CRISPR/Cas9 mediated knockout of microRNAs for precise cell engineering

Recent advances in the development of molecular tools available for cell line engineering has opened avenues for precise modulation of production cell lines to optimize cellular process relevant parameters. Among those, the CRISPR/Cas9 system represents a rapid and straightforward cell line engineering tool which allows for precise gene editing of host genomes to modify relevant signaling pathways. In addition, microRNAs (miRNAs) have proven to serve as versatile molecular tools to improve production cells regarding protein production and growth characteristics. These small non-coding RNA molecules are regulators of gene expression and regulate process relevant cellular pathways as growth, apoptosis, protein expression or stress. With an individual microRNA being able to regulate up to several hundred target genes, the modulation of miRNA expression may mimic simultaneous modification of several hundred target genes. Therefore, miRNAs may offer great potential to serve as targets for CRISPR/Cas9-mediated genome editing, as an individual microRNA can regulate up to several hundred target genes and therefore mimics multiplexing of hundreds of target genes in one single step. While the overexpression of miRNAs to improve the performance of biopharmaceutical production hosts has received major interest in the past years, effects of precise knockout of unfavorable miRNAs in Chinese hamster ovary (CHO) production cells have not been reported yet. Therefore, the current study aimed at applying a novel strategy to increase product yields by enhancing viability and culture longevity of CHO cells using CRISPR/Cas9-mediated deletion of a miRNA causing adverse effects. In a previous high content screen with more than 1000 miRNAs we were able to identify miR-744 as being involved in the regulation of apoptosis. After the apoptosis-inducing capacity of miR-744 in monoclonal antibody-producing CHO-mAb1 cells was revalidated, the genomic precursor sequence of miR-744 was deleted by two sgRNA-Cas9-mediated DNA double-strand breaks flanking the miR-744 gene in the CHO genome. A plasmid encoding both required sgRNAs in a tandem array, Cas9 and GFP was used to allow for simultaneous delivery of all required components and thereby reducing the screening effort for potential knockout (KO) cell lines. After fluorescent-activated cell sorting (FACS) seven putative, clonal miR-744-KO cell lines could be recovered. From these, three were identified and confirmed as miR-744-KOs by sequencing of their miR-744 locus respectively. In a subsequent growth experiment over a period of nine days considerable differences in the investigated parameters viable cell count, viability, apoptosis, necrosis and antibody titers of the miR-744-KO cell lines in comparison to untreated cells and non-targeting sgRNA transfected cells could be observed. Further experiments are conducted to analyze the observed effects. In summary, the present study elucidates the role of microRNAs, which bear adverse effects on production cells, as targets for the novel CRISPR/Cas9 genome editing technique in the context of cell line engineering. This approach contributes to the idea of a rational design of CHO production hosts with controlled modulation of signaling pathways or metabolic characteristics to enable the generation of optimized production cells

Start-up Firms of the self-employed: 9 Case studies

Because of its importance to a dynamic development of the economy, the formation of new firms has been viewed with rapidly increasing interest from the perspectives of practical economic policy at the national, regional and local levels and from the perspectives of private business associations, formal and informal networks. According to statements of the Swiss Federal Office for Vocational Education and Technology (BBT, Bern), there are more than 200 initiatives that aim at encouraging the formation of new firms in Switzerland. However, an accompanying scientific study of these initiatives is lacking. Serious knowledge gaps exist. The present research project attempts to make a contribution, in a systematic way, to fill some of these gaps. It is based on a systematic study of 20 case studies and of a large survey conducted in 1999 of 745 newly founded firms in Switzerland. In this paper the resutls of 9 case studies are presented concerning the following areas: (1) Portrait of firm founders. (2) Success indicators of firm formation. (3) Factors affecting success of firm formation. (4) Relationships between success and success factors.Formation of new firms; new enterprises; small firms; small-to-medium-sized enterprises; self-employed; start-ups; firm foundations; promoting start-ups; firm promotion; private sector promotion; Switzerland

Which aspects facilitate the adherence of patients with low back pain to physiotherapy? : a Delphi study

