Mechanisms of pluripotent cell state transitions

Cell state transitions enable the differentiation of stem and progenitor cells into more mature and specialized cell types and are, thus, fundamental to the formation of multicellular organisms. Developmental progression is largely a unidirectional process. However, expression of reprogramming factors is sufficient to de-differentiate mature somatic cells, suggesting that cellular plasticity persists even in terminally differentiated cell types. Multiple signaling pathways, epigenetic regulators, metabolic sensing cascades and transcription factors (TFs) contribute to differentiation and de differentiation. However, if reprogramming requires the reversion of naturally occurring developmental mechanisms remains unknown. A suitable model system to study cell state transitions in vitro are lineage-related mouse embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) which are derivatives of the pre implantation blastocyst and the post-implantation epiblast, respectively. Interconvertibility of ESCs and EpiSCs provides an experimental model to explore to which extent lineage progression and reprogramming overlap mechanistically. In a collaborative project, I contributed to the characterization of a novel ESC differentiation pathway: in a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) screen we identified multiple components of a conserved amino acid signaling pathway as crucial drivers of ESC progression. Mechanistically, the lysosome activity, the Ragulator protein complex, and the tumor-suppressor Folliculin (Flcn) enable the Rag GTPases C and D to bind and seclude the TF Tfe3 in the cytoplasm. Ectopic nuclear Tfe3 represses specific developmental and activates metabolic transcriptional programs which are associated with in vivo development. In collaboration with geneticists, we identified point mutations in a Tfe3 domain required for cytoplasmic inactivation as a potential cause of a human developmental disorder. This work reveals an instructive and biomedically relevant role for metabolic signaling in licensing embryonic cell fate transitions. In my main PhD project, we aimed to identify cell state transition regulators which both are required for exit from the ESC state and inhibit acquisition of the induced pluripotent cell (iPSC) identity upon reprogramming of EpiSCs. We therefore performed a large-scale loss-of-function reprogramming screen in sensitized EpiSCs. Comparison with ESC differentiation screens revealed the constitutively expressed TF Zfp281 as a unique bidirectional regulator of cell state interconversion. We identified the histone methyltransferase Ehmt1 and the zinc finger TF Zic2 as differentiation-specific protein interaction partners of Zfp281 and showed that subtle chromatin binding changes of Zfp281 during ESC progression translate into activation of Ehmt1 and stabilization of Zic2 on promoters and enhancers. Genetic gain- and loss-of function experiments confirmed a critical role of Ehmt1 and Zic2 downstream of Zfp281 both in driving exit from the ESC state, and in restricting reprogramming of EpiSCs. This study reveals that the cell type invariant chromatin association of Zfp281 provides an interaction platform for remodeling the cis-regulatory network underlying cellular plasticity

