Emc1 is essential for vision and zebrafish photoreceptor outer segment morphogenesis.

Inherited retinal diseases (IRDs) are a rare group of eye disorders characterized by progressive dysfunction and degeneration of retinal cells. In this study, we characterized the raifteirí (raf) zebrafish, a novel model of inherited blindness, identified through an unbiased ENU mutagenesis screen. A mutation in the largest subunit of the endoplasmic reticulum membrane protein complex, emc1 was subsequently identified as the causative raf mutation. We sought to elucidate the cellular and molecular phenotypes in the emc1-/- knockout model and explore the association of emc1 with retinal degeneration. Visual behavior and retinal electrophysiology assays demonstrated that emc1-/- mutants had severe visual impairments. Retinal histology and morphometric analysis revealed extensive abnormalities, including thinning of the photoreceptor layer, in addition to large gaps surrounding the lens. Notably, photoreceptor outer segments were drastically smaller, outer segment protein expression was altered and hyaloid vasculature development was disrupted. Transcriptomic profiling identified cone and rod-specific phototransduction genes significantly downregulated by loss of emc1. These data shed light on why emc1 is a causative gene in inherited retinal disease and how outer segment morphogenesis is regulated.

Novel marine metalloprotease-new approaches for inhibition of biofilm formation of Stenotrophomonas maltophilia.

Many marine organisms produce bioactive molecules with unique characteristics to survive in their ecological niches. These enzymes can be applied in biotechnological processes and in the medical sector to replace aggressive chemicals that are harmful to the environment. Especially in the human health sector, there is a need for new approaches to fight against pathogens like Stenotrophomonas maltophilia which forms thick biofilms on artificial joints or catheters and causes serious diseases. Our approach was to use enrichment cultures of five marine resources that underwent sequence-based screenings in combination with deep omics analyses in order to identify enzymes with antibiofilm characteristics. Especially the supernatant of the enrichment culture of a stony coral caused a 40% reduction of S. maltophilia biofilm formation. In the presence of the supernatant, our transcriptome dataset showed a clear stress response (upregulation of transcripts for metal resistance, antitoxins, transporter, and iron acquisition) to the treatment. Further investigation of the enrichment culture metagenome and proteome indicated a series of potential antimicrobial enzymes. We found an impressive group of metalloproteases in the proteome of the supernatant that is responsible for the detected anti-biofilm effect against S. maltophilia. KEY POINTS: • Omics-based discovery of novel marine-derived antimicrobials for human health management by inhibition of S. maltophilia • Up to 40% reduction of S. maltophilia biofilm formation by the use of marine-derived samples • Metalloprotease candidates prevent biofilm formation of S. maltophilia K279a by up to 20.

The basic helix-loop-helix transcription factor TCF4 impacts brain architecture as well as neuronal morphology and differentiation.

Germline mutations in the basic helix-loop-helix transcription factor 4 (TCF4) cause the Pitt-Hopkins syndrome (PTHS), a developmental disorder with severe intellectual disability. Here, we report findings from a new mouse model with a central nervous system-specific truncation of Tcf4 leading to severe phenotypic abnormalities. Furthermore, it allows the study of a complete TCF4 knockout in adult mice, circumventing early postnatal lethality of previously published mouse models. Our data suggest that a TCF4 truncation results in an impaired hippocampal architecture affecting both the dentate gyrus as well as the cornu ammonis. In the cerebral cortex, loss of TCF4 generates a severe differentiation delay of neural precursors. Furthermore, neuronal morphology was critically affected with shortened apical dendrites and significantly increased branching of dendrites. Our data provide novel information about the role of Tcf4 in brain development and may help to understand the mechanisms leading to intellectual deficits observed in patients suffering from PTHS.

Preclinical toxicity analyses of lentiviral vectors expressing the HIV-1 LTR-specific designer-recombinase Brec1.

