Large numbers of long noncoding RNAs have been discovered in recent years, but only a few have been characterized. NEAT1 (nuclear paraspeckle assembly transcript 1) is a mammalian long noncoding RNA that is important for the reproductive physiology of mice, cancer development, and the formation of subnuclear bodies termed paraspeckles. The two major isoforms of NEAT1 (3.7 kb NEAT1_1 and 23 kb NEAT1_2 in human) are generated from a common promoter and are produced through the use of alternative transcription termination sites. This gene structure has made the functional relationship between the two isoforms difficult to dissect. Here we used CRISPR-Cas9 genome editing to create several different cell lines: total NEAT1 knockout cells, cells that only express the short form NEAT1_1, and cells with twofold more NEAT1_2. Using these reagents, we obtained evidence that NEAT1_1 is not a major component of paraspeckles. In addition, our data suggest NEAT1_1 localizes in numerous nonparaspeckle foci we termed “microspeckles,” which may carry paraspeckle-independent functions. This study highlights the complexity of lncRNA and showcases how genome editing tools are useful in dissecting the structural and functional roles of overlapping transcripts.

Initially described as an early onset seizure variant of Rett syndrome, the CDKL5 disorder is now considered as an independent entity. However, little is currently known about the full spectrum of comorbidities that affect these patients and available literature is limited to small case series. This study aimed to use a large international sample to examine the prevalence in this disorder of comorbidities of epilepsy, gastrointestinal problems including feeding difficulties, sleep and respiratory problems and scoliosis and their relationships with age and genotype. Prevalence and onset were also compared with those occurring in Rett syndrome. Data for the CDKL5 disorder and Rett syndrome were sourced from the International CDKL5 Disorder Database (ICDD), InterRett and the Australian Rett syndrome Database (ARSD). Logistic regression (multivariate and univariate) was used to analyse the relationships between age group, mutation type and the prevalence of various comorbidities. Binary longitudinal data from the ARSD and the equivalent cross-sectional data from ICDD were examined using generalized linear models with generalized estimating equations. The Kaplan-Meier method was used to estimate the failure function for the two disorders and the log-rank test was used to compare the two functions. The likelihood of experiencing epilepsy, GI problems, respiratory problems, and scoliosis in the CDKL5 disorder increased with age and males were more vulnerable to respiratory and sleep problems than females. We did not identify any statistically significant relationships between mutation group and prevalence of comorbidities. Epilepsy, GI problems and sleep abnormalities were more common in the CDKL5 disorder than in Rett syndrome whilst scoliosis and respiratory problems were less prevalent. This study captured a much clearer picture of the CDKL5 disorder than previously possible using the largest sample available to date. There were differences in the presentation of clinical features occurring in the CDKL5 disorder and in Rett syndrome, reinforcing the concept that CDKL5 is an independent disorder with its own distinctive characteristics.

Overcoming the massive and rapid death of injected donor myoblasts is the primary hurdle for successful myoblast transfer therapy (MTT), designed as a treatment for the lethal childhood myopathy Duchenne muscular dystrophy. The injection of male myoblasts into female host mice and quantification of surviving male DNA using the Y-chromosome-specific (Y1) probe allows the speed and extent of death of donor myoblasts to be determined. Cultured normal C57BL/10Sn male donor myoblasts were injected into untreated normal C57BL/10Sn and dystrophic mdx female host mice and analyzed by slot blots using a ³² P-labeled Y1 probe. The amount of male DNA from donor myoblasts showed a remarkable decrease within minutes and by 1 h represented only about 10–18% of the 2.5 × 10 ⁵ cells originally injected (designated 100%). This declined further over 1 week to approximately 1–4%. The host environment (normal or dystrophic) as well as the extent of passaging in tissue culture (early “P3” or late “P15–20” passage) made no difference to this result. Modulation of the host response by CD4 ⁺ /CD8 ⁺ -depleting antibodies administered prior to injection of the cultured myoblasts dramatically enhanced donor myoblast survival in dystrophic mdx hosts (15-fold relative to untreated hosts after 1 week). NK1.1 depletion also dramatically enhanced donor myoblast survival in dystrophic mdx hosts (21-fold after 1 week) compared to untreated hosts. These results provide a strategic approach to enhance donor myoblast survival in clinical trials of MTT.

Irradiation in the red/near-infrared spectrum (R/NIR, 630-1000 nm) has been used to treat a wide range of clinical conditions, including disorders of the central nervous system (CNS), with several clinical trials currently underway for stroke and macular degeneration. However, R/NIR irradiation therapy (R/NIR-IT) has not been widely adopted in clinical practice for CNS injury or disease for a number of reasons, which include the following. The mechanism/s of action and implications of penetration have not been thoroughly addressed. The large range of treatment intensities, wavelengths and devices that have been assessed make comparisons difficult, and a consensus paradigm for treatment has not yet emerged. Furthermore, the lack of consistent positive outcomes in randomised controlled trials, perhaps due to sub-optimal treatment regimens, has contributed to scepticism. This review provides a balanced précis of outcomes described in the literature regarding treatment modalities and efficacy of R/NIR-IT for injury and disease in the CNS. We have addressed the important issues of specification of treatment parameters, penetration of R/NIR irradiation to CNS tissues and mechanism/s, and provided the necessary detail to demonstrate the potential of R/NIR-IT for the treatment of retinal degeneration, damage to white matter tracts of the CNS, stroke and Parkinson's disease.