discrete compartments, is essential for diverse cellular events, such as endocytosis and vesicle/granule biogenesis; however, the process remains unclear. The hemostatic protein von Willebrand factor is produced in vascular endothelial cells and packaged into specialized secretory granules, Weibel-Palade bodies (WPBs) at the trans-Golgi network (TGN). Here, we reported that V0a1, a V-ATPase component, is required for the membrane fission of WPBs. We identified two V0a isoforms in distinct populations of WPBs in cultured endothelial cells, V0a1 and V0a2, on mature and nascent WPBs, respectively. Although WPB buds were formed, WPBs could not separate from the TGN in the absence of V0a1. Screening using dominant-negative forms of known membrane fission regulators revealed protein kinase D (PKD) as an essential factor in biogenesis of WPBs. Further, we showed that the induction of wild-type PKDs in V0a1-depleted cells does not support the segregation of WPBs from the TGN; suggesting a primary role of V0a1 in the membrane fission of WPBs. The identification of V0a1 as a new membrane fission regulator should facilitate the understanding of molecular events that enable membrane fission.

Tryptase Regulates the Epigenetic Modification of Core Histones in Mast Cell Leukemia Cells

Mast cells are immune cells that store large amounts of mast cell-restricted proteases in their secretory granules, including tryptase, chymase and carboxypeptidase A3. In mouse mast cells, it has been shown that tryptase, in addition to its canonical location in secretory granules, can be found in the nuclear compartment where it can impact on core histones. Here we asked whether tryptase can execute core histone processing in human mast cell leukemia cells, and whether tryptase thereby can affect the epigenetic modification of core histones. Our findings reveal that triggering of cell death in HMC-1 mast cell leukemia cells is associated with extensive cleavage of core histone 3 (H3) and more restricted cleavage of H2B. Tryptase inhibition caused a complete blockade of such processing. Our data also show that HMC-1 cell death was associated with a major reduction of several epigenetic histone marks, including H3 lysine-4-mono-methylation (H3K4me1), H3K9me2, H3 serine-10-phosphorylation (H3S10p) and H2B lysine-16-acetylation (H2BK16ac), and that tryptase inhibition reverses the effect of cell death on these epigenetic marks. Further, we show that tryptase is present in the nucleus of both viable and dying mast cell leukemia cells. In line with a role for tryptase in regulating nuclear events, tryptase inhibition caused increased proliferation of the mast cell leukemia cells. Altogether, the present study emphasizes a novel principle for how epigenetic modification of core histones is regulated, and provides novel insight into the biological function of human mast cell tryptase.

MORPHOGENESIS OF THE WALL OF GLANDULAR PART OF THE STOMACH IN CHICKENS DURING POSTNATAL PERIOD OF ONTOGENESIS

Knowledge about the morphological features of the structure and functions of the digestive organs provide the basis for rational and effective use of feed, prevention and treatment of gastrointestinal diseases in poultry. In this regard, the study of the morphogenesis of the digestive system in birds and the mechanisms of their regulation is of great importance. The object of the study was the glandular part of the stomach (proventriculus) in chickens of Shaver 579 strain. The material for macro- and microscopic examinations was selected from birds at age of 1, 30, 60, 90, 120, 150, 180, 210, 240, 270, and 300 days and 1, 2, and 3 years; it was fixed in a 10% neutral formalin solution and embedded into paraffin according to conventional methods. For submicroscopic examinations, the material was selected from hens of this strain at age of 180 days. The structure of epitheliocytes in the superficial epithelium and secretory cells of the deep glands was studied in ultrathin sections. Digital indicators of research results were statistically processed by a personal computer using Microsoft Excel program. The glandular stomach in chickens is a direct extension of the esophagus and has the form of a thick-walled tube, the wall of which is formed by mucous, muscular, and serous membranes. The superficial epithelium of the mucosa is represented by cylindrical epithelial cells that are located within the basal membrane. They are linked to each other by different types of contacts and have a well-defined polar differentiation. The lobules of the deep glands are formed by cells with well-developed synthesizing organelles and secretory granules. The morphogenesis of the glandular part of the stomach in chickens according to age aspect is manifested by changes in morphometric parameters of the thickness and the area of the membranes of its wall. The wall thickness increases (between the folds 4223.23 ± 189.25 and in the area of the folds 5561.32 ± 45.01 μm) unevenly in chickens up to age of 240 days. The most developed membrane of the wall of the glandular stomach is the mucosa. Its area increases (by 82.14 ± 0.56%) in chickens up to age of 180 days, and area of muscular and serous membranes decreases (by 15.54 ± 0.65 and 2.32 ± 0.33%, respectively). In older birds, the thickness of the wall and the area of the membranes of the glandular stomach do not change significantly.

Inhibitory Effect of Astaxanthin on Testosterone-Induced Benign Prostatic Hyperplasia in Rats

This study investigates the inhibitory effect of astaxanthin (AST) on testosterone-induced benign prostatic hyperplasia (BPH) in rats. Except for the sham operation, BPH model rats were randomly assigned to five groups: the BPH model control rats, AST-treated BPH model rats (20 mg/kg, 40 mg/kg, and 80 mg/kg), and epristeride (EPR)-treated BPH model rats. After treatment, as compared with the BPH model control rats, the prostate and ventral prostate weights of the AST-treated rats decreased, while there was a marked decline in the 80 mg/kg AST-treated rats. The same effect was also observed in the prostate index and ventral prostate index. The proliferation characteristics of epithelia observed in the BPH model control group were gradually alleviated in the AST-treated rats. As compared with the BPH model control rats, lower epithelial thicknesses of prostates and fewer secretory granules in epithelia were observed in the AST-treated rats. The superoxide dismutase (SOD) activity of prostates increased in all the AST-treated rats with a significant increase in the 40 mg/kg and 80 mg/kg AST-treated rats. The testosterone (T) and dihydrotestosterone (DHT) levels of prostates in the AST-treated groups were lower than those in the BPH model control group, and a significant decline was found in the T level of prostates in the 40 g/kg and 80 mg/kg AST-treated rats and the DHT level of prostates in the 40 mg/kg AST-treated rats. These results indicate that AST might have an inhibitory effect on T-induced BPH in rats, possibly due to SOD activity regulation and T and DHT levels.

Microanalysis of the Intestinal Bulb of Grass Carp (Ctenopharyngodon Idella): Histological, Histochemical, Immunohistochemical, and Scanning Electron Microscopical Studies

Cyprinid fishes have one of the simplest types of gastrointestinal tract among vertebrates. Those fish species do not possess a true stomach that is replaced by a simple dilatation at the anterior part of the intestine called the intestinal bulb. Twenty adult specimens of grass carp were used in the present study to identify the cellular components as well as the immunohistochemical and surface architectural characteristics of the intestinal bulb. The mucosa of the intestinal bulb shows numerous, deep longitudinal folds arranged in zigzagging-like patterns. The epithelium is composed mainly of absorptive columnar cells covered by microvilli and mucous goblet cells. Spindle-shaped enteroendocrine cells and some migratory immune cells such as intraepithelial lymphocytes and rodlet cells could be identified between the absorptive cells. The epithelium also contains many secretory granules and large numbers of vacuoles containing digestive enzymes mostly in the basal part. The immunohistochemistry revealed that CD20-positive B-lymphocytes are immunolocalized mainly in the connective tissue core lamina propria of the mucosal folds. However, CD3-immunopositive T-lymphocytes are highly concentrated in the lamina propria. In addition, intraepithelial T-lymphocytes expressed immunopositivity to CD3. The current study presented many types of immune cells and suggests their essential immunological role for the intestinal blub.