Die Modellierung des wissenschaftlichen Denkens im Grundschulalter

Ausgehend von neueren entwicklungspsychologischen Forschungsergebnissen zu Basisfähigkeiten im wissenschaftlichen Denken bereits im Grundschulalter wurde ein Kompetenzmodell mit den Komponenten Wissenschaftsverständnis (Verständnis wissenschaftlicher Konzepte und der Ziele von Wissenschaften, Theorieverständnis) und Methodenkompetenz (Experimentierstrategien, Dateninterpretation) aufgestellt sowie ein gruppentestfähiges Testinstrument im schriftlichen Antwortformat entwickelt. Das Ziel dieser Arbeit war die Modellierung der latenten Struktur der Kompetenz wissenschaftliches Denken, die Beschreibung von Entwicklungsveränderungen im Grundschulalter sowie die Untersuchung von Zusammenhängen zwischen wissenschaftlichem Denken und kognitiven Fähigkeiten und motivationalen Orientierungen. In der Studie wurde an 285 Grundschulkindern (72 Zweitklässler, 58 Drittklässler und 155 Viertklässler) wissenschaftliches Denken, Leseverständnis, Intelligenz, Problemlösen, Inhibition, räumliches Denken, formal-operatorische Fähigkeiten, Einstellungen zu Naturwissenschaften, naturwissenschaftsbezogenes Fähigkeitsselbstkonzept sowie Interesse an forschenden Aktivitäten erfasst. Der Vergleich von ein- und mehrdimensionalen Rasch-Modellen ergab eine eindimensionale Struktur der Kompetenz wissenschaftliches Denken mit einer zufrieden stellenden Reliabilität. Zwischen den Teilkompetenzen des wissenschaftlichen Denkens wurden hohe latente Interkorrelationen und bidirektionale Beziehungen ermittelt. Es zeigten sich signifikante Leistungsverbesserungen von der zweiten zur vierten Klassenstufe. In der zweiten Klasse bestanden mehrheitlich Fehlvorstellungen, während in der vierten Klasse schon wissenschaftlich adäquate Vorstellungen vorherrschten. In der dritten Jahrgangsstufe traten markante Entwicklungsveränderungen auf. Innerhalb der Teilkompetenzen des wissenschaftlichen Denkens wurde ein sequenzieller Entwicklungsverlauf von einem Verständnis der Ziele von Wissenschaften über ein Verständnis von Experimentierstrategien und Dateninterpretation hin zu einem Theorieverständnis festgestellt. Darüber hinaus bestanden substantielle Zusammenhänge zu den kognitiven Fähigkeiten, jedoch konnte die Kompetenz wissenschaftliches Denken jeweils als separates Konstrukt abgegrenzt werden. In latenten hierarchischen Regressionsanalysen hatten Leseverständnis und Intelligenz einen signifikanten Einfluss auf die Kompetenz im wissenschaftlichen Denken. Daneben waren Problemlösefähigkeiten von herausragender prädiktiver Bedeutung, in geringerem Ausmaß auch räumliche Fähigkeiten und das naturwissenschaftsbezogene Fähigkeitsselbstkonzept. Besonders im frühen Grundschulalter zeigten sich positive Zusammenhänge zwischen der Kompetenz im wissenschaftlichen Denken und den motivationalen Variablen

DAY HOSPITAL FOR EARLY INTERVENTION FOR INDIVIDUALS WITH PSYCHOTIC DISORDERS

In long-term outcome studies on individuals with first-episode psychosis, improved remission and recovery rates perhaps reflect the improved treatment in dedicated early intervention program. The first episode is a critical period in which individuals with psychosis, as well as members of their families, are confronted with the illness for the first time. Until nowadays, treatment of first psychotic episodes in Croatia has usually been provided in hospital setting. The day hospital provides comprehensive therapeutic approach that refers to early systematic application of all available and effective therapeutic methods in the initial phases of psychotic disorders, and aims to attain and maintain remission and recovery, as well as insight and adherence to treatment. The day hospital is a time-limited structured program that comprises diagnostic procedures, treatment and rehabilitation based on various group psychotherapy and socio-therapy approaches. It is cheaper than hospital treatment and preferred by patients and their families. The importance of involving family members along with patients in the therapeutic process is recognized. The aim of this paper is to present the first day hospital for early intervention and treatment of individuals with psychotic disorder, established within Psychiatric hospital "Sveti Ivan", Zagreb, Croatia

Viral promoters can initiate expression of toxin genes introduced into Escherichia coli

BACKGROUND: The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. RESULTS: We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene. CONCLUSION: According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms

Quantitative Analysis of Protein Dynamics during Asymmetric Cell Division

SummaryIn dividing Drosophila sensory organ precursor (SOP) cells, the fate determinant Numb and its associated adaptor protein Pon localize asymmetrically and segregate into the anterior daughter cell, where Numb influences cell fate by repressing Notch signaling. Asymmetric localization of both proteins requires the protein kinase aPKC and its substrate Lethal (2) giant larvae (Lgl) [1–3]. Because both Numb and Pon localization require actin and myosin [4–6], lateral transport along the cell cortex has been proposed as a possible mechanism for their asymmetric distribution [5]. Here, we use quantitative live analysis of GFP-Pon and Numb-GFP fluorescence and fluorescence recovery after photobleaching (FRAP) to characterize the dynamics of Numb and Pon localization during SOP division. We demonstrate that Numb and Pon rapidly exchange between a cytoplasmic pool and the cell cortex and that preferential recruitment from the cytoplasm is responsible for their asymmetric distribution during mitosis. Expression of a constitutively active form of aPKC impairs membrane recruitment of GFP-Pon. This defect can be rescued by coexpression of nonphosphorylatable Lgl, indicating that Lgl is the main target of aPKC. We propose that a high-affinity binding site is asymmetrically distributed by aPKC and Lgl and is responsible for asymmetric localization of cell-fate determinants during mitosis

