Elastin‐like‐polypeptide based fusion proteins for osteogenic factor delivery in bone healing

Modern treatments of bone injuries and diseases are becoming increasingly dependent on the usage of growth factors to stimulate bone growth. Bone morphogenetic protein‐2 (BMP‐2), a potent osteogenic inductive protein, exhibits promising results in treatment models, but recently has had its practical efficacy questioned due to the lack of local retention, ectopic bone formation, and potentially lethal inflammation. Where a new delivery technique of the BMP‐2 is necessary, here we demonstrate the viability of an elastin‐like peptide (ELP) fusion protein containing BMP‐2 for delivery of the BMP‐2. This fusion protein retains the performance characteristics of both the BMP‐2 and ELP. The fusion protein was found to induce osteogenic differentiation of mesenchymal stem cells as evidenced by the production of alkaline phosphatase and extracellular calcium deposits in response to treatment by the fusion protein. Retention of the ELPs inverse phase transition property has allowed for expression of the fusion protein within a bacterial host (such as Escherichia coli) and easy and rapid purification using inverse transition cycling. The fusion protein formed self‐aggregating nanoparticles at human‐body temperature. The data collected suggests the viability of these fusion protein nanoparticles as a dosage‐efficient and location‐precise noncytotoxic delivery vehicle for BMP‐2 in bone treatment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1029–1037, 2016     

The Identity Objection to the future‐like‐ours argument

Some critics of Don Marquis's ‘future‐like‐ours’ anti‐abortion argument launch what has been called the Identity Objection. The upshot of this objection is that under a psychological theory of personal identity, a non‐sentient fetus lacks precisely what Marquis believes gives it a right to life – a future like ours. However, Eric Vogelstein, in a recent article, has argued that under this theory of personal identity a non‐sentient fetus, in fact, has a future like ours, which he believes dissolves the Identity Objection. But Vogelstein is mistaken. Even if he is correct that there is a sense in which a non‐sentient fetus has a future of value under a psychological theory of personal identity, the sense in which it has one is importantly different from the sense in which we have one, meaning that, under such a theory, a non‐sentient fetus does not have a future like ours.     

The future‐like‐ours argument,animalism, and mereological universalism

Which metaphysical theories are involved—whether presupposed or implied—in Marquis’ future‐like‐ours (FLO) argument against abortion? Vogelstein has recently argued that the supporter of the FLO argument faces a problematic dilemma; in particular, Marquis, the main supporter of the argument, seems to have to either (a) abandon diachronic universalism (DU) or (b) acquiesce and declare that contraception is morally wrong. I argue that the premises of Marquis’ argument can be reasonably combined with a form of unrestricted composition and that the FLO argument is better viewed as including animalism, i.e., the thesis that we are animals.     

Identification of β integrin‐like‐ and fibronectin‐like proteins in the bivalve mollusk Mytilus trossulus

Integrins play a key role in the intermediation and coordination between cells and extracellular matrix components. In this study, we first determined the presence of the β integrin‐like protein and its presumptive ligand, fibronectin‐like protein, during development and in some adult tissues of the bivalve mollusc Mytilus trossulus. We found that β integrin‐like protein expression correlated with the development and differentiation of the digestive system in larvae. Besides the presence of β integrin‐like protein in the digestive epithelial larval cells, this protein was detected in the hemocytes and some adult tissues of M. trossulus. The fibronectin‐like protein was detected firstly at the blastula stage and later, the FN‐LP‐immunoreactive cells were scattered in the trochophore larvae. The fibronectin‐like protein was not expressed in the β integrin‐positive cells of either the veliger stage larvae or the adult mussel tissues and the primary hemocyte cell culture. Despite the β integrin‐ and fibronectin‐like proteins being expressed in different cell types of mussel larvae, we do not exclude the possibility of direct interaction between these two proteins during M. trossulus development or in adult tissues.     

Simultaneous color‐coded imaging to distinguish cancer “stem‐like” and non‐stem cells in the same tumor

In this study, we demonstrate that the differential behavior, including malignancy and chemosensitivity, of cancer stem‐like and non‐stem cells can be simultaneously distinguished in the same tumor in real time by color‐coded imaging. CD133⁺ Huh‐7 human hepatocellular carcinoma (HCC) cells were considered as cancer stem‐like cells (CSCs), and CD133^? Huh‐7 cells were considered as non‐stem cancer cells (NSCCs). CD133⁺ cells were isolated by magnetic bead sorting after Huh‐7 cells were genetically labeled with green fluorescent protein (GFP) or red fluorescent protein (RFP). In this scheme, CD133⁺ cells were labeled with GFP and CD133^? cells were labeled with RFP. CSCs had higher proliferative potential compared to NSCCs in vitro. The same number of GFP CSCs and the RFP NSCCs were mixed and injected subcutaneously or in the spleen of nude mice. CSCs were highly tumorigenic and metastatic as well as highly resistant to chemotherapy in vivo compared to NSCCs. The ability to specifically distinguish stem‐like cancer cells in vivo in real time provides a visual target for prevention of metastasis and drug resistance. J. Cell. Biochem. 111: 1035–1041, 2010. © 2010 Wiley‐Liss, Inc.     

