A Microbe Associated with Sleep Revealed by a Novel Systems Genetic Analysis of the Microbiome in Collaborative Cross Mice.

The microbiome influences health and disease through complex networks of host genetics, genomics, microbes, and environment. Identifying the mechanisms of these interactions has remained challenging. Systems genetics in laboratory mice (Mus musculus) enables data-driven discovery of biological network components and mechanisms of host-microbial interactions underlying disease phenotypes. To examine the interplay among the whole host genome, transcriptome, and microbiome, we mapped QTL and correlated the abundance of cecal messenger RNA, luminal microflora, physiology, and behavior in a highly diverse Collaborative Cross breeding population. One such relationship, regulated by a variant on chromosome 7, was the association of Odoribacter (Bacteroidales) abundance and sleep phenotypes. In a test of this association in the BKS.Cg-Dock7m +/+ Leprdb/J mouse model of obesity and diabetes, known to have abnormal sleep and colonization by Odoribacter, treatment with antibiotics altered sleep in a genotype-dependent fashion. The many other relationships extracted from this study can be used to interrogate other diseases, microbes, and mechanisms

Cyclic stretch increases splicing noise rate in cultured human fibroblasts

BACKGROUND: Mechanical forces are known to alter the expression of genes, but it has so far not been reported whether they may influence the fidelity of nucleus-based processes. One experimental approach permitting to address this question is the application of cyclic stretch to cultured human fibroblasts. As a marker for the precision of nucleus-based processes, the number of errors that occur during co-transcriptional splicing can then be measured. This so-called splicing noise is found at low frequency in pre-mRNA splicing. FINDINGS: The amount of splicing noise was measured by RT-qPCR of seven exon skips from the test genes AATF, MAP3K11, NF1, PCGF2, POLR2A and RABAC1. In cells treated by altered uniaxial cyclic stretching for 18 h, a uniform and significant increase of splicing noise was found for all detectable exon skips. CONCLUSION: Our data demonstrate that application of cyclic stretch to cultured fibroblasts correlates with a reduced transcriptional fidelity caused by increasing splicing noise

Revertierung von Neurofibromatose Typ 1 Haploinsuffizienzeffekten durch Proteintransduktion in vitro

Enzyme replacement therapies have been performed since the 1970s to treat the lack of proteins in human blood or in lysosomes of human cells. Up to now, there are no comparable treatments to replace lacking proteins in the cytosol of cells of patients suffering from genetic diseases. Here, this approach is exemplarily studied in fibroblasts of neurofibromatosis type 1 (NF1) patients. In these cells, the amount of the cytosolic neurofibromin is reduced. This protein reduction leads to a hyperactivation of the mitogen activated protein kinase pathway and to an altered capability of the fibroblasts to orientate themselves on nanostructured surfaces. In cell cultures it could be shown that the missing amounts of neurofibromin in NF1 haploinsufficient fibroblasts can be replaced using protein transduction methods. Two test systems were used to illustrate that the transduced neurofibromin can assume the functions of the lacking proteins in these cells and revert cellular effects based on NF1 haploinsufficiency. The endolysosomal entrapment of transduced proteins is the bottleneck of cytosolic protein transduction. For the cell culture experiments, this problem was solved by performing photochemical internalization (PCI) treatments. PCI was used to translocate endosomal entrapped proteins to the cytosol of the transduced fibroblasts. For that systemic PCI treatments are not realizable in patients, future studies will have to examine protein modifications that allow a cytosolic delivery of transduced proteins without an interfering from an external source. Experiments using a neurofibromin fused to the protein transduction domain of HIV-TAT revealed promising evidence that modifications can replace secondary treatments like PCI. Hence, the cytosolic replacement of lacking intracellular proteins seems to be a possible treatment for patients with monogenic diseases like NF1

