CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis.

BACKGROUND: Transthyretin amyloidosis, also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent that is designed to treat ATTR amyloidosis by reducing the concentration of TTR in serum. It is based on the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system and comprises a lipid nanoparticle encapsulating messenger RNA for Cas9 protein and a single guide RNA targeting TTR. METHODS: After conducting preclinical in vitro and in vivo studies, we evaluated the safety and pharmacodynamic effects of single escalating doses of NTLA-2001 in six patients with hereditary ATTR amyloidosis with polyneuropathy, three in each of the two initial dose groups (0.1 mg per kilogram and 0.3 mg per kilogram), within an ongoing phase 1 clinical study. RESULTS: Preclinical studies showed durable knockout of TTR after a single dose. Serial assessments of safety during the first 28 days after infusion in patients revealed few adverse events, and those that did occur were mild in grade. Dose-dependent pharmacodynamic effects were observed. At day 28, the mean reduction from baseline in serum TTR protein concentration was 52% (range, 47 to 56) in the group that received a dose of 0.1 mg per kilogram and was 87% (range, 80 to 96) in the group that received a dose of 0.3 mg per kilogram. CONCLUSIONS: In a small group of patients with hereditary ATTR amyloidosis with polyneuropathy, administration of NTLA-2001 was associated with only mild adverse events and led to decreases in serum TTR protein concentrations through targeted knockout of TTR. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).

Global Transcriptome Analysis Reveals Acclimation-Primed Processes Involved in the Acquisition of Desiccation Tolerance in Boea hygrometrica.

Boea hygrometrica resurrection plants require a period of acclimation by slow soil-drying in order to survive a subsequent period of rapid desiccation. The molecular basis of this observation was investigated by comparing gene expression profiles under different degrees of water deprivation. Transcripts were clustered according to the expression profiles in plants that were air-dried (rapid desiccation), soil-dried (gradual desiccation), rehydrated (acclimated) and air-dried after acclimation. Although phenotypically indistinguishable, it was shown by principal component analysis that the gene expression profiles in rehydrated, acclimated plants resemble those of desiccated plants more closely than those of hydrated acclimated plants. Enrichment analysis based on gene ontology was performed to deconvolute the processes that accompanied desiccation tolerance. Transcripts associated with autophagy and α-tocopherol accumulation were found to be activated in both air-dried, acclimated plants and soil-dried non-acclimated plants. Furthermore, transcripts associated with biosynthesis of ascorbic acid, cell wall catabolism, chaperone-assisted protein folding, respiration and macromolecule catabolism were activated and maintained during soil-drying and rehydration. Based on these findings, we hypothesize that activation of these processes leads to the establishment of an optimal physiological and cellular state that enables tolerance during rapid air-drying. Our study provides a novel insight into the transcriptional regulation of critical priming responses to enable survival following rapid dehydration in B. hygrometrica.

Repeat protein engineering: creating functional nanostructures/biomaterials from modular building blocks.

There is enormous interest in molecular self-assembly and the development of biological systems to form smart nanostructures for biotechnology (so-called 'bottom-up fabrications'). Repeat proteins are ideal choices for development of such systems as they: (i) possess a relatively simple relationship between sequence, structure and function; (ii) are modular and non-globular in structure; (iii) act as diverse scaffolds for the mediation of a diverse range of protein-protein interactions; and (iv) have been extensively studied and successfully engineered and designed. In the present review, we summarize recent advances in the use of engineered repeat proteins in the self-assembly of novel materials, nanostructures and biosensors. In particular, we show that repeat proteins are excellent monomeric programmable building blocks that can be triggered to associate into a range of morphologies and can readily be engineered as stimuli-responsive biofunctional materials.

Implant and patient survival rates using Exeter Trauma Stem hemiarthroplasty in fracture neck of femur patients: The largest study to date.

INTRODUCTION: Exeter Trauma Stems (ETS) femoral hemiarthroplasties are based on Exeter THR stems with a few design changes. Little has been published on ETS survival rates to justify their high cost compared to other cheaper implants. This is the largest prospective study to assess ETS implant failure-free survival rates in fracture neck of femur patients (NOF). This non-developing-centre study examined whether these design differences have altered implant survival (compared with Exeter THR's published survival data). METHODS: Data were prospectively collected by independent audit officers. Dislocation, periprosthetic fracture, re-admission with severe hip pain, deep infection and revision surgery were considered events of interest in implant failure-free survival. RESULTS: This study assessed 1,123 ETS stems (36 patients received bilateral ETS) in NOF patients. The mean patient age at the time of operation was 83 years (range; 49 - 102 years). The mean observation period was 2.5 years (range; 0 days - 8 years). Only 29 implants failed. All failure events were reported within the first year. Stem failure-free survival was 97.2% at eight years (CI 95.9% - 98%). Dislocation occurred in 10 patients (1%), periprosthetic femoral fracture in 4 (0.4%), and deep infection in 11 patients (1.2%). Patient survival rates were 75% and 48% at one and five years respectively. CONCLUSION: ETS has high implant failure-free survival rates when used in hip fractures. ETS design changes have not altered ETS survival when used in hip fractures compared with the published literature of Exeter THR stem when used as a treatment for OA. Exeter Trauma Stems in NOF patients might last these elderly patients their entire short lifetime.

