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In addition, electrochemical stimulation methods such as electrotaxis are mentioned as promising techniques for biofabrication. Finally, future research directions in this field and the application prospects are highlighted. © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.Isoflavones are a class of flavonoids present in legumes and are called phytoestrogens because of their estrogen-like activity. Endogenous estrogen is well known to regulate mammary gland morphogenesis during pregnancy. Each isoflavone also has different physiological activities. However, it is difficult to investigate the direct effect of each isoflavone in mammary morphogenesis in vivo because isoflavones are metabolized into different isoflavones by enteric bacteria. In this study, investigated are the direct influences of coumestrol, daidzein, and genistein on mammary structure development and future milk production ability of mammary epithelial cells (MECs) using in vitro culture models. Mouse MECs are cultured in Matrigel with basic fibroblast growth factor and epidermal growth factor to induce ductal branching and alveolar formation, respectively. Coumestrol and genistein inhibit ductal branching and alveolar formation by affecting the proliferation and migration of MECs with the induction of apoptosis. Daidzein hardly influences mammary structure development. Furthermore, pretreatment with coumestrol adversely affects the induction of milk production ability of MECs. These results suggest that each isoflavone differentially influences mammary morphogenesis and future milk production by affecting MEC behaviors. These results also suggest that the culture models are effective to study mammary epithelial morphogenesis in vitro. © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.The rapid dissemination of non-conjugative virulence plasmids among non-K1/K2 types of Klebsiella pneumoniae poses an unprecedented threat to human health, yet the underlying mechanisms governing dissemination of such plasmids is unclear. In this study, a novel 68 581 bp IncFIA plasmid is discovered that can be fused to a hypervirulence-encoding plasmid to form a hybrid conjugative virulence plasmid in conjugation experiments; such fusion events involve homologous recombination between a 241 bp homologous region located in each of the two plasmids. The fusion hypervirulence-encoding plasmid can be conjugated to both classic and blaKPC-2 -bearing carbapenem-resistant K. pneumoniae strains through conjugation, enabling such strains to acquire the ability to express the hypervirulence phenotype. Dissemination of this fusion virulence plasmid will impose an enormous burden on current efforts to control and treat infections caused by multidrug resistant and hypervirulent K. pneumoniae. © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.Engineered promoters are key components in the cell-factory design, allowing precise and enhanced expression of genes. Promoters having exceptional strength are attractive candidates for designing metabolic engineering strategies for tailoring de novo production strategies that require directed evolution methods by engineering with de novo synthetic biology tools. Here, the custom-designed AOX1 hybrid-promoter architectures in coordination with targeted transcription factors are shown, transcriptionally rewired the expression over methanol-free substrate-utilization pathway(s) and converted methanol-dependent Pichia pastoris alcohol oxidase 1(AOX1) promoter (PAOX1 ) expression into a non-toxic carbon-source-regulated system. AOX1 promoter variants are engineered by replacing specified cis-regulatory DNA elements with synthetic Adr1, Cat8, and Aca2 cis-acting DNA elements for Mxr1, Cat8, and Aca1 binding, respectively. Applications of the engineered-promoters are validated for eGFP expression and extracellular human serum albumin production. The hybrid-promoter architecture designed with single Cat8 cis-acting DNA element deregulates the expression on ethanol. Compared with PAOX1 on methanol, the expression on ethanol is increased with i) PAOX1/Cat8-L3 (designed with single Cat8 cis-acting element) to 74%, ii) PAOX1/Adr1-L3/Cat8-L3 (designed with single- Cat8 and Adr1 cis-acting elements) to 85%, and for further consolidation of deregulated expression iii) PeAOX1 (designed with triplet- Cat8 and Adr1 cis-acting elements) 1.30-fold, at t = 20 h of batch cultivations. © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.Animals are thought to use only glucose polymers (glycogen) as energy reserve, whereas both glucose (starch) and fructose polymers (fructans) are used by microbes and plants. Here, it is reported that the gall midge Mayetiola destructor, and likely other herbivorous animal species, gained the ability to utilize dietary fructans directly as storage polysaccharides by a single horizontal gene transfer (HGT) of bacterial levanase/inulinase gene followed by gene expansion and differentiation. Multiple genes encoding levanases/inulinases have their origin in a single HGT event from a bacterium and they show high expression levels and enzymatic activities in different tissues of the gall midge, including nondigestive fat bodies and eggs, both of which contained significant amounts of fructans. This study provides evidence that animals can also use fructans as energy reserve by incorporating bacterial genes in their genomes. © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.The correct human brain function is dependent on the activity of non-neuronal cells called astrocytes. The bioelectrical properties of astrocytes in vitro do not closely resemble those displayed in vivo and the former are incapable of generating action potential; thus, reliable approaches in vitro for noninvasive electrophysiological recording of astrocytes remain challenging for biomedical engineering. Here it is found that primary astrocytes