Banks gauge and verify business potential when processing small business loan applications by conducting due diligence on the firm. Although research has examined the factors that influence small business financing, little attention has been given to understanding the due diligence process that informs credit decision making. We address this gap using signal theory to analyze and develop insights from 24 qualitative interviews. Our study identified 11 key hard and soft signals loan officers use to access small business credit risk during due diligence. The key signals include key person risk, change in leadership risk, articulation of company value, adaptation to change, separating business and personal finances, personal reputation, transparency and willingness to share information, data accuracy and completeness, regular account operation, faulty budget and turnover assumptions, and legitimacy of collateral. The implications for small and medium-sized enterprises finance theory and practice are discussed.

The backdated research dedicated to digital entrepreneurship education is immense, which makes it difficult to create an overview. Conversely, forward-thinking bibliometric visualization mapping and clustering can assist in visualizing and structuring difficult research literature. Hence, the goal of this mapping visualization study is to thoroughly discover and create clusters of EE to convey a taxonomic structure that can oblige as a basis for upcoming research. The analyzed data, which is drawn from Google Scholar through Publish or Perish tool, contain 1000 documents published between 2007 and 2022. This taxonomy should generate stronger bonds with digital entrepreneurial education research; on the other, it should stand in international research association to boost both interdisciplinary digital entrepreneurial education and its influence on a universal basis. This work strengthens student’s understanding of current digital entrepreneurial education research by classifying and decontaminating the most powerful knowledgeable relationship among its contributions and contributors. The bibliographic analysis includes ‘citation network’, ‘author’s research area’ and ‘paper content’ regarding the desired topic. In this paper, the above three mentioned terms are integrated which produces a bibliographic model of authors, titles of their papers, keywords and abstract by using Harzing’s Publish or Perish tool for extracting data from Google Scholar and further using VOSViewer to visualize networking map of co-authorship and term co-occurrence to administer the data for an instinctive and appropriate understanding of university students concerning ‘digital entrepreneurial intention’ research. This paper uses bibliometric analysis to analyze the keyword co-occurrence and co-authorship and VOSViewer is used for visualization.

This article explores the opportunities, challenges, as well as the activities of the Chinese governmental and commercial stakeholders to promote cross-border e-commerce trade between China and Africa, based on the classification and correlation analysis of the literature from 2011 to 2019. The results show that the biggest driver for the development of China-Africa cross-border e-commerce trade is the gap between the rapid growth of the African population, especially the middle class, and the limited local capability to satisfy their demand. The rapid development of the internet and mobile internet is another driving factor. The biggest challenge is the last mile delivery of logistics, and online payment issues in Africa. At the macro-level the Chinese government has promoted measures such as infrastructure investment, e-commerce test zones and the establishment of pilot projects. At the firm level, Chinese companies have focused on solving practical micro-level local operational problems such as logistics, online payment, and talent training. The results also show that the referred literature is still in its infancy, mostly theoretical and less practical, and requires more in-depth domain specific analysis in the future.

Until recently, studies regarding e-banking transactions have focused more on motivational factors that trigger the intention to accept and use the e-banking transaction, rather than the de-motivational factors that propel the action. However, in the developing countries like Sub-Sahara economies, the factors associated with the former have not been explored and are still rudimentary in the literature. Drawing from the Technology Threat Avoidance Theory (TTAT), the study seeks to examine the impact of online identity theft on customers' willingness to engage in e-banking transactions in Ghana. A quantitative survey of 393 valid responses from retail bank customers amongst two leading commercial banks in Ghana for the analyses. Results from the PLS-SEM showed that the research constructs; perceived online identity theft' positively and significantly predict "fear of financial loss", "fear of reputational damage", and "security and privacy concern" whilst the former has a negative mediated-relationship between perceived online identity theft and the intention to engage in e-banking transaction. This study is the first of its kind that has extended the application of the TTAT framework into the study of e-banking transactions. The study serves as a practical tool that will enable the banks in their quest to assess customers' restriction/aversion towards the use of Fintech while ensuring sustainable growth of e-banking transactions in an emerging economy context. The study is limited to only banking institutions in Ghana without considering other players in the financial sub-sector. Future research direction has been suggested in the concluding part of the paper.