A well‐preserved ‘charadriiform‐like’ fossil bird from the Early Eocene Fur Formation of Denmark

Abstract: We describe a new, exceptionally well‐preserved fossil bird recovered from marine deposits of the Early Eocene Fur Formation of Denmark. Morsoravis sedilis gen. et sp. nov. is known by a single specimen that consists of a three‐dimensional skull, vertebral column, ribs, pelvis, and left hindlimb and associated parts of the right hindlimb. Comparisons based on overall morphology and particularly characters of the skull, vertebrae and pelvis indicate that the new specimen is morphologically similar to charadriiform birds (the shorebirds and relatives). This similarity is also expressed by a phylogenetic analysis of higher neornithine (modern birds) taxa, which supports a close relationship between the new fossil and modern charadriiforms. The morphology of the hindlimbs, in particular, shows that the new fossil corresponds to a new taxon that is distinguishable from modern charadriiform clades. One interesting aspect of its morphology is the presence of hindlimb specializations that are most commonly found among perching birds – these suggest that ecologically the new Danish fossil bird may have differed from the wading habits typical of most charadriiforms.     

Recombinant PBD‐1 (porcine beta‐defensin 1) expressed in the milk by transplanting transgenic mES‐like‐derived cells into mouse mammary gland

ES (embryonic stem)‐derived cells have been investigated in many animal models of severe injury and degenerative disease. However, few studies have examined the ability of ES‐derived cells to improve functional outcome following partially damaged breast and also the modification of mammary tissue to produce costly proteins. This study investigates the feasibility of implanting mES‐dK (mouse ES‐derived keratinocytes‐like) cells stably transfected with a mammary gland special expression vector for the PBD‐1 (porcine beta‐defensin 1) in developing mammary glands. Our aim was to assess the ability of cell grafting to improve functional outcome following partial damage of the breast, also on the breast modification mammary tissue in mice for the production of PBD‐1 protein secreted in the milk. Our results showed that the ratios of the surviving cells labelled with the myoepithelial or luminal cell markers, EMA (epithelial membrane antigen) and CALLA, were 41.7±15.2% and 28.4±9.6%, respectively, which revealed that transplanted mES‐dK cells survived, integrated in vivo and differentiated into myoepithelial or luminal cells. In addition, Western blot analysis showed that 37.5% (3 out of 8) female transplanted mice had PBD‐1 expression in their milk and reached 0.4998, 0.5229 and 0.5195 μg/ml, respectively.     

Entomotoxic action of Sambucus nigra agglutinin i in Acyrthosiphon pisum aphids and Spodoptera exigua caterpillars through caspase‐3‐like‐dependent apoptosis

In this project, the toxicity and mechanism of action of the ricin‐B‐related lectin SNA‐I from elderberry (Sambucus nigra) in the pea aphid (Acyrthosiphon pisum) and the beet armyworm (Spodoptera exigua), two important pest insects in agriculture, were studied. SNA‐I is a chimeric lectin belonging to the class of ribosome‐inactivating proteins and consists of an A‐chain with N‐glycosidase activity and a carbohydrate‐binding B‐chain. Incorporation of 2 mg/ml of SNA‐I in the diet of neonates and adults of A. pisum caused 40–46% mortality within 2 days, while in third instars of S. exigua, the larval biomass was significantly reduced by 12% after feeding for 3 days on a diet containing 5 mg/g of SNA‐I. Interestingly, extracts of the (mid)gut of treated A. pisum and S. exigua demonstrated DNA fragmentation and this was accompanied with an increase in caspase‐3‐like activity. The involvement of cell death or apoptosis in the entomotoxicity of SNA‐I through induction of caspase‐3‐like activity was also confirmed by addition of the permeable caspase‐3 inhibitor III in the diet, leading to a rescue of the treated aphid neonates. Finally, similar to the chimeric lectin SNA‐I, the hololectin SNA‐II, consisting of two carbohydrate‐binding B‐chains caused high mortality to neonate A. pisum aphids with an LC₅₀ of 1.59 mg/ml, suggesting that the entomotoxic action of the lectins under study mainly relies on their carbohydrate‐binding activity. © 2010 Wiley Periodicals, Inc.     

