Description: The emergence and future of mammalian synthetic biology depends on technologies for orchestrating and custom tailoring complementary gene expression and signaling processes in a predictable manner. Here, we demonstrate for the first time multi-chromatic expression control in mammalian cells by differentially inducing up to three genes in a single cell culture in response to light of different wavelengths. To this end, we developed an ultraviolet B (UVB)-inducible expression system by designing a UVB-responsive split transcription factor based on the Arabidopsis thaliana UVB receptor UVR8 and the WD40 domain of COP1. The system allowed high (up to 800-fold) UVB-induced gene expression in human, monkey, hamster and mouse cells. Based on a quantitative model, we determined critical system parameters. By combining this UVB-responsive system with blue and red light-inducible gene control technology, we demonstrate multi-chromatic multi-gene control by differentially expressing three genes in a single cell culture in mammalian cells, and we apply this system for the multi-chromatic control of angiogenic signaling processes. This portfolio of optogenetic tools enables the design and implementation of synthetic biological networks showing unmatched spatiotemporal precision for future research and biomedical applications.

Description: X-linked adrenoleukodystrophy (X-ALD) is a fatal neurodegenerative disease caused by mutations in the ABCD1 gene, encoding a member of the peroxisomal ABC transporter family. The ABCD1 protein transports CoA-activated very long-chain fatty acids (VLCFAs) into peroxisomes for degradation via β-oxidation. In the severest form, X-ALD patients suffer from inflammatory demyelination of the brain. As the extent of the metabolic defect in the main immune cells is unknown, we explored their phenotypes concerning mRNA expression pattern of the three peroxisomal ABC transporters, VLCFA accumulation and peroxisomal β-oxidation. In controls, ABCD1 expression was high in monocytes, intermediate in B cells and low in T cells; ABCD2 expression was extremely low in monocytes, intermediate in B cells and highest in T cells; ABCD3 mRNA was equally distributed. In X-ALD patients, the expression patterns remained unaltered; accordingly, monocytes, which lack compensatory VLCFA transport by ABCD2, displayed the severest biochemical phenotype with a 6-fold accumulation of C26:0 and a striking 70% reduction in peroxisomal β-oxidation activity. In contrast, VLCFA metabolism was close to control values in B cells and T cells, supporting the hypothesis that sufficient ABCD2 is present to compensate for ABCD1 deficiency. Thus, the vulnerability of the main immune cell types is highly variable in X-ALD. Based on these results, we propose that in X-ALD the halt of inflammation after allogeneic hematopoietic stem cell transplantation relies particularly on the replacement of the monocyte lineage. Additionally, these findings support the concept that ABCD2 is a target for pharmacological induction as an alternative therapeutic strategy.

Description: Alternative splicing and polyadenylation were observed pervasively in eukaryotic messenger RNAs. These alternative isoforms could either be consequences of physiological regulation or stochastic noise of RNA processing. To quantify the extent of stochastic noise in splicing and polyadenylation, we analyzed the alternative usage of splicing and polyadenylation sites in Entamoeba histolytica using RNA-Seq. First, we identified a large number of rarely spliced alternative junctions and then showed that the occurrence of these alternative splicing events is correlated with splicing site sequence, occurrence of constitutive splicing events and messenger RNA abundance. Our results implied the majority of these alternative splicing events are likely to be stochastic error of splicing machineries, and we estimated the corresponding error rates. Second, we observed extensive microheterogeneity of polyadenylation cleavage sites, and the extent of such microheterogeneity is correlated with the occurrence of constitutive cleavage events, suggesting most of such microheterogeneity is likely to be stochastic. Overall, we only observed a small fraction of alternative splicing and polyadenylation isoforms that are unlikely to be solely stochastic, implying the functional relevance of alternative splicing and polyadenylation in E. histolytica is limited . Lastly, we revised the gene models and annotated their 3'UTR in AmoebaDB, providing valuable resources to the community.

Description: Background In early estrogen receptor (ER)-positive/HER2-negative breast cancer, the decision to administer chemotherapy is largely based on prognostic criteria. The combined molecular/clinical EndoPredict test (EPclin) has been validated to accurately assess prognosis in this population. In this study, the clinical relevance of EPclin in relation to well-established clinical guidelines is assessed. Patients and methods We assigned risk groups to 1702 ER-positive/HER2-negative postmenopausal women from two large phase III trials treated only with endocrine therapy. Prognosis was assigned according to National Comprehensive Cancer Center Network-, German S3-, St Gallen guidelines and the EPclin. Prognostic groups were compared using the Kaplan–Meier survival analysis. Results After 10 years, absolute risk reductions (ARR) between the high- and low-risk groups ranged from 6.9% to 11.2% if assigned according to guidelines. It was at 18.7% for EPclin. EPclin reassigned 58%–61% of women classified as high-/intermediate-risk (according to clinical guidelines) to low risk. Women reclassified to low risk showed a 5% rate of distant metastasis at 10 years. Conclusion The EPclin score is able to predict favorable prognosis in a majority of patients that clinical guidelines would assign to intermediate or high risk. EPclin may reduce the indications for chemotherapy in ER-positive postmenopausal women with a limited number of clinical risk factors.

