Sirtuin 2-mediated deacetylation of cyclin-dependent kinase 9 promotes STAT1 signaling in type I interferon responses.

Type I interferons (IFNs) induce expression of multiple genes that control innate immune responses to invoke both antiviral and antineoplastic activities. Transcription of these interferon-stimulated genes (ISGs) occurs upon activation of the canonical Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathways. Phosphorylation and acetylation are both events crucial to tightly regulate expression of ISGs. Here, using mouse embryonic fibroblasts and an array of biochemical methods including immunoblotting and kinase assays, we show that sirtuin 2 (SIRT2), a member of the NAD-dependent protein deacetylase family, is involved in type I IFN signaling. We found that SIRT2 deacetylates cyclin-dependent kinase 9 (CDK9) in a type I IFN-dependent manner and that the CDK9 deacetylation is essential for STAT1 phosphorylation at Ser-727. We also found that SIRT2 is subsequently required for the transcription of ISGs and for IFN-driven antiproliferative responses in both normal and malignant cells. These findings establish the existence of a previously unreported signaling pathway whose function is essential for the control of JAK-STAT signaling and the regulation of IFN responses. Our findings suggest that targeting sirtuin activities may offer an avenue in the development of therapies for managing immune-related diseases and cancer.

Mitochondria and the dynamic control of stem cell homeostasis.

The maintenance of cellular identity requires continuous adaptation to environmental changes. This process is particularly critical for stem cells, which need to preserve their differentiation potential over time. Among the mechanisms responsible for regulating cellular homeostatic responses, mitochondria are emerging as key players. Given their dynamic and multifaceted role in energy metabolism, redox, and calcium balance, as well as cell death, mitochondria appear at the interface between environmental cues and the control of epigenetic identity. In this review, we describe how mitochondria have been implicated in the processes of acquisition and loss of stemness, with a specific focus on pluripotency. Dissecting the biological functions of mitochondria in stem cell homeostasis and differentiation will provide essential knowledge to understand the dynamics of cell fate modulation, and to establish improved stem cell-based medical applications.

Concise Review: Induced Pluripotent Stem Cell-Based Drug Discovery for Mitochondrial Disease.

High attrition rates and loss of capital plague the drug discovery process. This is particularly evident for mitochondrial disease that typically involves neurological manifestations and is caused by nuclear or mitochondrial DNA defects. This group of heterogeneous disorders is difficult to target because of the variability of the symptoms among individual patients and the lack of viable modeling systems. The use of induced pluripotent stem cells (iPSCs) might significantly improve the search for effective therapies for mitochondrial disease. iPSCs can be used to generate patient-specific neural cell models in which innovative compounds can be identified or validated. Here we discuss the promises and challenges of iPSC-based drug discovery for mitochondrial disease with a specific focus on neurological conditions. We anticipate that a proper use of the potent iPSC technology will provide critical support for the development of innovative therapies against these untreatable and detrimental disorders. Stem Cells 2017;35:1655-1662.

Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9.

Human induced pluripotent stem cells (hiPSCs) represent an ideal in vitro platform to study human genetics and biology. The recent advent of programmable nucleases makes also the human genome amenable to experimental genetics through either the correction of mutations in patient-derived iPSC lines or the de novo introduction of mutations into otherwise healthy iPSCs. The production of specific and sometimes complex genotypes in multiple cell lines requires efficient and streamlined gene editing technologies. In this article we provide protocols for gene editing in hiPSCs. We presently achieve high rates of gene editing at up to three loci using a modified iCRISPR system. This system includes a doxycycline inducible Cas9 and sgRNA/reporter plasmids for the enrichment of transfected cells by fluorescence-activated cell sorting (FACS). Here we cover the selection of target sites, vector construction, transfection, and isolation and genotyping of modified hiPSC clones.

The CRISPR/Cas9 system sheds new lights on the biology of protozoan parasites.

