Comparing Differentiated Rod And Cone Transcriptomes Reveals Rax As A Controller Of A Defined Cone Gene Regulatory Program

PurposeRods and cones convert light stimuli into signaling information for brain- mediated visual perception. Specific gene mutations primarily affecting both photoreceptor (PRs) types lead to retinal dystrophies, including rod-cone (retinitis pigmentosa) and cone-rod degeneration. Rods and cones are generated from common precursor cells and their differentiation program is strongly regulated by transcription factors (TFs) that control gene networks during development transitions and in the adult to maintain their identity and function. The aim of this thesis was to investigate post-mitotic transcriptional regulation of rod- and cone-specific gene regulatory networks (GRNs). Specifically, retrieving transcription factor sets has biological relevance and may be instrumental for designing somatic gene transfer methodologies for both gene-based and regenerative therapeutic strategies. Methods We developed an Adeno-associated viral (AAV) vector-based transfer method to isolate rods and cones by fluorescence-based sorting. To isolate “pure” rod and cone populations from the same retinal samples we designed a double expression cassette (“double fluo”) encoding mCherry under the transcriptional control of the human G-protein coupled receptor Kinase 1 (GRK1) gene rods and cones-specific promoter elements of, and eGFP under the transcriptional control of rod-specific human Guanine Nucleotide Binding Protein Alpha Transducing (GNAT1) promoter elements. Porcine (Sus scrofa) retinae were injected with AAV 2/8 GRK1-mCherry_hGNAT1-eGFP, at the dose of 1x1012 genome copies (GC) per eye (n=3). The animals were sacrificed 15 days after injection to evaluate fluorescence reporter based rod and cone photoreceptor identities by histological and FACS analysis. We evaluated, on sorted populations, rods and cone specific transcriptomes by RNA sequencing (RNA- seq) using Illumina Hiseq 1500 (n=3). Moreover, after overexpression of human Retina And Anterior Neural Fold Homeobox (RAX), using subretinal injection of AAV2/8 hGNAT1-hRAX2aeGFP at a dose of 5 x 1011gc/eye we studied the GRN associated with RAX, by a second round of RNA-seq. Electroretinography (ERG) was used to correlate RAX-specific GRN with retina function. Results Histological analysis showed unambiguous expression of both mCherry and eGFP in rods, whereas cones showed exclusive expression of mCherry. Furthermore, cone arrestin (CAR) showed exclusive co-localization with the mCherry signal (cones), which was absent in red-green cells (rods). Photoreceptors specifically labeled were positive for both mCherry and eGFP (rods) or mCherry-only (cones) expressing Rhodopsin (RHO), Neural retina leucin zipper (NRL), Guanine Nucleotide Binding Protein Alpha Transducine 1 (GNAT1) (rod markers) and Short wavelength cone opsin (S-opsin), Medium wavelength cone opsin (M-opsin), Guanine Nucleotide Binding Protein Alpha Transducing 2 (GNAT2) (cone markers), respectively. RNA-seq differential expression analysis (DEA) showed 198 differential expressed genes (DEGs), of which 68 genes were up-regulated and 128 genes down-regulated in rod versus cone analysis. Furthermore, 8 TFs were found differentially expressed in rods and 8 in cones. In the cone-specific TFs set, out of 3 known TFs (Rax, Sall3 and Six6), Rax was selected for further analysis. Rax was ectopically expressed in rods (Rax-rod) to reconstruct its GRN. Differential expression analysis identified 386 DEGs, of which 224 genes (58%) were up- and 162 (42%) down regulated. Clustering for biological processes gene ontology (BP-GO), showed enrichment in action potential genes. Furthermore, ERG showed a different light response of the hRax-treated retina. In particular, a decrease of the mesopic (dark adapted) and an increase of the photopic (light adapted) responses were found. Conclusion We showed that the “double fluo” provided a robust method to isolate and cross-compare rod and cone photoreceptors in the adult porcine retina upon photoreceptor somatic gene transfer via AAV vectors. Furthermore, RNA-seq data set analysis showed that the cone and rod specific transcriptome differs quantitatively by about 200 transcripts and that 8 TFs are photoreceptor specific In addition, the data show that post-mitotic rods are able to express cone genes upon Rax ectopic expression. Furthermore the data support a role of Rax in controlling cone-specific action potential gene set

MHC-I genotype drives early immune selection of oncogenic mutations.

