DNA origami nanorulers and emerging reference structures

The DNA origami technique itself is considered a milestone of DNA nanotechnology and DNA origami nanorulers represent the first widespread application of this technique. DNA origami nanorulers are used to demonstrate the capabilities of techniques and are valuable training samples. They have meanwhile been developed for a multitude of microscopy methods including optical microscopy, atomic force microscopy, and electron microscopy, and their unique properties are further exploited to develop point-light sources, brightness references, nanophotonic test structures, and alignment tools for correlative microscopy. In this perspective, we provide an overview of the basics of DNA origami nanorulers and their increasing applications in fields of optical and especially super-resolution fluorescence microscopy. In addition, emerging applications of reference structures based on DNA origami are discussed together with recent developments

AluY-mediated germline deletion, duplication and somatic stem cell reversion in <i>UBE2T</i> defines a new subtype of Fanconi anemia

Fanconi anemia (FA) is a rare inherited disorder clinically characterized by congenital malformations, progressive bone marrow failure and cancer susceptibility. At the cellular level, FA is associated with hypersensitivity to DNA-crosslinking genotoxins. Eight of 17 known FA genes assemble the FA E3 ligase complex, which catalyzes monoubiquitination of FANCD2 and is essential for replicative DNA crosslink repair. Here, we identify the first FA patient with biallelic germline mutations in the ubiquitin E2 conjugase UBE2T. Both mutations were aluY-mediated: a paternal deletion and maternal duplication of exons 2-6. These loss-of-function mutations in UBE2T induced a cellular phenotype similar to biallelic defects in early FA genes with the absence of FANCD2 monoubiquitination. The maternal duplication produced a mutant mRNA that could encode a functional protein but was degraded by nonsense-mediated mRNA decay. In the patient's hematopoietic stem cells, the maternal allele with the duplication of exons 2-6 spontaneously reverted to a wild-type allele by monoallelic recombination at the duplicated aluY repeat, thereby preventing bone marrow failure. Analysis of germline DNA of 814 normal individuals and 850 breast cancer patients for deletion or duplication of UBE2T exons 2-6 identified the deletion in only two controls, suggesting aluY-mediated recombinations within the UBE2T locus are rare and not associated with an increased breast cancer risk. Finally, a loss-of-function germline mutation in UBE2T was detected in a high-risk breast cancer patient with wild-type BRCA1/2. Cumulatively, we identified UBE2T as a bona fide FA gene (FANCT) that also may be a rare cancer susceptibility gene.</p

Self-Regeneration and Self-Healing in DNA Origami Nanostructures

DNA nanotechnology and advances in the DNA origami technique have enabled facile design and synthesis of complex and functional nanostructures. Molecular devices are, however, prone to rapid functional and structural degradation due to the high proportion of surface atoms at the nanoscale and due to complex working environments. Besides stabilizing mechanisms, approach for the self‐repair of functional molecular devices are desirable. Here we exploit the self‐assembly and reconfigurability of DNA origami nanostructures to induce the self‐repair of defects of photoinduced and enzymatic damage. With different examples of repair in DNA nanostructures, we distinguish between unspecific self‐regeneration and damage specific self‐healing mechanisms. Using DNA origami nanorulers studied by atomic force and superresolution DNA PAINT microscopy, quantitative preservation of fluorescence properties is demonstrated with direct potential for improving nanoscale calibration samples

A new variant of Abernethy malformation treated by transhepatic interventional closure: a case report

BACKGROUND: Congenital portosystemic shunts (CPSS) are rare vascular malformations and can be classified into extrahepatic and intrahepatic shunts. Extrahepatic CPSS, also termed Abernethy malformations are associated with severe long-term complications including portopulmonary hypertension, liver atrophy, hyperammoniemia and hepatic encephalopathy. We report a hitherto undescribed variant of Abernethy malformation requiring an innovative approach for interventional treatment. CASE PRESENTATION: We describe a 31-year-old patient following surgical repair of atrioventricular septal defect at the age of 6 years. In the long-term follow-up he showed persistent pulmonary hypertension which deteriorated despite dual pulmonary vasodilative treatment. When he developed arterial desaturation and symptomatic hyperammoniemia detailed reassessment revealed as underlying cause a hitherto undescribed variant of Abernethy malformation connecting the portal vein with the right lower pulmonary vein. Following interdisciplinary discussions we opted for an interventional approach. Since the malformation was un-accessible to interventional closure via antegrade venous or retrograde arterial access, a transhepatic percutaneous puncture of the portal vein was performed. Temporary balloon occlusion of the malformation revealed only a slight increase in portal venous pressure. Interventional occlusion of the large vascular connection was achieved via this transhepatic approach by successive implantation of two large vascular occluding devices. The postinterventional course was unremarkable and both ammonia levels and arterial saturation normalized at follow-up of 12 months. CONCLUSIONS: Portal vein anomalies should be included in the differential diagnoses of pulmonary hypertension or pulmonary arterio-venous malformations. Based on careful assessment of the anatomy and testing of portal vein hemodynamics interventional therapy of complex Abernethy malformations can be performed successfully in specialized centers