The CRISPR/Cas9 system, a natural defence system of bacterial organisms, has recently been used to modify genomes of the most important protozoa parasites. Successful genome manipulations with the CRISPR/Cas9 system are changing the present view of genetics in parasitology. The application of this system offers a major chance to overcome the current restriction in culturing, maintaining and analysing protozoan parasites, and allows dynamic analysis of parasite genes functions, leading to a better understanding of pathogenesis. CRISPR/Cas9 system will have a significant influence on the process of developing novel drugs and treatment strategies against protozoa parasites.

Epigenetics of cell fate reprogramming and its implications for neurological disorders modelling.

The reprogramming of human induced pluripotent stem cells (hiPSCs) proceeds in a stepwise manner with reprogramming factors binding and epigenetic composition changes during transition to maintain the epigenetic landscape, important for pluripotency. There arises a question as to whether the aberrant epigenetic state after reprogramming leads to epigenetic defects in induced stem cells causing unpredictable long term effects in differentiated cells. In this review, we present a comprehensive view of epigenetic alterations accompanying reprogramming, cell maintenance and differentiation as factors that influence applications of hiPSCs in stem cell based technologies. We conclude that sample heterogeneity masks DNA methylation signatures in subpopulations of cells and thus believe that beside a genetic evaluation, extensive epigenomic screening should become a standard procedure to ensure hiPSCs state before they are used for genome editing and differentiation into neurons of interest. In particular, we suggest that exploitation of the single-cell composition of the epigenome will provide important insights into heterogeneity within hiPSCs subpopulations to fast forward development of reliable hiPSC-based analytical platforms in neurological disorders modelling and before completed hiPSC technology will be implemented in clinical approaches.

Transcriptome profiling of Staphylococci-infected cow mammary gland parenchyma.

BACKGROUND: Genome-wide gene expression profiling allows for identification of genes involved in the defense response of the host against pathogens. As presented here, transcriptomic analysis and bioinformatics tools were applied in order to identify genes expressed in the mammary gland parenchyma of cows naturally infected with coagulase-positive and coagulase-negative Staphylococci. RESULTS: In cows infected with coagulase-positive Staphylococci, being in 1st or 2nd lactation, 1700 differentially expressed genes (DEGs) were identified. However, examination of the 3rd or 4th lactations revealed 2200 DEGs. Gene ontology functional classification showed the molecular functions of the DEGs overrepresented the activity of cytokines, chemokines, and their receptors. In cows infected with coagulase-negative Staphylococci, in the 1st or 2nd lactations 418 DEGs, while in the 3rd or 4th lactations, 1200 DEGs were identified that involved in molecular functions such as protein, calcium ion and lipid binding, chemokine activity, and protein homodimerization. Gene network analysis showed DEGs associated with inflammation, cell migration, and immune response to infection, development of cells and tissues, and humoral responses to infections caused by both types of Staphylococci. CONCLUSION: A coagulase-positive Staphylococci infection caused a markedly stronger host response than that of coagulase-negative, resulting in vastly increased DEGs. A significant increase in the expression of the FOS, TNF, and genes encoding the major histocompatibility complex proteins (MHC) was observed. It suggests these genes play a key role in the synchronization of the immune response of the cow's parenchyma against mastitis-causing bacteria. Moreover, the following genes that belong to several physiological pathways (KEGG pathways) were selected for further studies as candidate genes of mammary gland immune response for use in Marker Assisted Selection (MAS): chemokine signaling pathway (CCL2, CXCL5, HCK, CCR1), cell adhesion molecules (CAMs) pathway (BOLA-DQA2, BOLA-DQA1, F11R, ITGAL, CD86), antigen processing and presentation pathway (CD8A, PDIA3, LGMN, IFI30, HSPA1A), and NOD-like receptor signaling pathway (TNF, IL8, IL18, NFKBIA).

Social stress increases expression of hemoglobin genes in mouse prefrontal cortex.

