The neo-open reading frame peptides that comprise the tumor framome are a rich source of neoantigens for cancer immunotherapy.

Identification of immunogenic cancer neoantigens as targets for therapy is challenging. Here, we integrate cancer whole genome and long-read transcript sequencing to identify the collection of novel open reading frame peptides (NOPs) expressed in tumors, termed the framome. NOPs represent tumor-specific peptides that are different from wild-type proteins and may be strongly immunogenic. We describe an uncharacterized class of hidden NOPs, which derive from structural genomic variants involving an upstream protein coding gene driving expression and translation of non-coding regions of the genome downstream of a rearrangement breakpoint. NOPs represent a vast amount of possible neoantigens particularly in tumors with many (complex) structural genomic variants and a low number of missense mutations. We show that NOPs are immunogenic and epitopes derived from NOPs can bind to MHC class I molecules. Finally, we provide evidence for the presence of memory T-cells specific for hidden NOPs in lung cancer patient peripheral blood.

Development of a Personalized Tumor Neoantigen Based Vaccine Formulation (FRAME-001) for Use in a Phase II Trial for the Treatment of Advanced Non-Small Cell Lung Cancer.

Stage III-IV non-small cell lung cancer (NSCLC) is a devastating disease characterized by a poor prognosis. NSCLC tumors carry genetic mutations, which can lead to the expression of altered protein sequences. Peptides originating from mutated proteins and bound to MHC molecules on the tumor cell surface are referred to as neoantigens, as they are tumor-specific and not expressed in normal cells. Due to their tumor specificity, neoantigens have a strong potential to induce an anti-tumor immune response and have been investigated for development of personalized therapeutic cancer vaccines. The current study describes the development of a clinical grade neoantigen vaccine formulation (FRAME-001) intended as immunotherapy in advanced NSCLC in combination with the immune checkpoint inhibitor pembrolizumab. The detection of aberrant tumor-specific transcripts as well as an algorithm to select immunogenic neoantigen peptides are described. Subsequently, selected neoantigen peptides were synthesized with a high throughput synthesis platform and aseptically formulated under good manufacturing practice (GMP) conditions into four aqueous peptides mixtures that each contained six neoantigen peptides. A validated stability-indicating analytical method was developed in which we considered the personalized nature of the formulation. An extensive stability study performed either at -25 °C or -80 °C showed that the formulation was stable for up to 32 weeks. The formulation was mixed with the vaccine adjuvant Montanide ISA 51 VG, which yielded the final vaccine emulsion. The stability of the vaccine emulsion was demonstrated using microscopic examination, differential light scattering, and the water-drop test. The presented data show that FRAME-001 is a feasible personalized vaccine formulation for the treatment of stage III-IV NSCLC. The presented data may give guidance in the development of novel personalized therapeutic vaccines since this formulation strategy could be used for any cancer indication.

Meningioma DNA methylation groups identify biological drivers and therapeutic vulnerabilities.

Meningiomas are the most common primary intracranial tumors. There are no effective medical therapies for meningioma patients, and new treatments have been encumbered by limited understanding of meningioma biology. Here, we use DNA methylation profiling on 565 meningiomas integrated with genetic, transcriptomic, biochemical, proteomic and single-cell approaches to show meningiomas are composed of three DNA methylation groups with distinct clinical outcomes, biological drivers and therapeutic vulnerabilities. Merlin-intact meningiomas (34%) have the best outcomes and are distinguished by NF2/Merlin regulation of susceptibility to cytotoxic therapy. Immune-enriched meningiomas (38%) have intermediate outcomes and are distinguished by immune infiltration, HLA expression and lymphatic vessels. Hypermitotic meningiomas (28%) have the worst outcomes and are distinguished by convergent genetic and epigenetic mechanisms driving the cell cycle and resistance to cytotoxic therapy. To translate these findings into clinical practice, we show cytostatic cell cycle inhibitors attenuate meningioma growth in cell culture, organoids, xenografts and patients.

Accurate prediction of chemical shifts for aqueous protein structure on "Real World" data.