Description: Aims Stroke is a leading cause of morbidity and mortality, and its incidence increases with age. Both in animals and in humans, oxidative stress appears to play an important role in ischaemic stroke, with or without reperfusion. The adaptor protein p66 Shc is a key regulator of reactive oxygen species (ROS) production and a mediator of ischaemia/reperfusion damage in ex vivo hearts. Hence, we hypothesized that p66 Shc may be involved in ischaemia/reperfusion brain damage. To this end, we investigated whether genetic deletion of p66 Shc protects from ischaemia/reperfusion brain injury. Methods and results Transient middle cerebral artery occlusion (MCAO) was performed to induce ischaemia/reperfusion brain injury in wild-type (Wt) and p66 Shc knockout mice (p66 Shc–/– ), followed by 24 h of reperfusion. Cerebral blood flow and blood pressure measurements revealed comparable haemodynamics in both experimental groups. Neuronal nuclear antigen immunohistochemical staining showed a significantly reduced stroke size in p66 Shc–/– when compared with Wt mice ( P 〈 0.05, n = 7–8). In line with this, p66 Shc–/– mice exhibited a less impaired neurological function and a decreased production of free radicals locally and systemically ( P 〈 0.05, n = 4–5). Following MCAO, protein levels of gp91phox nicotinamide adenine dinucleotide phosphate oxidase subunit were increased in brain homogenates of Wt ( P 〈 0.05, n = 4), but not of p66 Shc–/– mice. Further, reperfusion injury in Wt mice induced p66 Shc protein in the basilar and middle cerebral artery, but not in brain tissue, suggesting a predominant involvement of vascular p66 Shc . Conclusion In the present study, we show that the deletion of the ageing gene p66 Shc protects mice from ischaemia/reperfusion brain injury through a blunted production of free radicals. The ROS mediator p66 Shc may represent a novel therapeutical target for the treatment of ischaemic stroke.

Description: Purpose The American Joint Committee on Cancer (AJCC) staging manual has introduced a TNM staging classification for jejunal-ileal (midgut) neuroendocrine tumors (NETs). This classification has not been validated in a population consisting solely of midgut NETs. The purpose of this study was to test the prognostic validity of the classification in such a population. Methods Patients with jejunal and ileocecal NETs who were treated at the Moffitt Cancer Center between 2000 and 2010 were assigned stages (I through IV). Kaplan-Meier analyses for overall survival (OS) were performed on the basis of TNM stage and pathologic grade. Multivariate modeling was performed using Cox proportional hazards regression. Results We identified 691 patients with jejunal-ileocecal NETs. The AJCC classification in aggregate was highly prognostic for OS ( P 〈 .001). Five-year OS rates for stages I through IV were 100%, 100%, 91%, and 72%, respectively. The survival difference between stages III and IV was significant ( P 〈 .001); the difference between stages I/II versus III was not statistically significant ( P = .1). Among patients with stage IIIB tumors, 5-year survival rates were 95% for resectable tumors versus 78% for unresectable mesenteric tumors ( P = .02). A proliferative threshold of five mitoses per 10 high-power fields (HPF) was of greater prognostic value than a threshold of two mitoses per 10 HPF for discriminating between low- and intermediate-grade tumors. Conclusion Stage I and II midgut NETs are associated with identical survival rates. Stage IIIB tumors are heterogeneous, with significant differences in survival observed between resectable mesenteric lymph nodes versus unresectable masses in the root of the mesentery. A higher mitotic cutoff of five per 10 HPF may lead to improved prognostic differentiation between low- and intermediate-grade tumors. Revisions to the current AJCC staging and grading classification may be warranted.