The roles of circRFWD2 and circINO80 during NELL‐1‐induced osteogenesis

Bone defects caused heavy social and economic burdens worldwide. Nel‐like molecule, type 1 (NELL‐1) could enhance the osteogenesis and the repairment of bone defects, while the specific mechanism remains to be elucidated. Circular RNAs (circRNAs) have been found to play critical roles in the tissue development and serve as biomarkers for various diseases. However, it remains unclear that the expression patterns of circRNAs and the roles of them played in recombinant NELL‐1‐induced osteogenesis of human adipose‐derived stem cells (hASCs). In this study, we performed RNA‐sequencing to investigate the expression profiles of circRNAs in recombinant NELL‐1‐induced osteogenic differentiation and identified two key circRNAs, namely circRFWD2 and circINO80. These two circRNAs were confirmed to be up‐regulated during recombinant NELL‐1‐induced osteogenesis, and knockdown of them affected the positive effect of NELL‐1 on osteogenesis. CircRFWD2 and circINO80 could interact with hsa‐miR‐6817‐5p, which could inhibit the osteogenesis. Silencing hsa‐miR‐6817‐5p could partially reverse the negative effect of si‐circRFWD2 and si‐circINO80 on the osteogenesis. Therefore, circRFWD2 and circINO80 could regulate the expression of hsa‐miR‐6817‐5p and influence the recombinant NELL‐1‐induced osteogenic differentiation of hASCs. It opens a new window to better understanding the effects of NELL‐1 on the osteogenic differentiation of hASCs and provides potential molecular targets and novel methods for bone regeneration efficiently and safely.     

Microbiological changes in whiting (Merlangius merlangus Linneaus, 1758) fillets during short‐term cold storage and a traditional ‘pastrami‐like’ treatment

The objective of this study was to test the effects of salt‐dried whiting (Merlangius merlangus) fillet storage when treated with a special paste and stored covered. For this purpose whiting fillets were salt‐dried at 4–6°C for 15 days. A subsequent test series involved a paste mixture prepared from ground fenugreek, cumin seeds, black pepper, red pepper powder and garlic. The fillets were coated with this paste and air‐dried (15–20°C) for 5 days. All microbiological changes during this drying period were noted. The aerobic mesophilic bacterial counts decreased significantly (P < 0.05) from 5.08 ± 0.20 log cfu g^?1 to 3.24 ± 0.06 log cfu g^?1 after 15 days of salt‐drying. After then covering with paste and drying for 5 days (at 15–20°C), the aerobic mesophilic and lactic acid bacteria counts of the fillets increased to 6.05 ± 0.45 and 5.85 ± 0.06 log cfu g^?1, respectively. The pH values of dried whiting fillets changed after 15 days of dry salting (from 6.1 to 6.4), but after coating and drying with the paste, the pH values were 5.6 on day 5. Enterobactericeae were few in number at the start of salt‐drying (about 1.20 ± 0.15 log cfu g^?1), but their number decreased to <1.0 log cfu g^?1 after 15 days of dry‐salt storage. Staphylococcus aureus, Escherichia coli, mould and yeast were not detected at any time of drying. According to the resuts of the microbiological analyses, dried whiting fillet are considered safe for human consumption.     

Crystal structure of the archaeosine synthase QueF‐like—Insights into amidino transfer and tRNA recognition by the tunnel fold

The tunneling‐fold (T‐fold) structural superfamily has emerged as a versatile protein scaffold of diverse catalytic activities. This is especially evident in the pathways to the 7‐deazaguanosine modified nucleosides of tRNA queuosine and archaeosine. Four members of the T‐fold superfamily have been confirmed in these pathways and here we report the crystal structure of a fifth enzyme; the recently discovered amidinotransferase QueF‐Like (QueF‐L), responsible for the final step in the biosynthesis of archaeosine in the D‐loop of tRNA in a subset of Crenarchaeota. QueF‐L catalyzes the conversion of the nitrile group of the 7‐cyano‐7‐deazaguanine (preQ₀) base of preQ₀‐modified tRNA to a formamidino group. The structure, determined in the presence of preQ₀, reveals a symmetric T‐fold homodecamer of two head‐to‐head facing pentameric subunits, with 10 active sites at the inter‐monomer interfaces. Bound preQ₀ forms a stable covalent thioimide bond with a conserved active site cysteine similar to the intermediate previously observed in the nitrile reductase QueF. Despite distinct catalytic functions, phylogenetic distributions, and only 19% sequence identity, the two enzymes share a common preQ₀ binding pocket, and likely a common mechanism of thioimide formation. However, due to tight twisting of its decamer, QueF‐L lacks the NADPH binding site present in QueF. A large positively charged molecular surface and a docking model suggest simultaneous binding of multiple tRNA molecules and structure‐specific recognition of the D‐loop by a surface groove. The structure sheds light on the mechanism of nitrile amidation, and the evolution of diverse chemistries in a common fold. Proteins 2016; 85:103–116. © 2016 Wiley Periodicals, Inc.