BACKGROUND: In order to better understand the effects of social stress on the prefrontal cortex, we investigated gene expression in mice subjected to acute and repeated social encounters of different duration using microarrays. RESULTS: The most important finding was identification of hemoglobin genes (Hbb-b1, Hbb-b2, Hba-a1, Hba-a2, Beta-S) as potential markers of chronic social stress in mice. Expression of these genes was progressively increased in animals subjected to 8 and 13 days of repeated stress and was correlated with altered expression of Mgp (Mglap), Fbln1, 1500015O10Rik (Ecrg4), SLC16A10, and Mndal. Chronic stress increased also expression of Timp1 and Ppbp that are involved in reaction to vascular injury. Acute stress did not affect expression of hemoglobin genes but it altered expression of Fam107a (Drr1) and Agxt2l1 (Etnppl) that have been implicated in psychiatric diseases. CONCLUSIONS: The observed up-regulation of genes associated with vascular system and brain injury suggests that stressful social encounters may affect brain function through the stress-induced dysfunction of the vascular system.

Expression patterns of ß-defensin and cathelicidin genes in parenchyma of bovine mammary gland infected with coagulase-positive or coagulase-negative Staphylococci.

BACKGROUND: Mastitis is still considered to be the most economically important infectious disease in dairy cattle breeding. The immune response in mammary gland tissues could help in developing support strategies to combat this disease. The role of neutrophils and macrophages in the innate response of mammary gland is well known. However, the immune response in mammary gland tissues, including levels of antimicrobial peptide transcripts, has not been well recognized. Moreover, most studies are conducted in vitro, on cell cultures, or on artificially infected animals, with analysis being done within a several dozen hours after infection.The aim of the study was to examine the in vivo transcript levels of beta-defensin and cathelicidins genes in cow mammary gland secretory tissue (parenchyma) with the chronic, recurrent and incurable mammary gland inflammation induced by coagulase-positive or coagulase-negative Staphyloccoci vs. bacteria-free tissue. RESULTS: The mRNA of DEFB1, BNBD4, BNBD5, BNBD10 and LAP genes, but not of TAP gene, were detected in all investigated samples regardless of the animals' age and microbiological status of the mammary gland, but at different levels. The expression of most of the beta-defensin genes was shown to be much higher in tissues derived from udders infected with bacteria (CoPS or CoNS) than from bacteria-free udders, regardless of parity. Cathelicidins (CATH4, CATH5 and CATH6) showed expression patterns contrasting those of ß-defensins, with the highest expression in tissues derived from bacteria-free udders. CONCLUSION: Increased expression of genes encoding ß-defensins in the infected udder confirms their crucial role in the defense of the cow mammary gland against mastitis. On the other hand, the elevated cathelicidin transcripts in non-infected tissues indicate their role in the maintenance of healthy mammary tissues. The expression levels of investigated genes are likely to depend on the duration of the infection and type of bacteria.

Hepatic transcriptome profiling identifies differences in expression of genes associated with changes in metabolism and postnatal growth between Hereford and Holstein-Friesian bulls.

This study examined liver transcriptomic profiles of cattle distinctly different in meat and milk production capacity. It was performed on bulls of two different genetic backgrounds: Herefords (H), a meat breed, and Holstein-Friesians (HF), a dairy breed. Using bovine long oligo-microarrays and qPCR, we identified 128 genes that are differentially expressed between the two breeds. In H bulls, we observed up-regulation of genes involved in fatty acid biosynthesis and lipid metabolism (CD36, CAT, HSD3B1, FABP1, ACAA1) and involved in insulin signaling (INSR, INSIG2, NR4A1) and down-regulation of genes involved in somatotropic axis signaling (IGF1, GHR, IGFBP3) as compared to HF. Transcriptome profiling of these two breeds allowed us to pinpoint the transcriptional differences between Holstein and Hereford bulls at hepatic level associated with changes in metabolism and postnatal growth.

Novel human neurodevelopmental and neurodegenerative disease associated with IRF2BPL gene variants-mechanisms and therapeutic avenues.