Drug-based antiretroviral therapies (ART) efficiently suppress HIV replication in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. Importantly, ART cannot eliminate HIV from an infected individual, since it does not target the integrated provirus. Therefore, genome editing-based strategies that can inactivate or excise HIV genomes would provide the technology for novel curative therapies. In fact, the HIV-1 LTR-specific designer-recombinase Brec1 has been shown to remove integrated proviruses from infected cells and is highly efficacious on clinical HIV-1 isolates in vitro and in vivo, suggesting that Brec1 has the potential for clinical development of advanced HIV-1 eradication strategies in people living with HIV. In line with the preparation of a first-in-human advanced therapy medicinal product gene therapy trial, we here present an extensive preclinical evaluation of Brec1 and lentiviral vectors expressing the Brec1 transgene. This included detailed functional analysis of potential genomic off-target sites, assessing vector safety by investigating vector copy number (VCN) and the risk for potential vector-related insertional mutagenesis, as well as analyzing the potential of Brec1 to trigger an undesired strong T cell immune response. In conclusion, the antiviral designer-recombinase Brec1 is shown to lack any detectable cytopathic, genotoxic or T cell-related immunogenic effects, thereby meeting an important precondition for clinical application of the therapeutic lentiviral vector LV-Brec1 in novel HIV-1 curative strategies.

Cancer-Associated Fibroblasts Exert Proangiogenic Activity in Merkel Cell Carcinoma.

The tumor microenvironment is a complex niche enveloping a tumor formed by extracellular matrix, blood vessels, immune cells, and fibroblasts constantly interacting with cancer cells. Although tumor microenvironment is increasingly recognized as a major player in cancer initiation and progression in many tumor types, its involvement in Merkel cell carcinoma (MCC) pathogenesis is currently unknown. In this study, we provide a molecular and functional characterization of cancer-associated fibroblasts (CAFs), the major tumor microenvironment component, in patient-derived xenografts of patients with MCC. We show that subcutaneous coinjection of patient-derived CAFs and human MCC MKL-1 cells into severe combined immunodeficient mice significantly promotes tumor growth and metastasis. These fast-growing xenografts are characterized by areas densely populated with human CAFs, mainly localized around blood vessels. We provide evidence that the growth-promoting activity of MCC-derived CAFs is mediated by the aminopeptidase A/angiotensin II and III/angiotensin II type 1 receptor axis, with the expression of aminopeptidase A in CAFs being a triggering event. Together, our findings point to aminopeptidase A as a potential marker for MCC prognostic stratification and as a candidate for therapeutic intervention.

Merkel Cell Carcinoma and Immune Evasion: Merkel Cell Polyomavirus Small T-Antigen‒Induced Surface Changes Can Be Reverted by Therapeutic Intervention.

Merkel cell polyomavirus is the causative agent for most Merkel cell carcinomas (MCCs). This highly aggressive skin cancer shows rapid progression, with metastasis being a significant challenge for patient therapy. Virus-positive MCCs show low mutation rates, and tumor cell proliferation is dependent on viral oncoproteins small T antigen (sT) and large T antigen. Although the role of sT and large T antigen in early events of tumorigenesis has been extensively studied, their role in tumor progression has been scarcely addressed. In this study, we investigate the possible mechanisms of how Merkel cell polyomavirus oncoproteins, particularly sTs, contribute to metastasis. We show that sT specifically affects selectin ligand binding and processing by altering the presentation of multiple MCC surface molecules, thereby influencing initial metastasis events and tumor cell immune recognition. Furthermore, we show that sT regulates the surface antigen CD47, which inhibits phagocytosis by macrophages. By applying either sT short hairpin RNAs, CD47-targeted small interfering RNAs, or a therapeutic anti-CD47 antibody, we show that immune recognition of MCC cells can be restored. Thus, CD47 is a promising therapeutic target on MCC cells. Blocking the CD47‒SIRPα interaction effectively promotes phagocytosis of MCC cells and might be a promising combinatorial immunotherapy approach together with PD-1/PD-L1 axis in MCC treatment.