Researchers have long pondered on the online banking transaction adoption. Some of these studies focus primarily on the motivating factors that affect customers’ intention to adopt/accept these services (technologies). However, research into the constraining factors, in particular socio-economic factors, barely exist in the literature, especially in the context of sub-Saharan Africa. Against this background, the paper seeks to fill in this gap by: (1) assessing the socio-economic factors impeding the engagement of e-banking transactions among retail bank customers in Ghana, and (2) examining the moderating effect of ‘customer experience of Internet’ on the identified factors that inhibit the engagement in online banking in Ghana. The paper used a quantitative research approach to obtain data from two leading Ghanaian banks. Out of the 450 questionnaires distributed, 393 were valid for analysis. Data were analyzed with the aid of PLS-SEM (partial least squares and structural equation modeling). Findings revealed that perceived knowledge gap and the price of digital devices were directly important to the intention to disembark on e-banking transactions among Ghanaian bank customers. Whilst customer experience (frequent use of the Internet), as a moderator variable, has a significant effect on the interaction between perceived knowledge gap and the intent to disembark on e-banking transactions; and finance charges and the intent to disembark on e-banking transactions. Study implications and directions for future research are discussed in the paper. AcknowledgmentThis work was supported by the Internal Grant Agency of Tomas Bata University under the Projects no. IGA/FaME/2019/008 and IGA/FaME/2020/002. The authors would like to extend their appreciation to Prof. Boris Popesko (Vice-dean for Research and Business Liaison) at the Faculty of Management and Economics for facilitating the financial readiness of this project.

In this contribution we present the concept for creating an “International Chair” position at a German University of Applied Sciences and our experiences in its first implementation. Our primary goal was to increase the diversity of the university’s teaching personalities and enrich student education by including content, methods, examples and experiences from other cultures. This gives students an international and intercultural learning experience that is otherwise only acquired through studying abroad. We conclude that the International Chair is a valuable and powerful university tool for increasing international exposure to the departments, their staff and students.

Over the years, Chinese hamster ovary (CHO) cells have emerged as the major host for expressing biotherapeutic proteins. Traditional methods to generate high-producer cell lines rely on random integration(s) of the gene of interest but have thereby left the identification of bottlenecks as a challenging task. For comparison of different producer cell lines derived from various transfections, a system that provides control over transgene expression behavior is highly needed. This motivated us to develop a novel “DUKX-B11 F3/F” cell line to target different single-chain antibody fragments into the same chromosomal target site by recombinase-mediated cassette exchange (RMCE) using the flippase (FLP)/FLP recognition target (FRT) system. The RMCE-competent cell line contains a gfp reporter fused to a positive/negative selection system flanked by heterospecific FRT (F) variants under control of an external CMV promoter, constructed as “promoter trap”. The expression stability and FLP accessibility of the tagged locus was demonstrated by successive rounds of RMCE. As a proof of concept, we performed RMCE using cassettes encoding two different anti-HIV single-chain Fc fragments, 3D6scFv-Fc and 2F5scFv-Fc. Both targeted integrations yielded homogenous cell populations with comparable intracellular product contents and messenger RNA (mRNA) levels but product related differences in specific productivities. These studies confirm the potential of the newly available “DUKX-B11 F3/F” cell line to guide different transgenes into identical transcriptional control regions by RMCE and thereby generate clones with comparable amounts of transgene mRNA. This new host is a prerequisite for cell biology studies of independent transfections and transgenes.

Methods for generating induced pluripotent stem cells (iPSCs) for disease modeling and cell therapies have progressed from integrating vectors to transient delivery of reprogramming factors, avoiding permanent genomic modification. A major limitation of unmodified iPSCs is the assessment of their distribution and contribution to adverse reactions in autologous cell therapy. Here, we report that polycistronic lentiviral vectors with single Flp recombinase (Flp) recognition target (FRT) sites can be used to generate murine iPSCs that are devoid of the reprogramming cassette but carry an intergenic 300-bp long terminal repeat sequence. Performing quantitative polymerase chain reaction on this marker, we could determine genetic identity and tissue contribution of iPSC-derived teratomas in mice. Moreover, we generated iPSCs carrying heterospecific FRT twin sites, enabling excision and recombinase-mediated cassette exchange (RMCE) of the reprogramming cassette for another expression unit of choice. Following screening of iPSCs for "safe harbor" integration sites, expression cassettes were introduced by RMCE into various previously silenced loci of selected single-copy iPSCs. Analysis of DNA methylation showed that RMCE reverted the local epigenetic signature, which allowed transgene expression in undifferentiated iPSCs and in differentiated progeny. These findings support the concept of creating clonotypically defined exchangeable and traceable pluripotent stem cells for disease research and cell therapy.