Recently a broad range of phenotypic abnormalities related to the neurodevelopmental and neurodegenerative disorder NEDAMSS (Neurodevelopmental Disorder with Regression, Abnormal Movements, Loss of Speech, and Seizures) have been associated with rare single-nucleotide polymorphisms (SNPs) or insertion and deletion variants (Indel) in the intron-less gene IRF2BPL. Up to now, 34 patients have been identified through whole exome sequencing carrying different heterozygous pathogenic variants spanning the intron-less gene from the first polyglutamine tract at the N-terminus to the C3HC4 RING domain of the C-terminus of the protein. As a result, the phenotypic spectrum of the patients is highly heterogeneous and ranges from abnormal neurocognitive development to severe neurodegenerative courses with developmental and seizure-related encephalopathies. While the treatment of IRF2BPL-related disorders has focused on alleviating the patient's symptoms by symptomatic multidisciplinary management, there has been no prospect of entirely relieving the symptoms of the individual patients. Yet, the recent advancement of CRISPR-Cas9-derived gene editing tools, leading to the generation of base editors (BEs) and prime editors (PEs), provide an encouraging new therapeutic avenue for treating NEDAMSS and other neurodevelopmental and neurodegenerative diseases, which contain SNPs or smaller Indels in post-mitotic cell populations of the central nervous system, due to its ability to generate site-specific DNA sequence modifications without creating double-stranded breaks, and recruiting the non-homologous DNA end joining repair mechanism.

Stress susceptibility-specific phenotype associated with different hippocampal transcriptomic responses to chronic tricyclic antidepressant treatment in mice.

BACKGROUND: The effects of chronic treatment with tricyclic antidepressant (desipramine, DMI) on the hippocampal transcriptome in mice displaying high and low swim stress-induced analgesia (HA and LA lines) were studied. These mice displayed different depression-like behavioral responses to DMI: stress-sensitive HA animals responded to DMI, while LA animals did not. RESULTS: To investigate the effects of DMI treatment on gene expression profiling, whole-genome Illumina Expression BeadChip arrays and qPCR were used. Total RNA isolated from hippocampi was used. Expression profiling was then performed and data were analyzed bioinformatically to assess the influence of stress susceptibility-specific phenotypes on hippocampal transcriptomic responses to chronic DMI. DMI treatment affected the expression of 71 genes in HA mice and 41 genes in LA mice. We observed the upregulation of Igf2 and the genes involved in neurogenesis (HA: Sema3f, Ntng1, Gbx2, Efna5, and Rora; LA: Otx2, Rarb, and Drd1a) in both mouse lines. In HA mice, we observed the upregulation of genes involved in neurotransmitter transport, the termination of GABA and glycine activity (Slc6a11, Slc6a9), glutamate uptake (Slc17a6), and the downregulation of neuropeptide Y (Npy) and corticotropin releasing hormone-binding protein (Crhbp). In LA mice, we also observed the upregulation of other genes involved in neuroprotection (Ttr, Igfbp2, Prlr) and the downregulation of genes involved in calcium signaling and ion binding (Adcy1, Cckbr, Myl4, Slu7, Scrp1, Zfp330). CONCLUSIONS: Several antidepressant treatment responses are similar in individuals with different sensitivities to stress, including the upregulation of Igf2 and the genes involved in neurogenesis. However, the findings also reveal that many responses to antidepressant treatments, involving the action of individual genes engaged in neurogenesis, neurotransmitter transport and neuroprotection, depend on constitutive hippocampal transcriptomic profiles and might be genotype dependent. The results suggest that, when and if this becomes feasible, antidepressant treatment should take into consideration individual sensitivity to stress.