Background: The effectiveness of physiotherapy to reduce low back pain depends on patient adherence to treatment. Facilitators and barriers to patient adherence are multifactorial and include patient and therapist-related factors. This Delphi study aimed to identify an expert consensus on aspects facilitating the adherence of patients with back pain to physiotherapy. Method: International experts were invited to participate in a three-round standard Delphi survey. The survey contained 49 items (32 original and 17 suggested by experts) which were rated on 5-point Likert scales. The items were assigned to six domains. The consensus level was defined as 60%. Results: Of 38 invited experts, 15 followed the invitation and completed all three rounds. A positive consensus was reached on 62% of the 49 proposed items to facilitate adherence. The highest consensus was achieved in the domains “Influence of biopsychosocial factors” (89%) and “Influence of cooperation between physiotherapists and patients” (79%). Additional important domains were the “Influence of competencies of physiotherapists” (71%) and “Interdisciplinary congruence” (78%). “Administration aspects” and the “Use of digital tools” did not reach expert consensus. Conclusions: Biopsychosocial factors, therapeutic skills, and patient-physiotherapist collaboration should be considered in physiotherapy practice to facilitate adherence in patients with LBP. Future studies should prospectively evaluate the effectiveness of individual or combined identified aspects for their influence on patient adherence in longitudinal study designs

Selbständigerwerbende und ihre jungen Unternehmen: 9 Fallbeispiele

Because of its importance to a dynamic development of the economy, the formation of new firms has been viewed with rapidly increasing interest from the perspectives of practical economic policy at the national, regional and local levels and from the perspectives of private business associations, formal and informal networks. According to statements of the Swiss Federal Office for Vocational Education and Technology (BBT, Bern), there are more than 200 initiatives that aim at encouraging the formation of new firms in Switzerland. However, an accompanying scientific study of these initiatives is lacking. Serious knowledge gaps exist. The present research project attempts to make a contribution, in a systematic way, to fill some of these gaps. It is based on a systematic study of 20 case studies and of a large survey conducted in 1999 of 745 newly founded firms in Switzerland. In this paper the resutls of 9 case studies are presented concerning the following areas: (1) Portrait of firm founders. (2) Success indicators of firm formation. (3) Factors affecting success of firm formation. (4) Relationships between success and success factors

DJI-Kinderbetreuungsreport 2018. Inanspruchnahme und Bedarfe bei Kindern bis 14 Jahre aus Elternperspektive – ein Bundesländervergleich

Mit der DJI-Kinderbetreuungsstudie U15 (KiBS) wurde eine repräsentative Studie aufgelegt, die jährlich im Rahmen der Sozialberichterstattung die Betreuungsbedarfe der Eltern mit Kindern unter 15 Jahren aufzeigt. Das Altersspektrum, über das kontinuierlich berichtet wird, reicht von unter einjährigen Kindern bis hin zu den Schülerinnen und Schülern der Sekundarstufe I. Die Datenbasis für den Kinderbetreuungsreport 2018 bildet die Elternbefragung aus dem Jahr 2017. (DIPF/Orig.

Large-scale screening of HCMV-seropositive blood donors indicates that HCMV effectively escapes from antibodies by cell-associated spread

Immunoglobulins are only moderately effective for the treatment of human cytomegalovirus (HCMV) infections, possibly due to ineffectiveness against cell-associated virus spread. To overcome this limitation, we aimed to identify individuals with exceptional antibodies in their plasma that can efficiently block the cell-associated spread of HCMV. A Gaussia luciferase-secreting mutant of the cell-associated HCMV strain Merlin was generated, and luciferase activity evaluated as a readout for the extent of cell-associated focal spread. This reporter virus-based assay was then applied to screen plasma samples from 8400 HCMV-seropositive individuals for their inhibitory effect, including direct-acting antiviral drugs as positive controls. None of the plasmas reduced virus spread to the level of these controls. Even the top-scoring samples that partially reduced luciferase activity in the screening assay failed to inhibit focal growth when reevaluated with a more accurate, immunofluorescence-based assay. Selected sera with high neutralizing capacity against free viruses were analyzed separately, and none of them prevented the focal spread of three recent clinical HCMV isolates nor reduced the number of particles transmitted, as demonstrated with a fluorescent Merlin mutant. We concluded that donors with cell-to-cell-spread-inhibiting plasma are nonexistent or extremely rare, emphasizing cell-associated spread as a highly efficient immune escape mechanism of HCM

DJI-Kinderbetreuungsreport 2017. Inanspruchnahme und Bedarfe aus Elternperspektive im Bundesländervergleich. Bericht

