Non-Invasive Targeted Peripheral Nerve Ablation Using 3D MR Neurography and MRI-Guided High-Intensity Focused Ultrasound (MR-HIFU): Pilot Study in a Swine Model

Purpose

Ultrasound (US)-guided high intensity focused ultrasound (HIFU) has been proposed for noninvasive treatment of neuropathic pain and has been investigated in in-vivo studies. However, ultrasound has important limitations regarding treatment guidance and temperature monitoring. Magnetic resonance (MR)-imaging guidance may overcome these limitations and MR-guided HIFU (MR-HIFU) has been used successfully for other clinical indications. The primary purpose of this study was to evaluate the feasibility of utilizing 3D MR neurography to identify and guide ablation of peripheral nerves using a clinical MR-HIFU system.

Methods

Volumetric MR-HIFU was used to induce lesions in the peripheral nerves of the lower limbs in three pigs. Diffusion-prep MR neurography and T1-weighted images were utilized to identify the target, plan treatment and immediate post-treatment evaluation. For each treatment, one 8 or 12 mm diameter treatment cell was used (sonication duration 20 s and 36 s, power 160–300 W). Peripheral nerves were extracted < 3 hours after treatment. Ablation dimensions were calculated from thermal maps, post-contrast MRI and macroscopy. Histological analysis included standard H&E staining, Masson’s trichrome and toluidine blue staining.

Results

All targeted peripheral nerves were identifiable on MR neurography and T1-weighted images and could be accurately ablated with a single exposure of focused ultrasound, with peak temperatures of 60.3 to 85.7°C. The lesion dimensions as measured on MR neurography were similar to the lesion dimensions as measured on CE-T1, thermal dose maps, and macroscopy. Histology indicated major hyperacute peripheral nerve damage, mostly confined to the location targeted for ablation.

Conclusion

Our preliminary results indicate that targeted peripheral nerve ablation is feasible with MR-HIFU. Diffusion-prep 3D MR neurography has potential for guiding therapy procedures where either nerve targeting or avoidance is desired, and may also have potential for post-treatment verification of thermal lesions without contrast injection.     

Methylation of HOXA9 and ISL1 Predicts Patient Outcome in High-Grade Non-Invasive Bladder Cancer

Introduction

Inappropriate DNA methylation is frequently associated with human tumour development, and in specific cases, is associated with clinical outcomes. Previous reports of DNA methylation in low/intermediate grade non-muscle invasive bladder cancer (NMIBC) have suggested that specific patterns of DNA methylation may have a role as diagnostic or prognostic biomarkers. In view of the aggressive and clinically unpredictable nature of high-grade (HG) NMIBC, and the current shortage of the preferred treatment option (Bacillus:Calmette-Guerin), novel methylation analyses may similarly reveal biomarkers of disease outcome that could risk-stratify patients and guide clinical management at initial diagnosis.

Methods

Promoter-associated CpG island methylation was determined in primary tumour tissue of 36 initial presentation high-grade NMIBCs, 12 low/intermediate-grade NMIBCs and 3 normal bladder controls. The genes HOXA9, ISL1, NKX6-2, SPAG6, ZIC1 and ZNF154 were selected for investigation on the basis of previous reports and/or prognostic utility in low/intermediate-grade NMIBC. Methylation was determined by Pyrosequencing of sodium-bisulphite converted DNA, and then correlated with gene expression using RT-qPCR. Methylation was additionally correlated with tumour behaviour, including tumour recurrence and progression to muscle invasive bladder cancer or metastases.

Results

The ISL1 genes’ promoter-associated island was more frequently methylated in recurrent and progressive high-grade tumours than their non-recurrent counterparts (60.0% vs. 18.2%, p = 0.008). ISL1 and HOXA9 showed significantly higher mean methylation in recurrent and progressive tumours compared to non-recurrent tumours (43.3% vs. 20.9%, p = 0.016 and 34.5% vs 17.6%, p = 0.017, respectively). Concurrent ISL1/HOXA9 methylation in HG-NMIBC reliably predicted tumour recurrence and progression within one year (Positive Predictive Value 91.7%), and was associated with disease-specific mortality (DSM).