Where possible, developments enabling the establishment of cell lines with predictable, long-term stable expression capacity are based on single-copy integrations at safe genomic loci with predictable properties. Robust performance could be assigned to lentiviral transduction systems anchoring single LV-units at sites with adequate transcription potential. In the case of gene therapeutic vectors it is essential that the expression interval can be safely terminated following individual requirements, which has mostly been achieved by lox-mediated excision ("floxing"). To extend the spectrum of possible applications we replaced the common, phage-derived Cre/loxP-setup by modules derived from the yeast "Flp/FRT" site-specific recombination system. This change enables a variety of additional options, for instance by "multiplexing" strategies, which rely on a variety of heterospecific FRT-site variants (F'). If we provide lentiviral LTRs with a "twin-site", here an FF3 fusion, the presence of Flp-recombinase will effectively excise the expression cassette, leaving behind a single neutral, genomically anchored FF3 unit. This tag serves to identify the integration locus and to apply sequence- and structural (SIDD-) analyses to predict its functions. Candidate loci are then used to accommodate, at the given site, other genes of interest by "Recombinase-Mediated Twin Site Targeting" (RMTT), a contemporary extension of existing cassette exchange (RMCE-) routines. Supported by the fact that FF3 twins remain accessible within the host genome, RMTT provides access to certified cell lines as it complies with recently defined stringent genomic safe harbor criteria. Our discussion- and outlook-sections will cover lentiviral targeting strategies and current possibilities to enable their fine-tuning.

Starting in 1991, the advance of Tyr-recombinases Flp and Cre enabled superior strategies for the predictable insertion of transgenes into compatible target sites of mammalian cells. Early approaches suffered from the reversibility of integration routes and the fact that co-introduction of prokaryotic vector parts triggered uncontrolled heterochromatization. Shortcomings of this kind were overcome when Flp-Recombinase Mediated Cassette Exchange entered the field in 1994. RMCE enables enhanced tag-and-exchange strategies by precisely replacing a genomic target cassette by a compatible donor construct. After “gene swapping” the donor cassette is safely locked in, but can nevertheless be re-mobilized in case other compatible donor cassettes are provided (“serial RMCE”). These features considerably expand the options for systematic, stepwise genome modifications. The first decade was dominated by the systematic generation of cell lines for biotechnological purposes. Based on the reproducible expression capacity of the resulting strains, a comprehensive toolbox emerged to serve a multitude of purposes, which constitute the first part of this review. The concept per se did not, however, provide access to high-producer strains able to outcompete industrial multiple-copy cell lines. This fact gave rise to systematic improvements, among these certain accumulative site-specific integration pathways. The exceptional value of RMCE emerged after its entry into the stem cell field, where it started to contribute to the generation of induced pluripotent stem (iPS-) cells and their subsequent differentiation yielding a variety of cell types for diagnostic and therapeutic purposes. This topic firmly relies on the strategies developed in the first decade and can be seen as the major ambition of the present article. In this context an unanticipated, potent property of serial Flp-RMCE setups concerns the potential to re-open loci that have served to establish the iPS status before the site underwent the obligatory silencing process. Other relevant options relate to the introduction of composite Flp-recognition target sites (“heterospecific FRT-doublets”), into the LTRs of lentiviral vectors. These “twin sites” enhance the safety of iPS re-programming and -differentiation as they enable the subsequent quantitative excision of a transgene, leaving behind a single “FRT-twin”. Such a strategy combines the established expression potential of the common retro- and lentiviral systems with options to terminate the process at will. The remaining genomic tag serves to identify and characterize the insertion site with the goal to identify genomic “safe harbors” (GOIs) for re-use. This is enabled by the capacity of “FRT-twins” to accommodate any incoming RMCE-donor cassette with a compatible design.

Recombinant biotherapeutic proteins such as monoclonal antibodies are mostly produced in Chinese hamster ovary (CHO) cells and pharmaceutical companies are interested in an appropriate platform technology for the development of large-scale production processes. A major aim of our study was therefore to improve the secretion efficiency of a recombinant biotherapeutic antibody by optimizing signal peptides. Reporter molecules such as gaussia and vargula luciferase or secreted alkaline phosphatase are frequently used to this end. In striking contrast, we used a biotherapeutic antibody that was fused to 16 different signal peptides during our study. In this way, the secretion efficiency of the recombinant antibody has been analyzed by transient expression experiments in CHO cell lines. Compared to the control signal peptide, it was not possible to achieve higher efficiencies with signal peptides derived from a variety of species or even natural immunoglobulin G signal peptides. The best results were obtained with natural signal peptides derived from human albumin and human azurocidin. These results were confirmed by fed-batch experiments with stably transfected cell pools, in which cell-specific productivities up to 90 pg cell(-1) day(-1) and product concentrations up to 4 g L(-1) could be determined using the albumin signal peptide. Finally, the applicability of the identified signal peptides for both different antibodies and non-antibody products was demonstrated by transient expression experiments. In conclusion, it was found that signal peptides derived from human albumin and human azurocidin are most appropriate to generate cell lines with clearly improved production rates suitable for commercial purposes in a product-independent manner.