Der vorliegende Report enthält die zentralen Ergebnisse der DJI-Kinderbetreuungsstudie U15 (2016), einer jährlichen, bundesländerrepräsentativen Elternbefragung zur Betreuung von Kindern im Alter von unter 15 Jahren in Deutschland. Die Studie knüpft an die von 2012 bis 2015 am Deutschen Jugendinstitut durchgeführten KiföG-Länderstudien an. Deren Ziel war es, die Betreuungsbedarfe, bezogen auf institutionelle Kindertagesbetreuung, sowie die aktuelle Betreuungssituation von unter dreijährigen Kindern aus der Perspektive der Eltern auf Länder- und Bundesebene abzubilden. Dabei geht die DJI-Kinderbetreuungsstudie U15 über ihre Vorgängerstudie hinaus, indem sie zum ersten Mal auch Kinder von drei bis unter fünfzehn Jahren in den Fokus nimmt. (DIPF/Orig.

DJI-Kinderbetreuungsreport 2019. Inanspruchnahme und Bedarf aus Elternperspektive im Bundesländervergleich. Bericht

Der Report stellt wesentliche Befunde der DJI-Kinderbetreuungsstudie (KiBS) des Deutschen Jugendinstituts vor. KiBS ist eine durch das Bundesministerium für Familie, Senioren, Frauen und Jugend (BMFSFJ) finanzierte bundeslandrepräsentative Studie, die jährlich im Rahmen der Sozialberichterstattung die Betreuungsbedarfe der Eltern von Kindern ab Geburt und bis zum Ende des Grundschulalters erhebt. [...] Der DJI-Kinderbetreuungsreport greift jedes Jahr ein aktuelles Thema vertiefend auf. In diesem Jahr liegt der Schwerpunkt - mit Blick auf das sogenannte "Gute-Kita-Gesetz" - auf der elterlichen Einschätzung der Qualität der Betreuung. Dabei geht es bei den kleineren Kindern z. B. um das Vorhandensein pädagogisch qualifizierter Fachkräfte und einer festen Bezugsperson für das Kind. Bei den Schulkindern steht eher im Vordergrund, ob es eine Unterstützung bei der Erledigung der Hausaufgaben gibt und Bildungsangebote jenseits des Schulstoffs existieren. Daneben interessiert aber auch, inwiefern die Integration von Kindern unterschiedlicher Herkunft gefördert wird. (Verlag

The origins of redistributive policy preferences: political socialisation with and without a welfare state

Research on the impact of the macroeconomy on individual-level preferences for redistribution has produced varying results. This paper presents a new theory on the presence of an expansive welfare state during one’s formative years as a source of heterogeneity in the effect that macroeconomic conditions have on individuals’ preferences for redistributive policy. This theory is tested using cohort analysis via the British Social Attitudes surveys (1983–2010), with generations coming of age between the end of World War I and today. Findings confirm that cohorts that were socialised before and after the introduction of the welfare state react differently to economic crises: the former become less supportive of redistribution, while the latter become more supportive. The research sheds light on the long-term shifts of support for the welfare state due to generational replacement

Symptoms and Needs of Patients with Advanced Lung Cancer: Early Prevalence Assessment

Background: Little is known on symptom burden, psychosocial needs, and perception of prognosis in advanced lung cancer patients at the time of diagnosis, although early assessment is strongly recommended within the setting of daily routine care. Methods: Twelve study sites cross-sectionally assessed symptoms and psychosocial needs of patients suffering from newly diagnosed incurable lung cancer. Assessment comprised NCCN distress thermometer, FACT-L, SEIQoL-Q, PHQ-4, and shortened and modified SCNS-SF-34 questionnaires. Additional prognostic information from both patients and physicians were collected. Results: A total of 208 patients were evaluated. Mean age was 63.6 years, 58% were male, 84% suffered from stage IV lung cancer, and 71% had an ECOG performance status of 0–1. Mean distress level was 5.4 (SD 2.5), FACT-L total score was 86 (21.5), and TOI 50.5 (14.9). PHQ-4 was 4.6 (3.3), and shortened and modified SCNS-SF-34 showed 9 (8.7) unmet needs per patient. According to their physicians’ perspective, 98.1% of patients were reflecting on and 85.2% were accepting incurability, while 26.5% of patients considered the treatment to be of curative intent. Conclusion: Our findings emphasize substantial domains of symptom burden seen in newly diagnosed, incurable lung cancer patients. Oncologists should be aware of these features and address prognostic issues early in the disease trajectory to facilitate opportunities to improve coping, advance care planning, and appropriate integration of palliative care, thus improving quality of life