Generation of an induced pluripotent stem cell line from a Huntington's disease patient with a long HTT-PolyQ sequence.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an abnormal length of CAG repeats in the gene HTT, leading to an elongated poly-glutamine (poly-Q) sequence in huntingtin (HTT). We used non-integrative Sendai virus to reprogram fibroblasts from a patient with juvenile onset HD to induced pluripotent stem cells (iPSCs). Reprogrammed iPSCs expressed pluripotency-associated markers, exhibited a normal karyotype, and following directed differentiation generated cell types belonging to the three germ layers. PCR analysis and sequencing confirmed the HD patient-derived iPSC line had one normal HTT allele and one with elongated CAG repeats, equivalent to ≥180Q.

Genome-engineering technologies for modeling and treatment of cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease caused by defects in the CF transmembrane conductance regulator (CFTR) protein. Due to the genetic nature of the disease, interventions in the genome can target any underlying alterations and potentially provide permanent disease resolution. The current development of gene-editing tools, such as designer nuclease technology capable of genome correction, holds great promise for both CF and other genetic diseases. In recent years, Cas9-based technologies have enabled the generation of genetically defined human stem cell and disease models based on induced pluripotent stem cells (iPSC). In this article, we outline the potential and possibilities of using CRISPR/Cas9-based gene-editing technology in CF modeling.

Cathelicidins: family of antimicrobial peptides. A review.

Cathelicidins are small, cationic, antimicrobial peptides found in humans and other species, including farm animals (cattle, horses, pigs, sheep, goats, chickens, rabbits and in some species of fish). These proteolytically activated peptides are part of the innate immune system of many vertebrates. These peptides show a broad spectrum of antimicrobial activity against bacteria, enveloped viruses and fungi. Apart from exerting direct antimicrobial effects, cathelicidins can also trigger specific defense responses in the host. Their roles in various pathophysiological conditions have been studied in mice and humans, but there are limited information about their expression sites and activities in livestock. The aim of the present review is to summarize current information about these antimicrobial peptides in farm animals, highlighting peptide expression sites, activities, and future applications for human and veterinary medicine.

Moderate Effects of School-Based Time Increasing Physical Education Intervention on Physical Fitness and Activity of 7-Year Pupils-A Report from a Follow-Up of a HCSC Study.

We evaluated the effectiveness of a 15-week intervention that increased from three to five lessons of physical education (PE) a week on 7-year-old boys' and girls' physical fitness (PF), physical activity (PA) and sedentary behaviour on week and weekend days. A total of 212 first grade pupils (mean age 6.95 ± 0.43) from two urban schools in Poznan were randomly assigned to the experimental or control groups. The PF was measured with a battery of field tests, while health-related behaviours were assessed with the Healthy Children in Sound Communities questionnaire. There were some interaction effects noticed in the PF scores in the case of a 20-min run for boys (F2,196 = 5.29, p = 0.0058) and for girls (F2,220 = 3.31, p = 0.0382) and the sit-ups test for boys (F2,196 = 1.93, p = 0.1478) and for girls (F2,220 = 3.98, p = 0.0201) and for the sit and reach test in the case of girls (F2,220 = 3.98, p = 0.0201). In terms of outdoor PA levels, there were no major differences between any of the examined groups. Differences were found between girls from the experimental and control groups in the post-test (p = 0.0107) and follow-up (p = 0.0390) during the weekdays, with no differences between the groups of boys. Despite the moderate effects of the extended PE time programme right after the intervention, there were some indications of progress in the follow-up experiments.

Deletion in the Y chromosome of B10.BR-Ydel mice alters transcription from MSYq genes and has moderate effect on DNA methylation.