Molecular features and vulnerabilities of recurrent chordomas

Background!#!Tumor recurrence is one of the major challenges in clinical management of chordoma. Despite R0-resection, approximately 50% of chordomas recur within ten years after initial surgery. The underlying molecular processes are poorly understood resulting in the lack of associated therapeutic options. This is not least due to the absence of appropriate cell culture models of this orphan disease.!##!Methods!#!The intra-personal progression model cell lines U-CH11 and U-CH11R were compared using array comparative genomic hybridization, expression arrays, RNA-seq, and immunocytochemistry. Cell line origin was confirmed by short tandem repeat analysis. Inter-personal cell culture models (n = 6) were examined to validate whether the new model is representative. Cell viability after HOX/PBX complex inhibition with small peptides was determined by MTS assays.!##!Results!#!Using whole genome microarray analyses, striking differences in gene expression between primary and recurrent chordomas were identified. These expression differences were confirmed in the world's first intra-personal model of chordoma relapse consisting of cell lines established from a primary (U-CH11) and the corresponding recurrent tumor (U-CH11R). Array comparative genomic hybridization and RNA-sequencing analyses revealed profound genetic similarities between both cell lines pointing to transcriptomic reprogramming as a key mechanism of chordoma progression. Network analysis of the recurrence specific genes highlighted HOX/PBX signaling as a common dysregulated event. Hence, HOX/PBX complexes were used as so far unknown therapeutic targets in recurrent chordomas. Treating chordoma cell lines with the complex formation inhibiting peptide HXR9 induced cFOS mediated apoptosis in all chordoma cell lines tested. This effect was significantly stronger in cell lines established from chordoma relapses.!##!Conclusion!#!Clearly differing gene expression patterns and vulnerabilities to HOX/PBX complex inhibition in highly therapy resistant chordoma relapses were identified using the first intra-personal loco-regional and further inter-personal chordoma progression models. For the first time, HOX/PBX interference was used to induce cell death in chordoma and might serve as the basic concept of an upcoming targeted therapy for chordomas of all progression stages

CARD9 Forms an Alternative CBM Complex in Richter Syndrome

Richter syndrome (RS) is defined as the transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, mostly diffuse large B-cell lymphoma (DLBCL). Despite intensive therapy, patients with RS have an unfavorable clinical outcome. The detailed pathobiology of Richter transformation still needs to be elucidated. Here, we report high mRNA and protein levels of CARD9 in the RS cell line U-RT1. Co-immunoprecipitation revealed the assembly of a CBM complex using CARD9 instead of CARD11. CARD9 is known to be an activator of NF-кB signaling in myeloid cells. U-RT1 Western blot analyses showed phosphorylation of IκB as well as IKK, indicating a constitutively active canonical NF-кB pathway. This was further supported by the significant reduction in cell viability and CYLD cleavage products after CARD9 siRNA knockdown. We also showed immunostaining for CARD9 in 53% of cases analyzed in a series of RS tissue specimens, whereas other lymphomas rarely show CARD9 expression. This is the first report on ectopic expression and function of CARD9 in an aggressive B-cell lymphoma. Our findings suggest that CARD9 may contribute to the pathogenesis of RS

Prognostic Relevance and In Vitro Targeting of Concomitant PTEN and p16 Deficiency in Chordomas

Chordomas are rare bone tumors arising along the spine. Due to high resistance towards chemotherapy, surgical resection—often followed by radiation therapy—is currently the gold standard of treatment. So far, targeted systemic therapies have not been approved. The most frequent molecular alterations include the loss of PTEN and CDKN2A (encoding p16), being associated with poor prognoses in chordoma patients. Specific inhibitors of the PI3K/AKT/mTOR pathway as well as CDK4/6 have shown antitumor activity in preclinical studies and have recently been under investigation in phase II clinical trials; however, the clinical impacts and therapeutic consequences of concomitant PTEN and p16 deficiency have not yet been investigated in chordomas. In a cohort of 43 chordoma patients, 16% of the cases were immunohistochemically negative for both markers. The simultaneous loss of PTEN and p16 was associated with a higher KI-67 index, a tendency to metastasize, and significantly shorter overall survival. Additionally, 30% of chordoma cell lines (n = 19) were PTEN-/p16-negative. Treating these chordoma cells with palbociclib (CDK4/6 inhibitor), rapamycin (mTOR inhibitor) or the pan-PI3K inhibitor buparlisib significantly reduced cell viability. Synergistic effects were observed when combining palbociclib with rapamycin. In conclusion, we show that patients with PTEN-/p16-negative chordomas have poor prognoses and provide strong preclinical evidence that these patients might benefit from a Palbociclib/rapamycin combination treatment