Epstein Barr virus-mediated transformation of B cells from XIAP-deficient patients leads to increased expression of the tumor suppressor CADM1.

X-linked lymphoproliferative disease (XLP) is either caused by loss of the SLAM-associated protein (SAP; XLP-1) or the X-linked inhibitor of apoptosis (XIAP; XLP-2). In both instances, infection with the oncogenic human Epstein Barr virus (EBV) leads to pathology, but EBV-associated lymphomas only emerge in XLP-1 patients. Therefore, we investigated the role of XIAP during B cell transformation by EBV. Using humanized mice, IAP inhibition in EBV-infected mice led to a loss of B cells and a tendency to lower viral titers and lymphomagenesis. Loss of memory B cells was also observed in four newly described patients with XIAP deficiency. EBV was able to transform their B cells into lymphoblastoid cell lines (LCLs) with similar growth characteristics to patient mothers' LCLs in vitro and in vivo. Gene expression analysis revealed modest elevated lytic EBV gene transcription as well as the expression of the tumor suppressor cell adhesion molecule 1 (CADM1). CADM1 expression on EBV-infected B cells might therefore inhibit EBV-associated lymphomagenesis in patients and result in the absence of EBV-associated malignancies in XLP-2 patients.

Host KIR/HLA-C Genotypes Determine HIV-Mediated Changes of the NK Cell Repertoire and Are Associated With Vpu Sequence Variations Impacting Downmodulation of HLA-C.

NK cells play a pivotal role in viral immunity, utilizing a large array of activating and inhibitory receptors to identify and eliminate virus-infected cells. Killer-cell immunoglobulin-like receptors (KIRs) represent a highly polymorphic receptor family, regulating NK cell activity and determining the ability to recognize target cells. Human leukocyte antigen (HLA) class I molecules serve as the primary ligand for KIRs. Herein, HLA-C stands out as being the dominant ligand for the majority of KIRs. Accumulating evidence indicated that interactions between HLA-C and its inhibitory KIR2DL receptors (KIR2DL1/L2/L3) can drive HIV-1-mediated immune evasion and thus may contribute to the intrinsic control of HIV-1 infection. Of particular interest in this context is the recent observation that HIV-1 is able to adapt to host HLA-C genotypes through Vpu-mediated downmodulation of HLA-C. However, our understanding of the complex interplay between KIR/HLA immunogenetics, NK cell-mediated immune pressure and HIV-1 immune escape is still limited. Therefore, we investigated the impact of specific KIR/HLA-C combinations on the NK cell receptor repertoire and HIV-1 Vpu protein sequence variations of 122 viremic, untreated HIV-1+ individuals. Compared to 60 HIV-1- controls, HIV-1 infection was associated with significant changes within the NK cell receptor repertoire, including reduced percentages of NK cells expressing NKG2A, CD8, and KIR2DS4. In contrast, the NKG2C+ and KIR3DL2+ NK cell sub-populations from HIV-1+ individuals was enlarged compared to HIV-1- controls. Stratification along KIR/HLA-C genotypes revealed a genotype-dependent expansion of KIR2DL1+ NK cells that was ultimately associated with increased binding affinities between KIR2DL1 and HLA-C allotypes. Lastly, our data hinted to a preferential selection of Vpu sequence variants that were associated with HLA-C downmodulation in individuals with high KIR2DL/HLA-C binding affinities. Altogether, our study provides evidence that HIV-1-associated changes in the KIR repertoire of NK cells are to some extent predetermined by host KIR2DL/HLA-C genotypes. Furthermore, analysis of Vpu sequence polymorphisms indicates that differential KIR2DL/HLA-C binding affinities may serve as an additional mechanism how host genetics impact immune evasion by HIV-1.

ACTN2 Mutant Causes Proteopathy in Human iPSC-Derived Cardiomyocytes.