Controls on sediment delivery in coastal plain rivers.

Rivers crossing coastal plains are often inefficient conveyors of sediment, so that changes in upstream sediment dynamics are not evident at the river mouth. Extensive accommodation space and low stream power often result in extensive alluvial storage upstream of estuaries and correspondingly low sediment loads at the river mouth. However, gaging stations with sediment records are typically well upstream of the coast, and thus tend to overestimate sediment yields by under-representing the lower coastal plain and because there is often a net loss of sediment in lower coastal plain reaches. Studies of alluvial sediment storage have generally focused on accommodation space, but, using examples from Texas, we show that low transport capacity controlled largely by slope is a crucial factor.

Results of Conservative and Surgical Management in Children with Idiopathic and Nonidiopathic Os Odontoideum.

OBJECTIVE: The outcomes of conservative and operative treatment of os odontoideum in children remain unclear. Our objective was to study the outcomes of conservative and surgical treatment of idiopathic os odontoideum in children and compare these outcomes in age- and treatment-matched nonidiopathic children with os odontoideum. METHODS: A retrospective multicenter review identified 102 children with os odontoideum, of whom 44 were idiopathic with minimum 2-year follow-up. Ten patients were treated conservatively, and 34 underwent spinal arthrodesis. Both groups were matched with nonidiopathic patients by age and type of treatment. Cervical arthrodesis was recommended for patients with increased atlantoaxial distance or reduced space available for the cord in flexion-extension radiographs. RESULTS: All 20 children undergoing conservative treatment remained asymptomatic during follow-up, but 1 nonidiopathic patient developed cervical instability. The idiopathic group had significantly less severe radiographic cervical instability and less neurologic complications than the nonidiopathic group (P < 0.05 for all comparisons). Thirty-three (97%) patients in the idiopathic group and 32 (94%) patients in the nonidiopathic group (94%) had spinal fusion at final follow-up (P = 0.55). The risk of complications (15% vs. 41%; odds ratio 0.234, 95% confidence interval 0.072-0.757, P = 0.015) and nonunion (6% vs. 24%; odds ratio 0.203, 95% confidence interval 0.040-0.99, P = 0.040) were significantly lower in the idiopathic than in the nonidiopathic group. Idiopathic children undergoing rigid fixation achieved spinal fusion. CONCLUSIONS: Idiopathic patients with stable atlantoaxial joint at presentation remained asymptomatic and intact during conservative treatment. Idiopathic children with os odontoideum undergoing spinal arthrodesis had significantly fewer complications and nonunion than nonidiopathic children. LEVEL OF EVIDENCE: III.

Os Odontoideum in Children: Treatment Outcomes and Neurological Risk Factors.

BACKGROUND: Treatment outcomes and risk factors for neurological deficits in pediatric patients with an os odontoideum are unclear. METHODS: We reviewed the data for 102 children with os odontoideum who were managed at 11 centers between 2000 and 2016 and had a minimum duration of follow-up of 2 years. Thirty-one children had nonoperative treatment, and 71 underwent instrumented posterior cervical spinal arthrodesis for the treatment of C1-C2 instability. Nonoperative treatment consisted of observation (n = 29) or immobilization with a cervical collar (n = 1) or halo body jacket (n = 1). Surgical treatment consisted of atlantoaxial (n = 50) or occipitocervical (n = 21) arthrodesis. One patient also underwent transoral odontoidectomy. RESULTS: Thirty children (29%) presented with neurological deficits, 28 of whom had radiographic atlantoaxial instability (atlantoaxial distance >5 mm) or limited space (≤13 mm) available for the spinal cord (risk ratio, 7.8 [95% confidence interval, 2.0 to 31] compared with children with no radiographic risk factors). The 27 children without neurological deficits or atlantoaxial instability at presentation underwent nonoperative treatment and remained asymptomatic. Of the initial nonoperative cohort, one child developed atlantoaxial instability, and another had a persistent neurological deficit; both children underwent spinal arthrodesis during the study period. One child with cervical instability declined surgery and remained asymptomatic. Spinal fusion occurred in 68 patients in the surgical group by the end of the study period (mean, 3.7 years; range, 2.0 to 11.8 years). Surgical complications occurred in 21 children, including nonunion in 12, new neurological deficits in 4, cerebrospinal fluid leak in 2, symptomatic instrumentation requiring removal 2, and vertebral artery injury in 1. Nine children underwent revision surgery. In the surgical group, Japanese Orthopaedic Association neurological function scores improved significantly from preoperatively to the latest follow-up for the upper extremities (p = 0.026) and lower extremities (p = 0.007). CONCLUSIONS: The risk of developing a neurological deficit was strongly associated with atlantoaxial instability and limited space available for the spinal cord in children with os odontoideum. Nonoperative treatment was safe for asymptomatic patients without atlantoaxial instability. Spinal arthrodesis resolved the neurological deficits of children with symptomatic os odontoideum. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Design and characterization of tissue-mimicking gel phantoms for diffusion kurtosis imaging.