Here we report a new machine learning algorithm for protein chemical shift prediction that outperforms existing chemical shift calculators on realistic data that is not heavily curated, nor eliminates test predictions ad hoc. Our UCBShift predictor implements two modules: a transfer prediction module that employs both sequence and structural alignment to select reference candidates for experimental chemical shift replication, and a redesigned machine learning module based on random forest regression which utilizes more, and more carefully curated, feature extracted data. When combined together, this new predictor achieves state-of-the-art accuracy for predicting chemical shifts on a randomly selected dataset without careful curation, with root-mean-square errors of 0.31 ppm for amide hydrogens, 0.19 ppm for Hα, 0.84 ppm for C', 0.81 ppm for Cα, 1.00 ppm for Cß, and 1.81 ppm for N. When similar sequences or structurally related proteins are available, UCBShift shows superior native state selection from misfolded decoy sets compared to SPARTA+ and SHIFTX2, and even without homology we exceed current prediction accuracy of all other popular chemical shift predictors.

Training standards in implant dentistry for general dental practitioners. A report.

In December 2005, the General Dental Council (GDC) convened a small working group to consider training standards for general dental practitioners (GDPs) who wish to practise implant dentistry. The membership of this group is given at the end of this report. The secretariat for the group was provided jointly by the GDC and the Faculty of General Dental Practice (UK). The remit of the group (which was independent of any organisation) was to consider what training standards would be necessary for a GDP before practising implant dentistry, to publish those standards, and then periodically to review them in the light of developments in implant dentistry. Such standards can be used not only by practitioners but also by the GDC in the consideration of patient complaints against dental practitioners who, allegedly, practise implant dentistry beyond the limits of their competence.

Pharmaceutical prescribing for children. Part 4. Antifungal and antiviral therapy.

This is the fourth paper in a series on the prescribing of medicines for children by dentists working in primary dental care. It deals with the prescription of antifungals and antivirals for children.

Enterobacter sakazakii in the mouths of stroke patients and its association with aspiration pneumonia.

BACKGROUND: Previous studies by our group have identified a high prevalence of Gram-negative bacilli in the mouths of stroke patients. Eradication of such organisms by selective decontamination of the digestive tract (SDD) has not previously been studied. Enterobacter sakazakii is a rarely found organism that has previously been identified as a cause of neonatal meningitis and sepsis but that has rarely been reported as a pathogen in older people. The purpose of this study was to look specifically for E. sakazakii in the mouths of stroke patients and to correlate its isolation with response to SDD gel and the presence of clinical complications such as pneumonia. METHODS: Following an acute stroke, 203 patients were recruited for the study and received placebo or SDD in a 50:50 ratio. Oral swabs were obtained on nine separate occasions and E. sakazakii was cultured from them. Clinical data were prospectively collected and analysed. RESULTS: E. sakazakii was identified in 7 of 203 patients. The organism was almost exclusively removed by SDD gel, but in those patients in whom an abnormal swallow was present, pneumonia was found in two of the four cases. CONCLUSION: E. sakazakii may colonise the mouths of acute stroke patients. Since SDD gel is not routinely administered to such patients, this organism may be implicated in patients who subsequently develop aspiration pneumonia.

Inhibition of ribonucleotide reductase reduces neointimal formation following balloon injury.