grown on a device formed by a forest of randomly oriented gold coated-silicon nanowires, resembling the complex structural and functional phenotype expressed by astrocytes in vivo. The device enables noninvasive extracellular recording of the slow-frequency oscillations generated by differentiated astrocytes, while flat electrodes failed on recording signals from undifferentiated cells. Pathophysiological concentrations of extracellular potassium, occurring during epilepsy and spreading depression, modulate the power of slow oscillations generated by astrocytes. A reliable approach to study the role of astrocytes function in brain physiology and pathologies is presented. © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.With more than 50% of bacteria resistant to standard antibiotics, new strategies to treat bacterial infection and colonization are needed. Based on the concept of targeting the bacteria synergistically on various fronts, it is hypothesized that an electrical insult associated with antibacterial materials may be a highly effective means of killing bacteria. selleckchem In this work, an injectable conductive gel based on silk fibroin (SF) and silver nanoparticles (Ag-NPs) is synthesized, capable of coating a zone of injury, allowing the application of a low electrical current to decrease bacterial contamination. With a high conductivity of 1.5 S cm-1 , SF/Ag-NPs gels killed 80% of Escherichia coli in 1 min, no toxicity toward Chinese hamster ovary cells is observed. The mechanism of an electrical composite gel combined with electrical wound therapy is associated with silver ion (Ag+ ) release, and reactive oxygen species (ROS) production. The findings in the present study show a similar Ag+ release for treatment with gels and the combined effect, whereas ROS generation is 50% higher when a small electrical current is applied leading to a broad bactericidal effect. © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.Despite the avidin/biotin reaction being one of the most ubiquitous noncovalent immobilization and sensing strategies in scientific research, the ability to synthesize useful amounts of biotin-binding fusion constructs is hampered by poor solubility in bacterial expression systems. As such, there are few reports of successful genetic reporter fusions incorporating a biotin-binding partner. To address this, a sensitivity-enhanced, synthetically facile reporter fusion is developed to merge the bioluminescence output of Gaussia luciferase (Gluc) with the recently characterized biotin-binding ability of tamavidin 2 (TA2) for general and universal signaling applications in biological and analytical systems. This fusion construct enables direct bacterial expression of a reporter system incorporating two important functionalities in a 11 stoichiometric relationship that can provide detection of discrete events at low concentrations. Using a cold-shock expression system, highly concentrated construct can be obtained from standard culture volumes while retaining essentially native protein activity. To demonstrate feasibility and provide an example application, this fusion construct is then included in a standard target-bridged assay design for the sensitive detection of four miRNA targets. © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.One well-studied bacterial factor recognized by the host immune system is lipopolysaccharides (LPS) that stimulate host cells, resulting in cell inflammation. Although photobiomodulation (PBM) therapy demonstrates its potency on anti-inflammatory activity, the complete mechanism of action in the host-bacteria interaction model is still elusive. In addition, many studies were performed regarding a distance between the light source and biological sample (non-contact therapy) that may result in disparate reports on the efficacy of PBM therapy. Thus, it is critical to clearly understand the effect of this approach to maximize efficacy and minimize side effects. Here, a custom-built light-emitting diode (LED) platform that mimics near-contact therapy is developed. The effect and mechanism of PBM therapy on epithelial cells in response to LPS is systematically investigated under various conditions (wavelength, irradiation-time, pulse-frequency). The data show that the irradiation of near-infrared (NIR-LED) significantly improves the viability of inflamed cells. It reveals that NIR-LED inhibits the production of reactive oxygen species by regulating the Nox4-NF-κB pathway. Interestingly, however, high-pulse frequency stimulus causes the collapse of the mitochondrial membrane potential (ΔΨm) of cells, resulting in cell death. These results suggest that the optimized "PBM condition" is critical to assist the healthy immune system of the host against bacterial invasion. © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.A number of natural polymer biomaterial-based nerve guidance conduits (NGCs) are developed to facilitate repair of peripheral nerve injuries. Cross-linking ensures mechanical integrity and desired degradation properties of the NGCs; however, common methods such as formaldehyde are associated with cellular toxicity. Hence, there is an unmet clinical need for alternative nontoxic cross-linking agents. In this study, collagen-based NGCs with a collagen/chondroitin sulfate luminal filler are used to study the effect of cross-linking on mechanical and structural properties, degradation, biocompatibility, and immunological response. A simplified manufacturing method of genipin cross-linking is developed, by incorporating genipin into solution prior to freeze-drying the NGCs. This leads to successful cross-linking as demonstrated by higher cross-linking degree and similar tensile strength of genipin cross-linked conduits compared to formaldehyde cross-linked conduits. Genipin cross-linking also preserves NGC macro and microstructure as observed through scanning electron microscopy and spectral analysis.