Am 18. November 2009 spielten die Fusballnationalmannschaften Frankreichs und Irlands im Stade de France gegeneinander. Das Hinspiel war fur Frankreich 1:0 ausgegangen, nun stand im Ruckspiel die Entscheidung an, welches Team sich fur die Weltmeisterschaft 2010 in Sudafrika qualifizieren wurde.

Most biotherapeutic drugs are recombinant monoclonal antibodies which are mostly produced in monoclonal cell lines derived from Chinese hamster ovary (CHO) cells. Various clones expressing a monoclonal recombinant antibody were analyzed and a correlation of the antibody concentration and the relative mRNA level of calreticulin (CALR), glucose-regulated protein 78 and 94 kDa (GRP78, GRP94) and spliced X-box binding protein 1 (XPB1) was observed. By means of these results we were motivated to establish a novel selection system based on endoplasmic reticulum (ER) stress, which allows the rapid identification and isolation of high-expressing clones out of a pool mainly consisting of low- and medium-producing cells. Several ER stress responsive elements were tested with the aid of a recombinase mediated cassette exchange (RMCE) procedure. Very surprisingly, only GRP78 reporter constructs were strongly stimulated upon antibody expression. Furthermore we found that GRP78 reporter constructs are very suitable to reflect the level of antibody expression (IgG) in recombinant CHO cells. Based on these results, it is concluded, that the novel ER stress based selection system developed during this study is suitable to identify and isolate clones with a high level of antibody expression.

Evidence is presented for the involvement of the interplay between transcription factor Yin Yang 1 (YY1) and poly(ADP-ribose) polymerase-1 (PARP-1) in the regulation of mouse PARP-1 gene (muPARP-1) promoter activity. We identified potential YY1 binding motifs (BM) at seven positions in the muPARP-1 core-promoter (-574/+200). Binding of YY1 was observed by the electrophoretic supershift assay using anti-YY1 antibody and linearized or supercoiled forms of plasmids bearing the core promoter, as well as with 30 bp oligonucleotide probes containing the individual YY1 binding motifs and four muPARP-1 promoter fragments. We detected YY1 binding to BM1 (-587/-558), BM4 (-348/-319) and a very prominent association with BM7 (+86/+115). Inspection of BM7 reveals overlap of the muPARP-1 translation start site with the Kozak sequence and YY1 and PARP-1 recognition sites. Site-directed mutagenesis of the YY1 and PARP-1 core motifs eliminated protein binding and showed that YY1 mediates PARP-1 binding next to the Kozak sequence. Transfection experiments with a reporter gene under the control of the muPARP-1 promoter revealed that YY1 binding to BM1 and BM4 independently repressed the promoter. Mutations at these sites prevented YY1 binding, allowing for increased reporter gene activity. In PARP-1 knockout cells subjected to PARP-1 overexpression, effects similar to YY1 became apparent; over expression of YY1 and PARP-1 revealed their synergistic action. Together with our previous findings these results expand the PARP-1 autoregulatory loop principle by YY1 actions, implying rigid limitation of muPARP-1 expression. The joint actions of PARP-1 and YY1 emerge as important contributions to cell homeostasis.

Traditional DNA transduction routes used for the modification of cellular genomes are subject to unpredictable alterations, as the cell-intrinsic repair machinery may affect both the integrity of the transgene and the recipient locus. These problems are overcome by recombinase-mediated cassette exchange (RMCE) approaches enabling predictable expression patterns by the nondisruptive insertion of a gene cassette at a pre-characterized genomic locus. The destination is marked by a "tag" consisting of two heterospecific recombination target sites (RTs) at the flanks of a selection marker. Provided on a circular donor vector, an analogous cassette encoding the gene of interest can cleanly replace the resident cassette under the influence of a site-specific recombinase. RMCE was first based on the yeast integrase Flp but had to give way to the originally more active phage-derived Cre enzyme. To be effective, both Tyr-recombinases have to be applied at a considerable concentration, which, in the case of Cre, triggers endonucleolytic activities and therefore cellular toxicity. This review addresses the particularities of both recombination routes depending on the structure of the synaptic complex and on improved integrase and RT variants. While the performance of Flp-RMCE can now firmly rely on optimized Flp variants and multiple sets of functional target sites (FRTs), the Cre system suffers from the promiscuity of its RT mutants, which is explained in molecular terms. At present, RMCE enters applications in the stem cell field. Remarkable efforts are noted in the framework of various mouse mutagenesis programs, which, in their first phase, have targeted virtually all genes and now start to shift their emphasis from gene trapping to gene modification.