Delivery of RNA interfering effectors against HPV-16 E6 and E7 proteins in OSCC cells

Introduction: Safe and effective delivery of short interfering RNA/short hairpin RNA (siRNA/shRNA) molecules for the purpose of RNA interference was one of the biggest challenges in the past 20 years. Transkingdom RNA interference (tkRNAi) was suggested as an alternative to the traditional delivery protocols. This work aimed to evaluate the efficiency and the potency of an improved bacterial delivery system in inhibiting human papilloma virus 16 (HPV16)-E7-specific mRNA in oral squamous cell carcinoma (OSCC). Method and Results: Firstly, shRNA plasmids were constructed and transformed in the CEQ221 bacteria to construct the delivery vehicles. After bacterial transfection of the tumor cells, the ability of the modified bacteria to penetrate and collect in the tumor cells was confirmed by fluorescent microscopy. Efficiency of shRNA delivery and expression was confirmed by measuring the expression level of small siRNA and target gene mRNA via qRT-PCR; followed by a confirmation via western blot. Significant drop in the IC50 of the CEQ221-E7 targeting HPV16-E7 in oral squamous carcinoma cells as compared to their control counterparts CEQ221-GFP; and higher total apoptosis and activated caspase-3 activation confirmed the functional effect of the bacterial delivery-mediated inhibition of the HPV16-E7. Conclusion: Transkingdom RNA interference is a potent tool to inhibit specific target genes in mammalian cells. It was proved for the first time that tkRNAi is capable of inhibiting HPV16-E7 gene in oral squamous cell carcinoma triggering apoptosis and lowering proliferation activity and eventually leading to target cell death.Sicherer und effektiver Transfer von Short-interfering-RNA-/Short-hairpin-RNA-Molekülen (siRNA/shRNA) zum Zweck der RNA-Interferenz war eine der größten Herausforderungen der vergangenen 20 Jahre. Als Alternative zu den herkömmlichen Transferprotokollen wurde eine Transkingdom-RNA-Interferenz (tkRNAi) vorgeschlagen. Die hier vorgestellte Arbeit hat die Effizienz und Wirksamkeit eines verbesserten Bakterien-Übertragungssystems zur Hemmung der humanpapillomvirus-16-(HPV16)-E7-spezifischen mRNA oraler Plattenepithelkarzinom-Zellen (oral squamous cell carcinoma , OSCC) untersucht. Methodik und Ergebnisse: Zunächst wurden Plasmide konstruiert, welche neben einer therapeutischen shRNA zwei unterschiedliche Proteine kodieren. Das erste „Invasin“ verleiht nichtinvasiven, nichtpathogenen Bakterien einen invasiven Phänotyp. Das zweite Protein „Listeriolysin O“ zeigt sich dafür verantwortlich, dass die therapeutischen shRNA-Moleküle nach bakterieller Invasion in der Zielzelle freigesetzt werden. Nach bakterieller Transfektion der Tumorzellen, wurde die Fähigkeit der modifizierten Bakterien, in Tumorzellen einzudringen und sich dort zu sammeln mittels Fluoreszenzmikrokospie nachgewiesen. Die Effizienz des Transfers und der Expression der shRNA wurde durch die Messung des Expressionsniveaus von kleiner siRNA und der Zielgen-mRNA mit qRT-PCR bestimmt; bestätigt per Western-Blot. Ein signifikanter Abfall des IC50 von anti-HPV16-E7 CEQ221-E7 in oraler Plattenepithelkarzinom-Zellen im Vergleich zu CEQ221-GFP; und eine höhere Gesamtapoptose sowie eine aktivierte Caspase-3-Aktivierung bestätigten den funktionellen Effekt der durch die bakterielle Abgabe vermittelten Hemmung des HPV16-E7. Schlussfolgerungen: Transkingdom-RNA-Interferenz ist ein wirksames Instrument zur Hemmung spezifischer Zielgene in Säugetierzellen. Es wurde zum ersten Mal nachgewiesen, dass tkRNAi das HPV16-E7-Gen beim oralen Plattenepithelkarzinom hemmen, Apoptose auslösen und die Proliferationsaktivität senken kann, was schließlich zum Tod von Zielzellen führt