MHC-I exposes the intracellular contents to immune cells for surveillance of cellular health. Due to high genomic variation, individuals' immune systems differ in their ability to expose and eliminate cancer-causing mutations. These personalized immune blind spots create specific oncogenic mutation predispositions within patients and influence their prevalence across populations

The Conundrum of Success in Music: Playing it or Talking About it?

Nowadays social media are the main means for conducting discussions and sharing opinions. The huge amount of information generated by social media users is helpful for predicting outcomes of real-world events in different fields, including business, politics and the entertainment industry. In this paper, we studied the possibility of forecasting the success of music albums by analyzing heterogeneous data sources spanning from social media (Twitter, Instagram and Facebook) to mainstream American newspapers (e.g., New York Times, Rolling Stones). The idea is to exploit music albums' pre-release hype and post-release approval to predict the album's rank with reference to the well-known Billboard 200 album chart, which tabulates the weekly popularity of music albums in the USA. To predict the success of a music album, that is its rank in the chart, we identified metrics based on the messages' posting trend, the variation of the sentiment associated to such messages, the number of followers of the album's author, and the importance of the people who talk about it. To evaluate the effectiveness of the proposed metrics we have compared the prediction performances of several models based on supervised learning approaches among those most used in literature. As a result, we obtained that the Random Forest approach is able to predict the music album rank in the Billboard 200 Chart with an expected accuracy of 97%. As a further validation, using this specific model, we also conducted an additional real usage test obtaining an almost matching result (accuracy of 94%)

484. Preclinical Proof of Concept of Transcriptional Silencing and Replacement Strategy for Treatment of Retinitis Pigmentosa Due To RHODOPSIN Mutations

Silencing and replacement strategy is a promising approach to overcome mutational heterogeneity of genetic defects. In autosomal dominant retinitis pigmentosa (adRP) due to rhodopsin gene (RHO) approximately 200 different mutations have been described, posing a challenge for the design of effective therapeutics.We designed a silencing and replacement strategy based on transcriptional silencing through an artificial zinc finger DNA-binding protein lacking effector domains (ZF6DBD), and tested both efficacy and safety in two animal models.In a murine model of adRP, we show that AAV-mediate retinal delivery (AAV2/8-CMV-ZF6-DBD) is associated with selective transcriptional silencing of the human mutated allele resulting in morphological and functional (Electroretinography, ERG a-wave and b-wave responses) rescue. We then tested the effect of transcriptional silencing in the porcine large pre-clinical model. Delivery of a low dose (AAV2/8-CMV-ZF6-DBD, 1×10e10 vector genomes, vg) of the ZF6 transcriptional silencer to the porcine retina resulted in robust transcriptional silencing of the endogenous porcine RHO transcript. Cell sorting of transduced photoreceptors showed an almost complete RHO transcriptional silencing effect (90% RHO transcriptional repression), underscoring the potency of the system. To determine the safety of the zinc-finger silencer we performed extensive RNA-seq analysis on treated and control retinae. The data sets generated demonstrate selective RHO gene transcriptional repression and a remarkably low number of differential expressed genes (DEGs), supporting specificity and thus, safety. The co-administration to the porcine retina of the AAV-ZF6 silencer (AAV2/8-CMV-ZF6-DBD) and the AAV-RHO replacement (5×10e11 vg, AAV2/8-GNAT1-HumanRHO) constructs resulted in a balanced silencing and replacement effect. This data support the use of zinc-finger based RHO transcriptional silencing for the development of a clinical trial for adRP patients

320 transcriptional silencing via synthetic dna binding protein lacking canonical repressor domains as a potent tool to generate therapeutics

