Novel variants in KAT6B spectrum of disorders expand our knowledge of clinical manifestations and molecular mechanisms.

The phenotypic variability associated with pathogenic variants in Lysine Acetyltransferase 6B (KAT6B, a.k.a. MORF, MYST4) results in several interrelated syndromes including Say-Barber-Biesecker-Young-Simpson Syndrome and Genitopatellar Syndrome. Here we present 20 new cases representing 10 novel KAT6B variants. These patients exhibit a range of clinical phenotypes including intellectual disability, mobility and language difficulties, craniofacial dysmorphology, and skeletal anomalies. Given the range of features previously described for KAT6B-related syndromes, we have identified additional phenotypes including concern for keratoconus, sensitivity to light or noise, recurring infections, and fractures in greater numbers than previously reported. We surveyed clinicians to qualitatively assess the ways families engage with genetic counselors upon diagnosis. We found that 56% (10/18) of individuals receive diagnoses before the age of 2 years (median age = 1.96 years), making it challenging to address future complications with limited accessible information and vast phenotypic severity. We used CRISPR to introduce truncating variants into the KAT6B gene in model cell lines and performed chromatin accessibility and transcriptome sequencing to identify key dysregulated pathways. This study expands the clinical spectrum and addresses the challenges to management and genetic counseling for patients with KAT6B-related disorders.

Bi-allelic Variations of SMO in Humans Cause a Broad Spectrum of Developmental Anomalies Due to Abnormal Hedgehog Signaling.

The evolutionarily conserved hedgehog (Hh) pathway is essential for organogenesis and plays critical roles in postnatal tissue maintenance and renewal. A unique feature of the vertebrate Hh pathway is that signal transduction requires the primary cilium (PC) where major pathway components are dynamically enriched. These factors include smoothened (SMO) and patched, which constitute the core reception system for sonic hedgehog (SHH) as well as GLI transcription factors, the key mediators of the pathway. Here, we report bi-allelic loss-of-function variations in SMO in seven individuals from five independent families; these variations cause a wide phenotypic spectrum of developmental anomalies affecting the brain (hypothalamic hamartoma and microcephaly), heart (atrioventricular septal defect), skeleton (postaxial polydactyly, narrow chest, and shortening of long bones), and enteric nervous system (aganglionosis). Cells derived from affected individuals showed normal ciliogenesis but severely altered Hh-signal transduction as a result of either altered PC trafficking or abnormal activation of the pathway downstream of SMO. In addition, Hh-independent GLI2 accumulation at the PC tip in cells from the affected individuals suggests a potential function of SMO in regulating basal ciliary trafficking of GLI2 when the pathway is off. Thus, loss of SMO function results in abnormal PC dynamics of key components of the Hh signaling pathway and leads to a large continuum of malformations in humans.

An efficient TOF-SIMS image analysis with spatial correlation and alternating non-negativity-constrained least squares.

MOTIVATION: Advances in analytical instrumentation towards acquiring high-resolution images of mass spectrometry constantly demand efficient approaches for data analysis. This is particularly true of time-of-flight secondary ion mass spectrometry imaging where recent advances enable acquisition of high-resolution data in multiple dimensions. In many applications, the distribution of different species from a sampled surface is spatially continuous in nature and a model that incorporates the spatial correlation across the surface would be preferable to estimations at discrete spatial locations. A key challenge here is the capability to analyse the high-resolution multidimensional data to extract relevant information reliably and efficiently. RESULTS: We propose a framework based on alternating non-negativity-constrained least squares which accounts for the spatial correlation across the sample surface. The proposed method also decouples the computational complexity of the estimation procedure from the image resolution, which significantly reduces the processing time. We evaluate the performance of the algorithm with biochemical image datasets generated from mixture of metabolites.

Multifunctional receptor-targeted nanocomplexes for the delivery of therapeutic nucleic acids to the brain.