PURPOSE: The aim of this work was to create tissue-mimicking gel phantoms appropriate for diffusion kurtosis imaging (DKI) for quality assurance, protocol optimization, and sequence development. METHODS: A range of agar, agarose, and polyvinyl alcohol phantoms with concentrations ranging from 1.0% to 3.5%, 0.5% to 3.0%, and 10% to 20%, respectively, and up to 3 g of glass microspheres per 100 ml were created. Diffusion coefficients, excess kurtosis values, and relaxation rates were experimentally determined. RESULTS: The kurtosis values for the plain gels ranged from 0.05 with 95% confidence interval (CI) of (0.029,0.071) to 0.216(0.185,0.246), well below the kurtosis values reported in the literature for various tissues. The addition of glass microspheres increased the kurtosis of the gels with values up to 0.523(0.465,0.581) observed for gels with the highest concentration of microspheres. Repeat scans of some of the gels after more than 6 months of storage at room temperature indicate changes in the diffusion parameters of less than 10%. The addition of the glass microspheres reduces the apparent diffusion coefficients (ADCs) and increases the longitudinal and transverse relaxation rates, but the values remain comparable to those for plain gels and tissue, with ADCs observed ranging from 818(585,1053) × 10-6  mm2 /s to 2257(2118,2296) × 10-6  mm2 /s, R1 values ranging from 0.34(0.32,0.35) 1/s to 0.51(0.50,0.52) 1/s, and R2 values ranging from 9.69(9.34,10.04) 1/s to 33.07(27.10, 39.04) 1/s. CONCLUSIONS: Glass microspheres can be used to effectively modify diffusion properties of gel phantoms and achieve a range of kurtosis values comparable to those reported for a variety of tissues.

Development of passive CLARITY and immunofluorescent labelling of multiple proteins in human cerebellum: understanding mechanisms of neurodegeneration in mitochondrial disease.

CLARITY enables immunofluorescent labelling and imaging of large volumes of tissue to provide a better insight into the three dimensional relationship between cellular morphology and spatial interactions between different cell types. In the current study, we optimise passive CLARITY and immunofluorescent labelling of neurons and mitochondrial proteins in mouse and human brain tissues to gain further insights into mechanisms of neurodegeneration occurring in mitochondrial disease. This is the first study to utilise human cerebellum fixed in paraformaldehyde and cryoprotected in conjunction with formalin-fixed tissues opening up further avenues for use of archived tissue. We optimised hydrogel-embedding and passive clearance of lipids from both mouse (n = 5) and human (n = 9) cerebellum as well as developing an immunofluorescent protocol that consistently labels different neuronal domains as well as blood vessels. In addition to visualising large structures, we were able to visualise mitochondrial proteins in passively cleared tissues to reveal respiratory chain deficiency associated with mitochondrial disease. We also demonstrate multiple use of tissues by stripping antibodies and re-probing the cerebellum. This technique allows interrogation of large volumes intact brain samples for better understanding of the complex pathological changes taking place in mitochondrial disease.

The role of systemic chemotherapy and multidisciplinary management in improving the overall survival of patients with metastatic squamous cell carcinoma of the anal canal.

Metastatic squamous cell carcinoma (SCCA) of the anal canal is a rare malignancy for which no standard treatment algorithm exists. To determine the best approach, all patients diagnosed with metastatic SCCA of the anal canal treated at a single institution were evaluated for choice of chemotherapy and treatment outcome. A retrospective study from January 2000 to May 2012 was conducted. Electronic medical records were reviewed for diagnosis of metastatic SCCA of the anal canal. All patients were treatment naïve for metastatic disease and completed all radiographic imaging at our institution. The purpose of this study was to evaluate outcomes among patients who received systemic chemotherapy and if appropriate were referred for multidisciplinary intervention (e.g., surgery, radiofrequency ablation, etc.). Seventy-seven patients fulfilled eligibility criteria. Forty-two patients (55%) received 5-fluorouracil (5-FU) + cisplatin (PF); 24 patients (31%) received carboplatin + paclitaxel (CP); 11 patients (14%) received an alternative regimen. After a median follow-up of 42 months, the median progression-free survival (PFS) for all patients was 7 months; the median overall survival (OS) was 22 months. Thirty-three patients (43%) underwent multidisciplinary management for metastatic disease resulting in a median PFS of 16 months (95% CI: 9.2 -22.8) and median OS of 53 months (95% CI: 28.3 - 77.6). Systemic chemotherapy provides durable survival for patients with surgically unresectable metastatic SCCA of the anal canal. Multidisciplinary management for select patients with metastatic disease effectively improves survival and should be considered whenever possible.