Percutaneous transluminal coronary angioplasty (PTCA) has greatly benefited patients with occluded coronary arteries, but its benefits have been undermined by a high incidence of restenosis. The introduction of coronary stents has significantly improved the short and long term outcome but restenosis still occurs in approximately 15 to 30% of patients within 6 months. Research efforts are now being directed toward combination stenting and drug delivery. Among the therapeutic targets being pursued are agents that can impede smooth muscle cell migration and proliferation, as these processes are critical components of restenosis injury. We propose that inhibiting the conversion of ribonucleotides to deoxyribonucleotides will impede cell proliferation and, as such, limit the degree of restenosis. Therefore, we tested whether the potent ribonucleotide reductase inhibitors Didox (3,4-dihydroxybenzohydraxamic acid) and Imidate (ethyl-3,4,5-hydroxybenzimidate) can limit the neointimal proliferation associated with restenosis using a rat carotid model of balloon dilatation injury. Results demonstrated that both Didox and Imidate significantly reduced intimal thickening, resulting in a 71 and 62% decrease in the intima/media ratio, respectively. Similar efficacy was seen with the commercially available ribonucleotide reductase inhibitor hydroxyurea, demonstrating the importance of this enzyme in vascular remodeling. Results from cell proliferation studies suggest that the mechanism of protection is inhibition of smooth muscle cell (SMC) proliferation. In addition, Didox and Imidate (100 microM) are potent inhibitors of SMC migration, which may also contribute to their vascular protective effects. These results suggest that inhibition of ribonucleotide reductase may provide a potent strategy to prevent post-PTCA restenosis.

Fluoridated elastomers: effect on the microbiology of plaque.

The objective of this study was to investigate the effect of fluoridated elastomeric ligatures on the microbiology of local dental plaque in vivo. This randomized, prospective, longitudinal, clinical trial had a split-mouth crossover design. The subjects were 30 patients at the beginning of their treatment with fixed orthodontic appliances in the orthodontic departments of the Liverpool and the Sheffield dental hospitals in the United Kingdom. The study consisted of 2 experimental periods of 6 weeks with a washout period between. Fluoridated elastomers were randomly allocated at the first visit to be placed around brackets on tooth numbers 12, 11, 33 or 22, 21, 43. Nonfluoridated elastomers were placed on the contralateral teeth. Standard nonantibacterial fluoridated toothpaste and mouthwash were supplied. After 6 weeks (visit 2), the elastomers were removed, placed in transport media, and plated on agar within 2 hours. Nonfluoridated elastomers were placed on all brackets for 1 visit to allow for a washout period. At visit 3, fluoridated elastomers were placed on the teeth contralateral to those that received them at visit 1. At visit 4, the procedures at visit 2 were repeated. Samples were collected on visits 2 and 4. A logistic regression was performed, with the presence or absence of streptococcal or anaerobic growth as the dependent variable. A mixed-effects analysis of variance was carried out with the percentage of streptococcal or anaerobic bacterial count as the dependent variable. The only significant independent variables were the subject variable (P =<.001) for the percentage of streptococcal and anaerobic bacterial count and the visit variable for the percentage of streptococcal count (P =<.001). The use of fluoridated or nonfluoridated elastomers was not significant for percentage of either streptococcal (P =.288) or anaerobic count (P =.230). Fluoridated elastomers are not effective at reducing local streptococcal or anaerobic bacterial growth after a clinically relevant time in the mouth.

The incidence of oral Gram-negative bacteria in patients with Parkinson's disease.

Background: Parkinson's disease is a common neurodegenerative disorder that affects an increasing number of older people every year. Dysphagia is not only a common feature, but one that results in poor nutrition and an increased risk of bronchopneumonia. Previous work has suggested that the oral flora is altered in patients with oral pathology. Methods: Fifty patients were assessed to quantify the incidence of oral Gram-negative bacteria. Results: Sixteen of the patients with Parkinson's disease were found to have six different Gram-negative bacilli in their oral cavities. The 20 different Gram-negative bacteria present were Escherichia coli (n=7), Klebsiella spp. (n=3), Kluyvera spp. (n=3), Serratia spp. (n=3), Proteus spp. (n=2) and Enterobacter spp. (n=2). We found that the oral cavity of 16 (32%) of the patients with Parkinson's disease was abnormally colonised with Gram-negative bacteria and that Gram-negative bacteria were more likely to occur in those patients in whom oromuscular dysfunction was present (88% vs. 21%; p<0.05). Conclusion: Further work is required to determine the association between oral flora and the pathogenic organisms found in aspiration pneumonia as well as work on innovative treatments to reduce oral Gram-negative bacteria in those patients at particular risk of aspiration pneumonia.