Description: Purpose To report the long-term results of the Intergroup Radiation Therapy Oncology Group 91-11 study evaluating the contribution of chemotherapy added to radiation therapy (RT) for larynx preservation. Patients and Methods Patients with stage III or IV glottic or supraglottic squamous cell cancer were randomly assigned to induction cisplatin/fluorouracil (PF) followed by RT (control arm), concomitant cisplatin/RT, or RT alone. The composite end point of laryngectomy-free survival (LFS) was the primary end point. Results Five hundred twenty patients were analyzed. Median follow-up for surviving patients is 10.8 years. Both chemotherapy regimens significantly improved LFS compared with RT alone (induction chemotherapy v RT alone: hazard ratio [HR], 0.75; 95% CI, 0.59 to 0.95; P = .02; concomitant chemotherapy v RT alone: HR, 0.78; 95% CI, 0.78 to 0.98; P = .03). Overall survival did not differ significantly, although there was a possibility of worse outcome with concomitant relative to induction chemotherapy (HR, 1.25; 95% CI, 0.98 to 1.61; P = .08). Concomitant cisplatin/RT significantly improved the larynx preservation rate over induction PF followed by RT (HR, 0.58; 95% CI, 0.37 to 0.89; P = .0050) and over RT alone ( P 〈 .001), whereas induction PF followed by RT was not better than treatment with RT alone (HR, 1.26; 95% CI, 0.88 to 1.82; P = .35). No difference in late effects was detected, but deaths not attributed to larynx cancer or treatment were higher with concomitant chemotherapy (30.8% v 20.8% with induction chemotherapy and 16.9% with RT alone). Conclusion These 10-year results show that induction PF followed by RT and concomitant cisplatin/RT show similar efficacy for the composite end point of LFS. Locoregional control and larynx preservation were significantly improved with concomitant cisplatin/RT compared with the induction arm or RT alone. New strategies that improve organ preservation and function with less morbidity are needed.

Description: TDP-43 is linked to neurodegenerative diseases including frontotemporal dementia and amyotrophic lateral sclerosis. Mostly localized in the nucleus, TDP-43 acts in conjunction with other ribonucleoproteins as a splicing co-factor. Several RNA targets of TDP-43 have been identified so far, but its role(s) in pathogenesis remains unclear. Using Affymetrix exon arrays, we have screened for the first time for splicing events upon TDP-43 knockdown. We found alternative splicing of the ribosomal S6 kinase 1 (S6K1) Aly/REF-like target (SKAR) upon TDP-43 knockdown in non-neuronal and neuronal cell lines. Alternative SKAR splicing depended on the first RNA recognition motif (RRM1) of TDP-43 and on 5'-GA-3’ and 5'-UG-3' repeats within the SKAR pre-mRNA. SKAR is a component of the exon junction complex, which recruits S6K1, thereby facilitating the pioneer round of translation and promoting cell growth. Indeed, we found that expression of the alternatively spliced SKAR enhanced S6K1-dependent signaling pathways and the translational yield of a splice-dependent reporter. Consistent with this, TDP-43 knockdown also increased translational yield and significantly increased cell size. This indicates a novel mechanism of deregulated translational control upon TDP-43 deficiency, which might contribute to pathogenesis of the protein aggregation diseases frontotemporal dementia and amyotrophic lateral sclerosis.

Description: Trigger-inducible transcription-control devices that reversibly fine-tune transgene expression in response to molecular cues have significantly advanced the rational reprogramming of mammalian cells. When designed for use in future gene- and cell-based therapies the trigger molecules have to be carefully chosen in order to provide maximum specificity, minimal side-effects and optimal pharmacokinetics in a mammalian organism. Capitalizing on control components that enable Caulobacter crescentus to metabolize vanillic acid originating from lignin degradation that occurs in its oligotrophic freshwater habitat, we have designed synthetic devices that specifically adjust transgene expression in mammalian cells when exposed to vanillic acid. Even in mice transgene expression was robust, precise and tunable in response to vanillic acid. As a licensed food additive that is regularly consumed by humans via flavoured convenience food and specific fresh vegetable and fruits, vanillic acid can be considered as a safe trigger molecule that could be used for diet-controlled transgene expression in future gene- and cell-based therapies.

Description: Integrins are transmembrane adhesion molecules that mediate cell–cell and cell–extracellular matrix attachment. Integrins regulate cell growth, proliferation, migration and apoptosis and as a consequence, can have a potential role in tumour progression and metastasis. In this study, we investigated 19 non-synonymous variants in the coding region of the human integrin genes representing 3 beta subunits and 13 alpha subunits, for their potential role in melanoma susceptibility and survival. The variants were selected on the basis of probable functional relevance and theoretical predictions. Our data showed that no genetic variant was significantly associated with survival. However, the variants in ITGA10 and ITGA6 genes showed association with decreased risk, and variants in ITGA2 , ITGAE and ITGAM were associated with increased risk of melanoma. The haplotype analysis revealed association of CA haplotype of ITGAE and TAC haplotype of ITGAX with the risk modulation. A prediction analysis of functional effect, homology modelling and multiple sequence alignments of integrin sequences from different species supported our data for linkage of variants in the ITGA2 and ITGAE genes with susceptibility. The amino acid changes in each of these integrin proteins could affect intramolecularly and/or the interaction of the heterodimers. Our experimental data indicated a possible role for some of the variant alleles and/or haplotypes of the integrin genes in melanoma susceptibility, which is augmented by the theoretical analysis performed.