Transcription factors (TFs) function by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs). Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain entails complete and gene-specific transcriptional silencing. To silence RHODOPSIN (RHO) gain-of-function mutations, we engineered a synthetic DNA-binding protein lacking canonical repressor domains and targeted to the regulatory region of the RHO gene. AAV-mediate retinal delivery at a low dose (AAV2/8-CMV-ZF6-DBD, 1×10e10 vector genomes, vg) in the porcine retina resulted in selective transcriptional silencing of RHO expression. The rod photoreceptors (the RHO expressing cells) transduced cells when isolated by FACS-sorting showed the remarkable 90% RHO transcriptional repression. To evaluate genome-wide transcriptional specificity, we analyzed the porcine retina transcriptome by RNA sequencing (RNA-Seq). The differentially expressed genes (DEGs) analysis showed that only 19 genes were perturbed. In this study, we describe a system based on a synthetic DNA binding protein enabling targeted transcriptional silencing of the RHO gene by in vivo gene transfer. The high rate of transcriptional silencing occurring in transduced cells supports applications of this regulatory genomic interference with a synthetic trans-acting factor for diseases requiring gene silencing in a large number of affected cells, including for instance a number of neurodegeneration disorders. The result support a novel mode of gene targeted silencing with a DNA-binding protein lacking intrinsic activity

Diagnosis and treatment of pulmonary embolism: a multidisciplinary approach

The diagnosis of pulmonary embolism (PE) is frequently considered in patients presenting to the emergency department or when hospitalized. Although early treatment is highly effective, PE is underdiagnosed and, therefore, the disease remains a major health problem. Since symptoms and signs are non specific and the consequences of anticoagulant treatment are considerable, objective tests to either establish or refute the diagnosis have become a standard of care. Diagnostic strategy should be based on clinical evaluation of the probability of PE. The accuracy of diagnostic tests for PE are high when the results are concordant with the clinical assessment. Additional testing is necessary when the test results are inconsistent with clinical probability. The present review article represents the consensus-based recommendations of the Interdisciplinary Association for Research in Lung Disease (AIMAR) multidisciplinary Task Force for diagnosis and treatment of PE. The aim of this review is to provide clinicians a practical diagnostic and therapeutic management approach using evidence from the literature

Differential Responses of Colorectal Cancer Cell Lines to Enterococcus faecalis' Strains Isolated from Healthy Donors and Colorectal Cancer Patients

The metabolites produced by the host’s gut microbiota have an important role in the maintenance of intestinal homeostasis, but can also act as toxins and induce DNA damage in colorectal epithelial cells increasing the colorectal cancer (CRC) chance. In this scenario, the impact of some of the components of the natural human gastrointestinal microbiota, such as Enterococcus faecalis (E. faecalis), at the onset of CRC progression remains controversial. Since under dysbiotic conditions it could turn into a pathogen, the aim of this study was to compare the effect of E. faecalis’ strains (isolated from CRC patients and healthy subjects’ stools) on the proliferation of different colorectal cells lines. First, we isolated and genotyping characterized the Enterococcus faecalis’ strains. Then, we analyzed the proliferation index (by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay) of three tumor and one normal intestinal cell lines, previously exposed to E. faecalis strains pre-cultured medium. Stool samples of CRC patients demonstrated a reduced frequency of E. faecalis compared to healthy subjects. In addition, the secreted metabolites of E. faecalis’ strains, isolated from healthy donors, decreased the human ileocecal adenocarcinoma cell line HCT-8 and human colon carcinoma cell line HCT-116 cell proliferation without effects on human colorectal adenocarcinoma cell line SW620 and on normal human diploid cell line CLR-1790. Notably, the metabolites of the strains isolated from CRC patients did not influence the cell growth of CRC cell lines. Our results demonstrated a new point of view in the investigation of E. faecalis’ role in CRC development, which raises awareness of the importance of not only associating the presence/absence of a unique microorganism, but also in defining the specific characteristics of the different investigated strains

Civiltà della Campania. Anno II, n. 3 (agosto-ottobre 1975)