TELEPSYCHIATRY IN THE TIME OF THE COVID-19 AND EARTHQUAKE IN ZAGREB AS ODYSSEUS BETWEEN SCYLLA AND CHARYBDIS

In this paper we would like to reveal some of the many challenges during the time of the twofold simultaneous trauma; the COVID-19 pandemic and the devastating earthquake in Zagreb. We described the functioning mode of two Day Hospitals for Early Intervention and Psychotic Disorders at Psychiatric Hospital “Sveti Ivan” during the outbreak of the COVID-19 pandemic. We tried to find ways to ensure the necessary continuation of treatment and to provide continuity in times of uncertainty. The vulnerable group of people treated for mental illnesses faced exceptional psychological demands and was in need for care in these moments. Telemedicine, more specifically telepsychiatry, through online therapy and telephone communication made it possible for people treated for psychotic disorders not to feel isolated and rejected. Health care professionals, in a state of distress themselves, should understand, support, be able to reduce anxiety, and provide stability and constancy. The psychotherapeutic approach and the capacity for mentalization allowed us to turn challenges into opportunities. Rapid changes without delay extended our scope of practice in these extraordinarily difficult times

Patients with Cancer and Family Caregivers: Management of symptoms caused by Cancer or Cancer Therapy at Home

People are diagnosed with cancer sooner nowadays thanks to increased awareness and improvements in cancer screenings. Patients are able to live longer due to cancer treatment regimens; however, they suffer the consequences of living with cancer and therapy-related symptoms. Symptom management is challenging for both patients and family caregivers. Therefore, family members must be integrated in the patient’s care plan. For this review, a literature search was conducted to determine what types of interventions were available that involved family members of cancer patients with the management of cancer and therapy-related symptoms. The following interventions were found that were designed for the family caregivers or both the patient and caregiver to aide with symptom management: pain intervention program, massage therapy, telephone intervention, self-efficacy improvement, coping enhancement and a multidimensional intervention. A positive effect was noted in all the studies, but several had no significance in the patient intervention group but did in the caregiver intervention group. However, studies indicated decreased symptom intensity for various symptoms, decreased symptom distress for both the patient and caregiver, increased self-efficacy of the family member, and increased satisfaction with certain interventions. Further research should be conducted on both existing interventions to better determine their effect and on family symptom management of cancer patients as they need support from healthcare professionals as well

A Genetic Screen for Dominant Enhancers of the Cell-Cycle Regulator α-Endosulfine Identifies Matrimony as a Strong Functional Interactor in Drosophila

The coordination of cell-cycle events with developmental processes is essential for the reproductive success of organisms. In Drosophila melanogaster, meiosis is tightly coupled to oocyte development, and early embryos undergo specialized S-M mitoses that are supported by maternal products. We previously showed that the small phosphoprotein α-endosulfine (Endos) is required for normal oocyte meiotic maturation and early embryonic mitoses in Drosophila. In this study, we performed a genetic screen for dominant enhancers of endos00003 and identified several genomic regions that, when deleted, lead to impaired fertility of endos00003/+ heterozygous females. We uncovered matrimony (mtrm), which encodes a Polo kinase inhibitor, as a strong dominant enhancer of endos. mtrm126 +/+ endos00003 females are sterile because of defects in early embryonic mitoses, and this phenotype is reverted by removal of one copy of polo. These results provide compelling genetic evidence that excessive Polo activity underlies the strong functional interaction between endos00003 and mtrm126. Moreover, we show that endos is required for the increased expression of Mtrm in mature oocytes, which is presumably loaded into early embryos. These data are consistent with the model that maternal endos antagonizes Polo function in the early embryo to ensure normal mitoses through its effects on Mtrm expression during late oogenesis. Finally, we also identified genomic deletions that lead to loss of viability of endos00003/+ heterozygotes, consistent with recently published studies showing that endos is required zygotically to regulate the cell cycle during development