Convection enhanced delivery (CED) is a method of direct injection to the brain that can achieve widespread dispersal of therapeutics, including gene therapies, from a single dose. Non-viral, nanocomplexes are of interest as vectors for gene therapy in the brain, but it is essential that administration should achieve maximal dispersal to minimise the number of injections required. We hypothesised that anionic nanocomplexes administered by CED should disperse more widely in rat brains than cationics of similar size, which bind electrostatically to cell-surface anionic moieties such as proteoglycans, limiting their spread. Anionic, receptor-targeted nanocomplexes (RTN) containing a neurotensin-targeting peptide were prepared with plasmid DNA and compared with cationic RTNs for dispersal and transfection efficiency. Both RTNs were labelled with gadolinium for localisation in the brain by MRI and in brain sections by LA-ICP-MS, as well as with rhodamine fluorophore for detection by fluorescence microscopy. MRI distribution studies confirmed that the anionic RTNs dispersed more widely than cationic RTNs, particularly in the corpus callosum. Gene expression levels from anionic formulations were similar to those of cationic RTNs. Thus, anionic RTN formulations can achieve both widespread dispersal and effective gene expression in brains after administration of a single dose by CED.

Views of recently first-certified US child neurologists on their residency training.

We surveyed child neurologists first certified in "Neurology with Special Qualification in Child Neurology" by the American Board of Psychiatry and Neurology (ABPN) between 2001 and 2010 using a 24-item questionnaire. Respondents (n = 204, 54% response rate) were between the ages of 30 and 59 years (54% male), and 68% completed adult neurology training in a 10- to 12-month, primarily inpatient block. Sixty-two percent of the sample completed subspecialty fellowship training and 82% currently reported practicing within a hospital or hospital-based/owned clinic. Current practice data showed just 3% provide general neurology services to adults. A majority reported using adult neurology residency training "less than weekly" and believed the ideal model for residency training in diagnosis and management of both common and rare neurologic conditions would involve less time in adult neurology and more time (mean 6 months) in child neurology, most prominently in genetics and developmental and behavioral areas.

Lipid peptide nanocomplexes for gene delivery and magnetic resonance imaging in the brain.

Gadolinium-labelled nanocomplexes offer prospects for the development of real-time, non-invasive imaging strategies to visualise the location of gene delivery by MRI. In this study, targeted nanoparticle formulations were prepared comprising a cationic liposome (L) containing a Gd-chelated lipid at 10, 15 and 20% by weight of total lipid, a receptor-targeted, DNA-binding peptide (P) and plasmid DNA (D), which electrostatically self-assembled into LPD nanocomplexes. The LPD formulation containing the liposome with 15% Gd-chelated lipid displayed optimal peptide-targeted, transfection efficiency. MRI conspicuity peaked at 4h after incubation of the nanocomplexes with cells, suggesting enhancement by cellular uptake and trafficking. This was supported by time course confocal microscopy analysis of transfections with fluorescently-labelled LPD nanocomplexes. Gd-LPD nanocomplexes delivered to rat brains by convection-enhanced delivery were visible by MRI at 6 h, 24 h and 48 h after administration. Histological brain sections analysed by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) confirmed that the MRI signal was associated with the distribution of Gd(3+) moieties and differentiated MRI signals due to haemorrhage. The transfected brain cells near the injection site appeared to be mostly microglial. This study shows the potential of Gd-LPD nanocomplexes for simultaneous delivery of contrast agents and genes for real-time monitoring of gene therapy in the brain.

Synthesis and characterization of a theranostic vascular disrupting agent for in vivo MR imaging.

Colchicine, a known tubulin binding agent and vascular disrupting agent, causes rapid vascular shut down and central necrosis in tumors. The binding of tubulin results in tubulin destabilization, with characteristic cell shape changes and inhibition of cell division, and results in cell death. A gadolinium(III) labeled derivative of colchicine (Gd·DOTA·Colchicinic acid) was synthesized and characterized as a theranostic agent (enabling simultaneous diagnostic/real time MRI contrast imaging). In vitro, Gd·DOTA·Colchicinic acid was shown to initiate cell changes characteristic of tubulin-destabilization in both OVCAR-3 and IGROV-1 ovarian carcinoma cell lines in vitro over a period of 24 h, while maintaining the qualities of the MR imaging tracer. In vivo, Gd·DOTA·Colchicinic acid (200 mg/kg) was shown to induce the formation of central necrosis, which was confirmed ex vivo by histology, in OVCAR-3 subcutaneous tumor xenografts, while simultaneously acting as an imaging agent to promote a significant reduction in the MR relaxation time T(1) (p < 0.05) of tumors 24 h post-administration. Morphological changes within the tumor which corresponded with areas derived from the formation of central necrosis were also present on MR images that were not observed for the same colchicine derivate that was not complexed with gadolinium that also presented with central necrosis ex vivo. However, Gd·DOTA·Colchicinic acid accumulation in the liver, as shown by changes in liver T(1) (p < 0.05), takes place within 2 h. The implication is that Gd·DOTA·Colchicinic acid distributes to tissues, including tumors, within 2 h, but enters tumor cells to lower T(1) times and promotes cell death over a period of up to 24 h. As the biodistribution/pharmacokinetic and pharmacodynamics data provided here is similar to that of conventional colchicines derivatives, such combined data are a potentially powerful way to rapidly characterize the complete behavior of drug candidates in vivo.