Conclusions

In this study we report methylation differences and similarities between clinical sub-types of high-grade NMIBC. We report the potential ability of methylation biomarkers, at initial diagnosis, to predict tumour recurrence and progression within one year of diagnosis. We found that specific biomarkers reliably predict disease outcome and therefore may help guide patient treatment despite the unpredictable clinical course and heterogeneity of high-grade NMIBC. Further investigation is required, including validation in a larger patient cohort, to confirm the clinical utility of methylation biomarkers in high-grade NMIBC.     

Molecular organization and dynamics of the melatonin MT1 receptor/RGS20/Gi protein complex reveal asymmetry of receptor dimers for RGS and Gi coupling

Functional asymmetry of G‐protein‐coupled receptor (GPCR) dimers has been reported for an increasing number of cases, but the molecular architecture of signalling units associated to these dimers remains unclear. Here, we characterized the molecular complex of the melatonin MT₁ receptor, which directly and constitutively couples to G_i proteins and the regulator of G‐protein signalling (RGS) 20. The molecular organization of the ternary MT₁/G_i/RGS20 complex was monitored in its basal and activated state by bioluminescence resonance energy transfer between probes inserted at multiple sites of the complex. On the basis of the reported crystal structures of G_i and the RGS domain, we propose a model wherein one G_i and one RGS20 protein bind to separate protomers of MT₁ dimers in a pre‐associated complex that rearranges upon agonist activation. This model was further validated with MT₁/MT₂ heterodimers. Collectively, our data extend the concept of asymmetry within GPCR dimers, reinforce the notion of receptor specificity for RGS proteins and highlight the advantage of GPCRs organized as dimers in which each protomer fulfils its specific task by binding to different GPCR‐interacting proteins.     

Tobacco mutants with reduced microtubule dynamics are less susceptible to TMV

A panel of seven SR1 tobacco mutants (ATER1 to ATER7) derived via T‐DNA activation tagging and screening for resistance to a microtubule assembly inhibitor, ethyl phenyl carbamate, were used to study the role of microtubules during infection and spread of tobacco mosaic virus (TMV). In one of these lines, ATER2, α‐tubulin is shifted from the tyrosinylated into the detyrosinated form, and the microtubule plus‐end marker GFP–EB1 moves significantly slower when expressed in the background of the ATER2 mutant as compared with the SR1 wild type. The efficiency of cell‐to‐cell movement of TMV encoding GFP‐tagged movement protein (MP‐GFP) is reduced in ATER2 accompanied by a reduced association of MP‐GFP with plasmodesmata. This mutant is also more tolerant to viral infection as compared with the SR1 wild type, implying that reduced microtubule dynamics confer a comparative advantage in face of TMV infection.     

6-Phenyl-1H-imidazo[4,5-c]pyridine-4-carbonitrile as cathepsin S inhibitors

6-Phenyl-1H-imidazo[4,5-c]pyridine-4-carbonitrile analogues were identified as potent and selective cathepsin S inhibitor against both purified enzyme and in human JY cell based cellular assays. This core has a very stable thio-trapping nitrile war-head in comparison with the well reported pyrimidine-2-carbonitrile cysteine cathepsin inhibitors. Compound 47 is also very potent in in vivo mouse spleenic Lip10 accumulation assays.     

Role of Spatial and Temporal Refuges in the Evolution of Pest Resistance to Toxic Crops

Toxic plants have been used for years in agriculture to control major crop pests. However, the continuous exposure of targeted pests to toxins dramatically increases the rate of resistance evolution (Gassman et al. in Annu Rev Entomol 54:147–163, 2009a; Tabashnik et al. Nat Biotechnol 26:199–202, 2008). To prevent or delay resistance, non toxic host plants can be used as refuges. Our study considers spatial and temporal refuges that are respectively implemented concurrently or alternatively a toxic crop. A conceptual model based on impulsive differential equations is proposed to describe the dynamics of the susceptible and resistant pest populations over time. The mathematical study enlightens threshold values of the proportion of the spatial refuge and key parameters that should help to understand evolution of pest resistance to toxic crop.