A. II, n.3 (agosto-ottobre 1975): Il messaggio dell’Assessore Emilio de Feo, P. 3 ; M. Parrilli, Continuità nel turismo regionale, P. 3 ; Napoli nei secoli, P. 5 ; G. Galasso, Tumulti ed elezioni del ’600, P. 6 ; N. Cilento, Nella città medioevale, P. 18 ; B. Gatta, Capri tra Napoleone e Murat, P. 24 ; R. Causa, Gioacchino Toma a Napoli, P. 30 ; A. Assante, Napoli e il suo porto, P. 34 ; G. Grimaldi, Messaggio di fede dell’Anno Santo, P. 40 ; R. Vlad, Musica all’aperto, P. 50 ; M. Stefanile, Viaggio nella storia di Amalfi, P. 52 ; D. Rea, Mappa minore, P. 60 ; M. Prisco , Incontro con la Badia, P. 68 ; P. Amos e A. Gambardella, Il villaggio di Albori, P. 74 ; R. Virtuoso, Giovanni Cuomo ritorna tra i giovani, P. 76 ; V. Panebianco, Il turismo venuto dalla storia, P. 80 ; A.P. Carbone, Le grotte di Pertosa, P. 84 ; F. de Ciuceis, Il mare di Caserta, P. 88 ; E. Tirone, Riti settennali a Guardia Sanframondi, P. 92 ; F. Calabro, Turismo e cultura a Capri, P. 98 ; F. de Ciuceis, Settembre al Borgo, P. 102 ; I. Santoro, Teggiano citta museo, P. 104 ; Notiziario, P. 108

Helminth Communities of Owls (Strigiformes) Indicate Strong Biological and Ecological Differences from Birds of Prey (Accipitriformes and Falconiformes) in Southern Italy

We compared the helminth communities of 5 owl species from Calabria (Italy) and evaluated the effect of phylogenetic and ecological factors on community structure. Two host taxonomic scales were considered, i.e., owl species, and owls vs. birds of prey. The latter scale was dealt with by comparing the data here obtained with that of birds of prey from the same locality and with those published previously on owls and birds of prey from Galicia (Spain). A total of 19 helminth taxa were found in owls from Calabria. Statistical comparison showed only marginal differences between scops owls (Otus scops) and little owls (Athene noctua) and tawny owls (Strix aluco). It would indicate that all owl species are exposed to a common pool of 'owl generalist' helminth taxa, with quantitative differences being determined by differences in diet within a range of prey relatively narrow. In contrast, birds of prey from the same region exhibited strong differences because they feed on different and wider spectra of prey. In Calabria, owls can be separated as a whole from birds of prey with regard to the structure of their helminth communities while in Galicia helminths of owls represent a subset of those of birds of prey. This difference is related to the occurrence in Calabria, but not Galicia, of a pool of 'owl specialist' species. The wide geographical occurrence of these taxa suggest that local conditions may determine fundamental differences in the composition of local communities. Finally, in both Calabria and Galicia, helminth communities from owls were species-poor compared to those from sympatric birds of prey. However, birds of prey appear to share a greater pool of specific helmith taxa derived from cospeciation processes, and a greater potential exchange of parasites between them than with owls because of phylogenetic closeness

Beta-Blocker Use in Older Hospitalized Patients Affected by Heart Failure and Chronic Obstructive Pulmonary Disease: An Italian Survey From the REPOSI Register

Beta (β)-blockers (BB) are useful in reducing morbidity and mortality in patients with heart failure (HF) and concomitant chronic obstructive pulmonary disease (COPD). Nevertheless, the use of BBs could induce bronchoconstriction due to β2-blockade. For this reason, both the ESC and GOLD guidelines strongly suggest the use of selective β1-BB in patients with HF and COPD. However, low adherence to guidelines was observed in multiple clinical settings. The aim of the study was to investigate the BBs use in older patients affected by HF and COPD, recorded in the REPOSI register. Of 942 patients affected by HF, 47.1% were treated with BBs. The use of BBs was significantly lower in patients with HF and COPD than in patients affected by HF alone, both at admission and at discharge (admission, 36.9% vs. 51.3%; discharge, 38.0% vs. 51.7%). In addition, no further BB users were found at discharge. The probability to being treated with a BB was significantly lower in patients with HF also affected by COPD (adj. OR, 95% CI: 0.50, 0.37-0.67), while the diagnosis of COPD was not associated with the choice of selective β1-BB (adj. OR, 95% CI: 1.33, 0.76-2.34). Despite clear recommendations by clinical guidelines, a significant underuse of BBs was also observed after hospital discharge. In COPD affected patients, physicians unreasonably reject BBs use, rather than choosing a β1-BB. The expected improvement of the BB prescriptions after hospitalization was not observed. A multidisciplinary approach among hospital physicians, general practitioners, and pharmacologists should be carried out for better drug management and adherence to guideline recommendations