A low molecular weight folate receptor targeted contrast agent for magnetic resonance tumor imaging.

PURPOSE: This study aims to develop a low molecular weight folate receptor (FR) contrast agent for MR tumor imaging. PROCEDURES: Gadolinium-tetraazacyclododecane tetraacetic acid (Gd.DOTA) was conjugated to folic acid to create Gd.DOTA.Folate. The efficacy of Gd.DOTA.Folate to bind FR was evaluated in vitro by inductively coupled mass spectrometry (ICP-MS) and in vivo by magnetic resonance imaging (MRI) tumor enhancement over 14 h, utilizing an overexpressing α-FR cell line (IGROV-1), compared to an α-FR-negative cell line (OVCAR-3). Gd.DOTA.Folate localization ex vivo was verified by laser ablation ICP-MS. RESULTS: ICP-MS confirmed Gd.DOTA.Folate uptake by IGROV-1 cells and competitive binding with free folic acid inhibited binding. IGROV-1 tumors showed an increase in R (1) at 2 h, which increased significantly over 14 h post-Gd.DOTA.Folate with clear enhancement on MR images. This was not observed in controls. CONCLUSION: These data support the use of FR-targeted small molecular weight MRI contrast agents for tumor imaging in vivo.

Manganese enhancement in non-CNS organs.

Manganese-enhanced magnetic resonance imaging (MEMRI) is a novel imaging technique capable of monitoring calcium influx, in vivo. Manganese (Mn2+) ions, similar to calcium ions (Ca2+), are taken up by activated cells where their paramagnetic properties afford signal enhancement in T(1)-weighted MRI methodologies. In this study we have assessed Mn2+ distribution in mice using magnetization-prepared rapid gradient echo (MP-RAGE) based MRI, by measuring changes in T(1)-effective relaxation times (T(1)-eff), effective R(1)-relaxation rates (R(1)-eff) and signal intensity (SI) profiles over time. The manganese concentration in the tissue was also determined using inductively coupled plasma atomic emission spectrometry (ICP-AES). Our results show a strong positive correlation between infused dose of MnCl2 and the tissue manganese concentration. Furthermore, we demonstrate a linear relationship between R(1)-eff and tissue manganese concentration and tissue-specific Mn2+ distribution in murine tissues following dose-dependent Mn2+ administration. This data provides an optimized MnCl2 dose regimen for an MP-RAGE based sequence protocol for specific target organs and presents a potential 3D MRI technique for in vivo imaging of Ca2+ entry during Ca2+-dependent processes in a wide range of tissues.

Imaging of gadolinium spatial distribution in tumor tissue by laser ablation inductively coupled plasma mass spectrometry.

PURPOSE: Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was utilized in postmortem imaging of gadolinium (Gd) spatial distribution in a mouse tumor model postadministration of PEGylated Gd liposomal nanoparticles. PROCEDURES: PEGylated liposomal nanoparticles were formulated using a paramagnetic lipid incorporating Gd, in addition to a fluorescent lipid, and injected intravenously into Balb/C nude mice bearing IGROV-1 tumors. At postinjection (2 h), the tumors and selective organs were imaged by magnetic resonance imaging (MRI) and, after excision, by histology and LA-ICP-MS. RESULTS: The presence of Gd within tumor tissue was confirmed by LA-ICP-MS and when correlated to histology was found to be prevalent in regions of higher vascularity. The presence of Gd in the kidneys was also confirmed. CONCLUSIONS: We have demonstrated, in a novel manner, the use of LA-ICP-MS for the spatial detection of Gd in tumor tissue. LA-ICP-MS is valuable in providing spatio-specific information of MRI contrast agents and more importantly Gd in tumor tissue.