MYT1L haploinsufficiency in human neurons and mice causes autism-associated phenotypes that can be reversed by genetic and pharmacologic intervention.

MYT1L is an autism spectrum disorder (ASD)-associated transcription factor that is expressed in virtually all neurons throughout life. How MYT1L mutations cause neurological phenotypes and whether they can be targeted remains enigmatic. Here, we examine the effects of MYT1L deficiency in human neurons and mice. Mutant mice exhibit neurodevelopmental delays with thinner cortices, behavioural phenotypes, and gene expression changes that resemble those of ASD patients. MYT1L target genes, including WNT and NOTCH, are activated upon MYT1L depletion and their chemical inhibition can rescue delayed neurogenesis in vitro. MYT1L deficiency also causes upregulation of the main cardiac sodium channel, SCN5A, and neuronal hyperactivity, which could be restored by shRNA-mediated knockdown of SCN5A or MYT1L overexpression in postmitotic neurons. Acute application of the sodium channel blocker, lamotrigine, also rescued electrophysiological defects in vitro and behaviour phenotypes in vivo. Hence, MYT1L mutation causes both developmental and postmitotic neurological defects. However, acute intervention can normalise resulting electrophysiological and behavioural phenotypes in adulthood.

Nuclear envelope rupture and NET formation is driven by PKCα-mediated lamin B disassembly.

The nuclear lamina is essential for the structural integration of the nuclear envelope. Nuclear envelope rupture and chromatin externalization is a hallmark of the formation of neutrophil extracellular traps (NETs). NET release was described as a cellular lysis process; however, this notion has been questioned recently. Here, we report that during NET formation, nuclear lamin B is not fragmented by destructive proteolysis, but rather disassembled into intact full-length molecules. Furthermore, we demonstrate that nuclear translocation of PKCα, which serves as the kinase to induce lamin B phosphorylation and disassembly, results in nuclear envelope rupture. Decreasing lamin B phosphorylation by PKCα inhibition, genetic deletion, or by mutating the PKCα consensus sites on lamin B attenuates extracellular trap formation. In addition, strengthening the nuclear envelope by lamin B overexpression attenuates NET release in vivo and reduces levels of NET-associated inflammatory cytokines in UVB-irradiated skin of lamin B transgenic mice. Our findings advance the mechanistic understanding of NET formation by showing that PKCα-mediated lamin B phosphorylation drives nuclear envelope rupture for chromatin release in neutrophils.

Predicting genotoxicity of viral vectors for stem cell gene therapy using gene expression-based machine learning.

Hematopoietic stem cell gene therapy is emerging as a promising therapeutic strategy for many diseases of the blood and immune system. However, several individuals who underwent gene therapy in different trials developed hematological malignancies caused by insertional mutagenesis. Preclinical assessment of vector safety remains challenging because there are few reliable assays to screen for potential insertional mutagenesis effects in vitro. Here we demonstrate that genotoxic vectors induce a unique gene expression signature linked to stemness and oncogenesis in transduced murine hematopoietic stem and progenitor cells. Based on this finding, we developed the surrogate assay for genotoxicity assessment (SAGA). SAGA classifies integrating retroviral vectors using machine learning to detect this gene expression signature during the course of in vitro immortalization. On a set of benchmark vectors with known genotoxic potential, SAGA achieved an accuracy of 90.9%. SAGA is more robust and sensitive and faster than previous assays and reliably predicts a mutagenic risk for vectors that led to leukemic severe adverse events in clinical trials. Our work provides a fast and robust tool for preclinical risk assessment of gene therapy vectors, potentially paving the way for safer gene therapy trials.

Protein Interactions during the Flavivirus and Hepacivirus Life Cycle.

Protein-protein interactions govern biological functions in cells, in the extracellular milieu, and at the border between cells and extracellular space. Viruses are small intracellular parasites and thus rely on protein interactions to produce progeny inside host cells and to spread from cell to cell. Usage of host proteins by viruses can have severe consequences e.g. apoptosis, metabolic disequilibria, or altered cell proliferation and mobility. Understanding protein interactions during virus infection can thus educate us on viral infection and pathogenesis mechanisms. Moreover, it has led to important clinical translations, including the development of new therapeutic and vaccination strategies. Here, we will discuss protein interactions of members of the Flaviviridae family, which are small enveloped RNA viruses. Dengue virus, Zika virus and hepatitis C virus belong to the most prominent human pathogenic Flaviviridae With a genome of roughly ten kilobases encoding only ten viral proteins, Flaviviridae display intricate mechanisms to engage the host cell machinery for their purpose. In this review, we will highlight how dengue virus, hepatitis C virus, Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus, yellow fever virus, and Zika virus proteins engage host proteins and how this knowledge helps elucidate Flaviviridae infection. We will specifically address the protein composition of the virus particle as well as the protein interactions during virus entry, replication, particle assembly, and release from the host cell. Finally, we will give a perspective on future challenges in Flaviviridae interaction proteomics and why we believe these challenges should be met.