The Architecture of the Persian Period in the Levant

The earliest scholars were not concerned about preparing extensive investigations linking the Persian-period building remains excavated in the entire Levant together. Moreover, the research interests of scholars caused some impediments to the study of this period viz in the last decades; the Achaemenid period has been neglected by the scholars who -in turn- focused on the earlier and later periods for religious reasons. Too, while some regions have been studied abundantly, but it was not the case in other areas, which makes our knowledge is incomplete. From the explanation side, some scholars try to interpret the architectural remains from an ethnic perspective or unsubstantiated personal fancies, so their arguments were utterly lacking any objectivity. This thesis explores what are the Persian architectural and ornamental impacts on the Levantine architecture and the relations between Persian-period sites in Syria-Palestine region. Too, the architectural remains and their contents benefited us to clarify the settlement patterns in the regions being discussed. The author analyzed the ground plans of the buildings and their architectural features and ornamental motifs by conducting a descriptive, analytical, and interpretative study. He also conducted comparisons with similar buildings outside the Levant, especially in Fars to obtain a more comprehensive and systematic study, and then extracting any direct or indirect Persian influences. This has given us a better understanding of the nature of the social, political, and religious life in the entire Levant and the knowledge gap has been bridged to a satisfying extent. This study has demonstrated a few of the Achaemenid impacts, especially on the northern coastline of the Levant

Can montelukast correct immune dysregulation in preschool children with mild persistent asthma?

Background: Asthma is the most common inflammatory disorder among preschool and school-age children. Regulation of immune cells and their cytokines is essential to control asthma. Montelukast is a leukotriene receptor antagonist that suppresses inflammatory cell proliferation, and reduces cytokines and mediator secretion. Objective: The research team's goal was to study the immunological parameters among mild  asthmatic patients before and after the treatment with Montelukast. Methods: Forty preschool children with mild persistent asthma and twenty healthy, non-allergic children were included in the study. Blood eosinophil count, total IgE, serum IL-4, IL-10, and IL-13 levels were  assessed. T helper (CD3+CD4+) and T regulatory (CD4+CD25+) cell counts were measured using flow cytometry; for mild asthmatics before and after six weeks of treatment with Montelukast and for the control group. Results: Asthmatic children have shown a significant elevation of serum levels of IgE, IL4 and IL13, and also an increase of eosinophils, total lymphocyte T cells and T helper cell count. However; serum levels of IL10 and Treg cell count was lower in asthmatics compared to control. Following six weeks of Montelukast treatment, all immunological parameters improved. There was a significant elevation of serum levels of IL10 and Treg cell count, with a decrease in serum levels of IgE, IL4 and IL13; eosinophil counts, and helper T cells. Conclusion: Montelukast treatment improves the impaired immunological balance of mild asthmatic children through the increase of serum IL-10, T regulatory cell counts that have anti-inflammatory and immunoregulatory effects. It also decreases T helper cells and their proinflammatory cytokines

La Bibbia nelle letterature germaniche medievali

È certamente un luogo comune affermare che la Bibbia è il testo fondamentale della cultura occidentale. Il fatto che si tratti di un luogo comune, tuttavia, non rende l’affermazione meno vera: per tutta l’epoca medievale il testo biblico è fondamento imprescindibile delle credenze socialmente accettate, è modello di comportamento etico, è repertorio di storie esemplari che permettono di assegnare un senso agli eventi storici come al vissuto quotidiano. La partecipazione dei popoli germanici alla costruzione del sistema culturale europeo, dalla tarda antichità fino all'età moderna, è percorsa e pervasa dai processi di traduzione, rielaborazione e riuso dei libri dell’Antico e del Nuovo Testamento. I contributi raccolti in questo volume offrono apporti interessanti e spesso originali ai molti ambiti di discussione sul testo biblico, inserendo così la riflessione dei filologi germanici italiani nel più ampio dibattito internazionale e contribuendo in misura significativa a svilupparlo e ad approfondirlo

Prognostication in Stargardt disease using Fundus Autofluorescence: Improving Patient Care