Enhancing Endosomal Escape of Transduced Proteins by Photochemical Internalisation

<div><p>Induced internalisation of functional proteins into cultured cells has become an important aspect in a rising number of <em>in vitro</em> and <em>in vivo</em> assays. The endo-lysosomal entrapment of the transduced proteins remains the major problem in all transduction protocols. In this study we compared the efficiency, cytotoxicity and protein targeting of different commercially available transduction reagents by transducing a well-studied fluorescently labelled protein (Atto488-bovine serum albumin) into cultured human sarcoma cells. The amount of internalised protein and toxicity differed between the different reagents, but the percentage of transduced cells was consistently high. Furthermore, in all protocols the signals of the transduced Atto488-BSA were predominantly punctual consistent with an endosomal localisation. To overcome the endosomal entrapment, the transduction protocols were combined with a photochemical internalisation (PCI) treatment. Using this combination revealed that an endosomal disruption is highly effective in cell penetrating peptide (CPP) mediated transduction, whereas lipid-mediated transductions lead to a lower signal spreading throughout the cytosol. No change in the signal distribution could be achieved in treatments using non-lipid polymers as a transduction reagent. Therefore, the combination of protein transduction protocols based on CPPs with the endosomolytic treatment PCI can facilitate protein transduction experiments <em>in vitro.</em></p> </div

Restoring functional neurofibromin by protein transduction

In Neurofibromatosis 1 (NF1) germ line loss of function mutations result in reduction of cellular neurofibromin content (NF1+/−, NF1 haploinsufficiency). The Ras-GAP neurofibromin is a very large cytoplasmic protein (2818 AA, 319 kDa) involved in the RAS-MAPK pathway. Aside from regulation of proliferation, it is involved in mechanosensoric of cells. We investigated neurofibromin replacement in cultured human fibroblasts showing reduced amount of neurofibromin. Full length neurofibromin was produced recombinantly in insect cells and purified. Protein transduction into cultured fibroblasts was performed employing cell penetrating peptides along with photochemical internalization. This combination of transduction strategies ensures the intracellular uptake and the translocation to the cytoplasm of neurofibromin. The transduced neurofibromin is functional, indicated by functional rescue of reduced mechanosensoric blindness and reduced RasGAP activity in cultured fibroblasts of NF1 patients or normal fibroblasts treated by NF1 siRNA. Our study shows that recombinant neurofibromin is able to revert cellular effects of NF1 haploinsuffiency in vitro, indicating a use of protein transduction into cells as a potential treatment strategy for the monogenic disease NF1

Change of signal distribution after PCI treatment.

<p>The median of the pixel gray values was calculated in fluorescence images of Atto488-BSA transduced sarcoma cells using different transduction reagents. Single cells were defined as region of interest in the ImageJ software and the median of the gray values was compared between cells before and after PCI treatment as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052473#s2" target="_blank">Methods</a>. The means of the medians of the gray values before and after PCI treatment are given. Error bars represent the 95% confidence intervals (n = 10). CPP-based transduction reagents (Proteoducin and Chariot) show high signal distribution changes. Reagents based on lipids (Proteofectene, Lipodin-Pro, ProJect and Pro-DeliverIN) also show changes but with partially lower significance. Reagents described as non-lipid or cationic polymers (TransPassP and TurboFect) did not lead to a signal spreading as a reason of a PCI treatment. The cytosolic signals produced by transduction with the endosomolytic reagent Endo-Porter was increased significantly by further disruption of endo- and lysosomes by PCI treatment.</p