Let's Talk About Sex: Tyrolean Online Study on Reproductive Health Issues.

Introduction: The way pupils and university students talk about sex does not reflect their real understanding of reproductive health issues. Therefore, we developed a survey for pupils and students in Tyrol, Austria, to evaluate the current state of knowledge. Methods: Two questionnaires with 39 items for pupils (n = 369) and 53 items for university students (n = 537) were developed, and an online survey was carried out in Tyrol, Austria, between April and July 2022. A sum score for each correct answer to ten identical items in both questionnaires was used to compare groups (range: 0-11 points), with analysis performed using independent sample t-test, analyses of variance (ANOVAs) and chi-square test. Results: With regards to reproductive health issues and fertility awareness, female vs. male participants (p < 0.001), university students vs. pupils (p < 0.001) and medical students vs. other students (p < 0.001) had greater knowledge. Participants who had already had sexual intercourse showed a broader knowledge of contraception and fertility (p < 0.001).The age at which the decline of female fertility becomes relevant was misstated by the adolescents, who gave a mean age of 42.6 years compared to university students who asserted a mean age of 35.9 years (p < 0.001). Overall, the lowest rate of correct answers was found with respect to emergency contraception (30.7%), while knowledge of contraceptive methods was comparatively high (99.2%). Conclusion: Substantial lack of knowledge of reproductive health issues exists, with differences found between pupils and university students, between genders, and according to field of study. Future health and fertility awareness programs at school and university should focus on gender-specific aspects to prevent unplanned pregnancies, sexually transmitted diseases, and childlessness.

Isolation and Neuronal Reprogramming of Mouse Embryonic Fibroblasts.

Forced expression of specific neuronal transcription factors in mouse embryonic fibroblasts (MEFs) can lead to their direct conversion into functional neurons. Direct neuronal reprogramming has become a powerful tool to characterize individual factors and molecular mechanisms involved in forced and normal neurogenesis and to generate neuronal cell types for in vitro studies. Here we provide a detailed protocol for the isolation of MEFs devoid of neural tissue and their direct reprogramming into functional neurons by overexpression of neuronal reprogramming factors (Ascl1, Brn2, and Myt1l) using lentiviral vectors. This method enables quick and efficient generation of mouse neurons in vitro for versatile functional and mechanistic characterization.

Combining Cell Fate Reprogramming and Protein Engineering to Study Transcription Factor Functions.

Gene expression regulation by transcription factors plays a central role in determining and maintaining cell fate during normal development as well as induced cell fate reprogramming. Induction of cell identity-determining gene regulatory networks by reprogramming factors that act as transcriptional activators is key to induce desired cell fates. Conversely, repression of unwanted genetic programs by transcriptional repressors is equally important to ensure cell fate fidelity. Here we describe engineering techniques to create fusion proteins that allow exploration of the major transcriptional contribution (activation or repression) of specific neuronal reprogramming factors during direct cell fate conversion. This method can be extended to every reprogramming regime to enable the functional categorization of any transcription factor.

The Role of Type III Interferons in Hepatitis C Virus Infection and Therapy.

The human interferon (IFN) response is a key innate immune mechanism to fight virus infection. IFNs are host-encoded secreted proteins, which induce IFN-stimulated genes (ISGs) with antiviral properties. Among the three classes of IFNs, type III IFNs, also called IFN lambdas (IFNLs), are an essential component of the innate immune response to hepatitis C virus (HCV). In particular, human polymorphisms in IFNL gene loci correlate with hepatitis C disease progression and with treatment response. To date, the underlying mechanisms remain mostly elusive; however it seems clear that viral infection of the liver induces IFNL responses. As IFNL receptors show a more restricted tissue expression than receptors for other classes of IFNs, IFNL treatment has reduced side effects compared to the classical type I IFN treatment. In HCV therapy, however, IFNL will likely not play an important role as highly effective direct acting antivirals (DAA) exist. Here, we will review our current knowledge on IFNL gene expression, protein properties, signaling, ISG induction, and its implications on HCV infection and treatment. Finally, we will discuss the lessons learnt from the HCV and IFNL field for virus infections beyond hepatitis C.