Genetic variants in α-actinin-2 (ACTN2) are associated with several forms of (cardio)myopathy. We previously reported a heterozygous missense (c.740C>T) ACTN2 gene variant, associated with hypertrophic cardiomyopathy, and characterized by an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we created with CRISPR/Cas9 genetic tools two heterozygous functional knock-out hiPSC lines with a second wild-type (ACTN2wt) and missense ACTN2 (ACTN2mut) allele, respectively. We evaluated their impact on cardiomyocyte structure and function, using a combination of different technologies, including immunofluorescence and live cell imaging, RNA-seq, and mass spectrometry. This study showed that ACTN2mut presents a higher percentage of multinucleation, protein aggregation, hypertrophy, myofibrillar disarray, and activation of both the ubiquitin-proteasome system and the autophagy-lysosomal pathway as compared to ACTN2wt in 2D-cultured hiPSC-CMs. Furthermore, the expression of ACTN2mut was associated with a marked reduction of sarcomere-associated protein levels in 2D-cultured hiPSC-CMs and force impairment in engineered heart tissues. In conclusion, our study highlights the activation of proteolytic systems in ACTN2mut hiPSC-CMs likely to cope with ACTN2 aggregation and therefore directs towards proteopathy as an additional cellular pathology caused by this ACTN2 variant, which may contribute to human ACTN2-associated cardiomyopathies.

Transcriptional behavior of the HIV-1 promoter in context of the BACH2 prominent proviral integration gene.

Chronic infection with human immunodeficiency virus (HIV)-1 is characterized by accumulation of proviral sequences in the genome of target cells. Integration of viral DNA in patients on long-term antiretroviral therapy selectively persists at preferential loci, suggesting site-specific crosstalk of viral sequences and human genes. This crosstalk likely contributes to chronic HIV disease through modulation of host immune pathways and emergence of clonal infected cell populations. To systematically interrogate such effects, we undertook genome engineering to generate Jurkat cell models that replicate integration of HIV-1 long terminal repeat (LTR) sequences at the BTB and CNC Homolog 2 (BACH2) integration locus. This locus is a prominent HIV-1 integration gene in chronic infection, found in 30 % of long-term treated patients with mapped proviral integrations. Using five clonal models carrying an LTR-driven reporter at different BACH2 intergenic regions, we here show that LTR transcriptional activity is repressed in BACH2 regions associated with proviral-DNA integrations in vivo but not in a control region. Our data indicates that this repression is in part epigenetically regulated, particularly through DNA methylation. Importantly, we demonstrate that transcriptional activity of the LTR is independent of BACH2 gene transcription and vice versa in our models. This suggests no transcriptional interference of endogenous and HIV-1 promoters. Taken together, our study provides first insights into how activity of HIV-1 LTR sequences is regulated at the BACH2 locus as prominent example for a recurrently-detected integration gene in chronic infection. Given the importance of integration-site dependent virus/host crosstalk for chronic HIV disease, our findings for the BACH2 locus have potential implications for future therapeutic strategies.

Mutation of a serine near the catalytic site of the choline acetyltransferase a gene almost completely abolishes motility of the zebrafish embryo.

In zebrafish, the gene choline acetyltransferase a (chata) encodes one of the two ChAT orthologs responsible for the synthesis of acetylcholine. Acetylcholine (ACh) is essential for neuromuscular transmission and its impaired synthesis by ChAT can lead to neuromuscular junction disorders such as congenital myasthenic syndromes in humans. We have identified a novel mutation in the chata gene of zebrafish, chatatk64, in a collection of uncharacterised ENU-induced mutants. This mutant carries a missense mutation in the codon of a highly conserved serine changing it to an arginine (S102R). This serine is conserved among ChATs from zebrafish, rat, mice and chicken to humans. It resides within the catalytic domain and in the vicinity of the active site of the enzyme. However, it has not been reported so far to be required for enzymatic activity. Modelling of the S102R variant change in the ChAT protein crystal structure suggests that the change affects protein structure and has a direct impact on the catalytic domain of the protein which abolishes embryo motility almost completely.