PURPOSE: To explore fundus autofluorescence (FAF) imaging as an alternative to electroretinogram (ERG), as a non-invasive, quick, and readily interpretable method to predict disease progression in Stargardt disease (STGD). DESIGN: Retrospective case series of patients who attended Moorfields Eye Hospital (London, UK). SUBJECTS: Patients with STGD who met the following criteria were included: (i) biallelic disease-causing variants in ABCA4, (ii) ERG testing performed inhouse with an unequivocal ERG group classification, and (iii) ultra-widefield (UWF) FAF imaging performed up to 2 years before or after the ERG. METHODS: Patients were divided into three ERG groups based on retinal function and three FAF groups according to the extent of the hypoautofluorescence and their retinal background appearance. FAF imaging of 30 and 55° were also subsequently reviewed. MAIN OUTCOME MEASURES: ERG/FAF concordance and its association with baseline visual acuity and genetics. RESULTS: 234 patients were included in the cohort. 170 patients (73%) had the same ERG and FAF group, 33 (14%) had a milder FAF than ERG group, and 31 (13%) had a more severe FAF than ERG group. Children under the age of 10 (n=23) had the lowest ERG/FAF concordance, 57% (9 out of the 10 with discordant ERG/FAF had milder FAF than ERG), and adults with adult onset had the highest (80%). Missense genotypes were more commonly seen in the mildest phenotypes. In 97% and 98% of the cases, respectively, 30° and 55° FAF imaging matched with the group defined by UWF FAF. CONCLUSIONS: We demonstrate that FAF imaging is an effective modality to determine the extent of retinal involvement and thereby inform prognostication, by comparing FAF to the current gold standard of ERG testing to determine retinal involvement and thereby prognosis. In 80% of patients in our large molecularly proven cohort we were able to predict if the disease was confined to the macula or also affected the peripheral retina. Children assessed at a young age, with at least one null variant, early disease onset, and/or poor initial VA may have wider retinal involvement than predicted by FAF alone and/or progress to a more severe FAF phenotype over time

Humoral Immunogenicity and Efficacy of a Single Dose of ChAdOx1 MERS Vaccine Candidate in Dromedary Camels

MERS-CoV seronegative and seropositive camels received a single intramuscular dose of ChAdOx1 MERS, a replication-deficient adenoviral vectored vaccine expressing MERS-CoV spike protein, with further groups receiving control vaccinations. Infectious camels with active naturally acquired MERS-CoV infection, were co-housed with the vaccinated camels at a ratio of 1:2 (infected:vaccinated); nasal discharge and virus titres were monitored for 14 days. Overall, the vaccination reduced virus shedding and nasal discharge (p = 0.0059 and p = 0.0274, respectively). Antibody responses in seropositive camels were enhancedby the vaccine; these camels had a higher average age than seronegative. Older seronegative camels responded more strongly to vaccination than younger animals; and neutralising antibodies were detected in nasal swabs. Further work is required to optimise vaccine regimens for younger seronegative camels

Characterization of greater middle eastern genetic variation for enhanced disease gene discovery

The Greater Middle East (GME) has been a central hub of human migration and population admixture. The tradition of consanguinity, variably practiced in the Persian Gulf region, North Africa, and Central Asia1-3, has resulted in an elevated burden of recessive disease4. Here we generated a whole-exome GME variome from 1,111 unrelated subjects. We detected substantial diversity and admixture in continental and subregional populations, corresponding to several ancient founder populations with little evidence of bottlenecks. Measured consanguinity rates were an order of magnitude above those in other sampled populations, and the GME population exhibited an increased burden of runs of homozygosity (ROHs) but showed no evidence for reduced burden of deleterious variation due to classically theorized ‘genetic purging’. Applying this database to unsolved recessive conditions in the GME population reduced the number of potential disease-causing variants by four- to sevenfold. These results show variegated genetic architecture in GME populations and support future human genetic discoveries in Mendelian and population genetics

Bi-allelic loss-of-function variants in PPFIBP1 cause a neurodevelopmental disorder with microcephaly, epilepsy, and periventricular calcifications

PPFIBP1 encodes for the liprin-β1 protein, which has been shown to play a role in neuronal outgrowth and synapse formation in Drosophila melanogaster. By exome and genome sequencing, we detected nine ultra-rare homozygous loss-of-function variants in 16 individuals from 12 unrelated families. The individuals presented with moderate to profound developmental delay, often refractory early-onset epilepsy, and progressive microcephaly. Further common clinical findings included muscular hyper- and hypotonia, spasticity, failure to thrive and short stature, feeding difficulties, impaired vision, and congenital heart defects. Neuroimaging revealed abnormalities of brain morphology with leukoencephalopathy, ventriculomegaly, cortical abnormalities, and intracranial periventricular calcifications as major features. In a fetus with intracranial calcifications, we identified a rare homozygous missense variant that by structural analysis was predicted to disturb the topology of the SAM domain region that is essential for protein-protein interaction. For further insight into the effects of PPFIBP1 loss of function, we performed automated behavioral phenotyping of a Caenorhabditis elegans PPFIBP1/hlb-1 knockout model, which revealed defects in spontaneous and light-induced behavior and confirmed resistance to the acetylcholinesterase inhibitor aldicarb, suggesting a defect in the neuronal presynaptic zone. In conclusion, we establish bi-allelic loss-of-function variants in PPFIBP1 as a cause of an autosomal recessive severe neurodevelopmental disorder with early-onset epilepsy, microcephaly, and periventricular calcifications