B10.BR-Ydel male mice with large deletion in the male-specific region of the Y chromosome long arm (MSYq) are very useful experimental model which requires, however, more detailed characterization. In the present study, the influence of the deletion on transcript levels of MSYq genes (Ssty1, Ssty2, Sly, Srsy, Asty, Orly) and homologous to them X-linked genes (Sstx, Slx, Slxl1, Srsx) was assessed. Quantitative PCR analysis showed that in testes of B10.BR-Ydel males activity of Ssty1 is unchanged, but transcription from all other MSYq genes is highly reduced and reaches from 59 % to only 5 % of the control levels. The decrease in expression of MSYq genes is accompanied by the two-fold increase in expression of Slx and Slxl1 genes. This is the first functional characterization of the deletion in B10.BR-Ydel strain. Another aim of the study was to reveal the mechanism through which deleted Y chromosome of B10.BR-Ydel males could alter phenotype of their female progeny, what was documented in our previous works. Epigenetic inheritance hypothesis was tested by microarray analysis of DNA methylation in B10.BR-Ydel and control B10.BR sperm. The assessment revealed moderate differences and allowed concluding that the mutated Y chromosome can influence traits of females from the next generation partially through altering sperm DNA methylation, but probably some additional mechanisms are engaged here. Breeding data indicate that feminization of pre- and neonatal environment in which next generation females develop is one of such additional mechanisms.

Generation of induced pluripotent stem cells from three individuals with Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by abnormal glutamine (Q) expansion in the huntingtin protein due to elongated CAG repeats in the gene HTT. We used non-integrative episomal plasmids to generate induced pluripotent stem cells (iPSCs) from three individuals affected by HD: CH1 (58Q), and two twin brothers CH3 (44Q) and CH4 (44Q). The iPSC lines exhibited one healthy HTT allele and one with elongated CAG repeats, as confirmed by PCR and sequencing. All iPSC lines expressed pluripotency markers, exhibited a normal karyotype, and generated cells of the three germ layers in vitro.

Mechanisms of IRF2BPL-related disorders and identification of a potential therapeutic strategy.

The recently discovered neurological disorder NEDAMSS is caused by heterozygous truncations in the transcriptional regulator IRF2BPL. Here, we reprogram patient skin fibroblasts to astrocytes and neurons to study mechanisms of this newly described disease. While full-length IRF2BPL primarily localizes to the nucleus, truncated patient variants sequester the wild-type protein to the cytoplasm and cause aggregation. Moreover, patient astrocytes fail to support neuronal survival in coculture and exhibit aberrant mitochondria and respiratory dysfunction. Treatment with the small molecule copper ATSM (CuATSM) rescues neuronal survival and restores mitochondrial function. Importantly, the in vitro findings are recapitulated in vivo, where co-expression of full-length and truncated IRF2BPL in Drosophila results in cytoplasmic accumulation of full-length IRF2BPL. Moreover, flies harboring heterozygous truncations of the IRF2BPL ortholog (Pits) display progressive motor defects that are ameliorated by CuATSM treatment. Our findings provide insights into mechanisms involved in NEDAMSS and reveal a promising treatment for this severe disorder.

Generation of Human iPSC-derived Neural Progenitor Cells (NPCs) as Drug Discovery Model for Neurological and Mitochondrial Disorders.

The high attrition rate in drug development processes calls for additional human-based model systems. However, in the context of brain disorders, sampling live neuronal cells for compound testing is not applicable. The use of human induced pluripotent stem cells (iPSCs) has revolutionized the field of neuronal disease modeling and drug discovery. Thanks to the development of iPSC-based neuronal differentiation protocols, including tridimensional cerebral organoids, it is now possible to molecularly dissect human neuronal development and human brain disease pathogenesis in a dish. These approaches may allow dissecting patient-specific treatment efficacy in a disease-relevant cellular context. For drug discovery approaches, however, a highly reproducible and cost-effective cell model is desirable. Here, we describe a step-by-step process for generating robust and expandable neural progenitor cells (NPCs) from human iPSCs. NPCs generated with this protocol are homogeneous and highly proliferative. These features make NPCs suitable for the development of high-throughput compound screenings for drug discovery. Human iPSC-derived NPCs show a metabolism dependent on mitochondrial activity and can therefore be used also to investigate neurological disorders in which mitochondrial function is affected. The protocol covers all steps necessary for the preparation, culture, and characterization of human iPSC-derived NPCs. Graphic abstract: Schematic of the protocol for the generation of human iPSC-derived NPCs.