A clinicogenomic model including GARD predicts outcome for radiation treated patients with HPV+ oropharyngeal squamous cell carcinoma

BACKGROUND: Treatment decision-making in oropharyngeal squamous cell carcinoma (OPSCC) includes clinical stage, HPV status, and smoking history. Despite improvements in staging with separation of HPV-positive and -negative OPSCC in AJCC 8th edition (AJCC8), patients are largely treated with a uniform approach, with recent efforts focused on de-intensification in low-risk patients. We have previously shown, in a pooled analysis, that the genomic adjusted radiation dose (GARD) is predictive of radiation treatment benefit and can be used to guide RT dose selection. We hypothesize that GARD can be used to predict overall survival (OS) in HPV-positive OPSCC patients treated with radiotherapy (RT). METHODS: Gene expression profiles (Affymetrix Clariom D) were analyzed for 234 formalin-fixed paraffin-embedded samples from HPV-positive OPSCC patients within an international, multi-institutional, prospective/retrospective observational study including patients with AJCC 7th edition stage III-IVb. GARD, a measure of the treatment effect of RT, was calculated for each patient as previously described. In total, 191 patients received primary RT definitive treatment (chemoradiation or RT alone, and 43 patients received post-operative RT. Two RT dose fractionations were utilized for primary RT cases (70 Gy in 35 fractions or 69.96 Gy in 33 fractions). Median RT dose was 70 Gy (range 50.88-74) for primary RT definitive cases and 66 Gy (range 44-70) for post-operative RT cases. The median follow up was 46.2 months (95% CI, 33.5-63.1). Cox proportional hazards analyses were performed with GARD as both a continuous and dichotomous variable and time-dependent ROC analyses compared the performance of GARD with the NRG clinical nomogram for overall survival. RESULTS: Despite uniform radiation dose utilization, GARD showed significant heterogeneity (range 30-110), reflecting the underlying genomic differences in the cohort. On multivariable analysis, each unit increase in GARD was associated with an improvement in OS (HR = 0.951 (0.911, 0.993), p = 0.023) compared to AJCC8 (HR = 1.999 (0.791, 5.047)), p = 0.143). ROC analysis for GARD at 36 months yielded an AUC of 80.6 (69.4, 91.9) compared with an AUC of 73.6 (55.4, 91.7) for the NRG clinical nomogram. GARD=64.2 was associated with improved OS (HR = 0.280 (0.100, 0.781), p = 0.015). In a virtual trial, GARD predicts that uniform RT dose de-escalation results in overall inferior OS but proposes two separate genomic strategies where selective RT dose de-escalation in GARD-selected populations results in clinical equipoise. CONCLUSIONS: In this multi-institutional cohort of patients with HPV-positive OPSCC, GARD predicts OS as a continuous variable, outperforms the NRG nomogram and provides a novel genomic strategy to modern clinical trial design. We propose that GARD, which provides the first opportunity for genomic guided personalization of radiation dose, should be incorporated in the diagnostic workup of HPV-positive OPSCC patients

Immune-based classification of HPV-associated oropharyngeal cancer with implications for biomarker-driven treatment de-intensification.

BACKGROUND: There is significant interest in treatment de-escalation for human papillomavirus-associated (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients given the generally favourable prognosis. However, 15-30% of patients recur after primary treatment, reflecting a need for improved risk-stratification tools. We sought to develop a molecular test to risk stratify HPV+ OPSCC patients. METHODS: We created an immune score (UWO3) associated with survival outcomes in six independent cohorts comprising 906 patients, including blinded retrospective and prospective external validations. Two aggressive radiation de-escalation cohorts were used to assess the ability of UWO3 to identify patients who recur. Multivariate Cox models were used to assess the associations between the UWO3 immune class and outcomes. FINDINGS: A three-gene immune score classified patients into three immune classes (immune rich, mixed, or immune desert) and was strongly associated with disease-free survival in six datasets, including large retrospective and prospective datasets. Pooled analysis demonstrated that the immune rich group had superior disease-free survival compared to the immune desert (HR&nbsp;=&nbsp;9.0, 95% CI: 3.2-25.5, P&nbsp;=&nbsp;3.6&nbsp;×&nbsp;10-5) and mixed (HR&nbsp;=&nbsp;6.4, 95% CI: 2.2-18.7, P&nbsp;=&nbsp;0.006) groups after adjusting for age, sex, smoking status, and AJCC8 clinical stage. Finally, UWO3 was able to identify patients from two small treatment de-escalation cohorts who remain disease-free after aggressive de-escalation to 30&nbsp;Gy radiation. INTERPRETATION: With additional prospective validation, the UWO3 score could enable biomarker-driven clinical decision-making for patients with HPV+ OPSCC based on robust outcome prediction across six independent cohorts. Prospective de-escalation and intensification clinical trials are currently being planned. FUNDING: CIHR, European Union, and the NIH