Multilayered stable 2D nano-sheets of Ti2NTx MXene: synthesis, characterization, and anticancer activity.

BACKGROUND: The biological activity of MXenes has been studied for several years because of their potential biomedical applications; however, investigations have so far been limited to 2D titanium carbides. Although monolayered Ti2NTx MXene has been expected to have biological activity, experimental studies revealed significant difficulties due to obstacles to its synthesis, its low stability and its susceptibility to oxidation and decomposition. RESULTS: In this paper, we report our theoretical calculations showing the higher likelihood of forming multilayered Ti2NTx structures during the preparation process in comparison to single-layered structures. As a result of our experimental work, we successfully synthesized multilayered Ti2NTx MXene that was suitable for biological studies by the etching of the Ti2AlN MAX phase and further delamination. The biocompatibility of Ti2NTx MXene was evaluated in vitro towards human skin malignant melanoma cells, human immortalized keratinocytes, human breast cancer cells, and normal human mammary epithelial cells. Additionally, the potential mode of action of 2D Ti2NTx was investigated using reactive oxygen tests as well as SEM observations. Our results indicated that multilayered 2D sheets of Ti2NTx showed higher toxicity towards cancerous cell lines in comparison to normal ones. The decrease in cell viabilities was dose-dependent. The generation of reactive oxygen species as well as the internalization of the 2D sheets play a decisive role in the mechanisms of toxicity. CONCLUSIONS: We have shown that 2D Ti2NTx in the form of multilayered nanoflakes exhibits fair stability and can be used for in vitro studies. These results show promise for its future applications in biotechnology and nanomedicine.

A homozygous I684T in GLE1 as a novel cause of arthrogryposis and motor neuron loss.

Mutations in GLE1, RNA export mediator (GLE1) gene have previously been shown to cause motor neuron diseases such as lethal congenital contracture syndrome 1 (LCCS1) and lethal arthrogryposis with anterior horn cell disease (LAAHD), including arthrogryposis, fetal akinesis and motor neuron loss as common clinical features. The homozygous FinMajor mutation p.T144_E145insPFQ has been described as one of the causes for LCCS1 whereas LAAHD is caused by a heterocompound FinMajor mutation together with p.R569H, p.V617M or p.I684T missense mutation. None of these heterocompound missense mutations have previously been reported as homozygous states. Here we present the clinical features of 2 siblings with a homozygous p.I684T mutation in GLE1. The patients suffered from similar, but milder symptoms than in LCCS1 and LAAHD, surviving up to 6 months before they died due to a progressive disease course including respiratory failure. Arthrogryposis, lack of spontaneous movements, and epilepsy were notable in both cases and lack of anterior horn cells was identified in autopsy samples. Our studies on patient-derived fibroblasts show that the homozygous p.I684T impairs the nuclear localization of GLE1 further confirming the pathogenic role of this mutation.

Recessive mutations in NDUFA2 cause mitochondrial leukoencephalopathy.

Deficiencies of mitochondrial respiratory chain complex I frequently result in leukoencephalopathy in young patients, and different mutations in the genes encoding its subunits are still being uncovered. We report 2 patients with cystic leukoencephalopathy and complex I deficiency with recessive mutations in NDUFA2, an accessory subunit of complex I. The first patient was initially diagnosed with a primary systemic carnitine deficiency associated with a homozygous variant in SLC22A5, but also exhibited developmental regression and cystic leukoencephalopathy, and an additional diagnosis of complex I deficiency was suspected. Biochemical analysis confirmed a complex I deficiency, and whole-exome sequencing revealed a homozygous mutation in NDUFA2 (c.134A>C, p.Lys45Thr). Review of a biorepository of patients with unsolved genetic leukoencephalopathies who underwent whole-exome or genome sequencing allowed us to identify a second patient with compound heterozygous mutations in NDUFA2 (c.134A>C, p.Lys45Thr; c.225del, p.Asn76Metfs*4). Only 1 other patient with mutations in NDUFA2 and a different phenotype (Leigh syndrome) has previously been reported. This is the first report of cystic leukoencephalopathy caused by mutations in NDUFA2.

Whole-exome sequencing is a valuable diagnostic tool for inherited peripheral neuropathies: Outcomes from a cohort of 50 families.

The inherited peripheral neuropathies (IPNs) are characterized by marked clinical and genetic heterogeneity and include relatively frequent presentations such as Charcot-Marie-Tooth disease and hereditary motor neuropathy, as well as more rare conditions where peripheral neuropathy is associated with additional features. There are over 250 genes known to cause IPN-related disorders but it is estimated that in approximately 50% of affected individuals a molecular diagnosis is not achieved. In this study, we examine the diagnostic utility of whole-exome sequencing (WES) in a cohort of 50 families with 1 or more affected individuals with a molecularly undiagnosed IPN with or without additional features. Pathogenic or likely pathogenic variants in genes known to cause IPN were identified in 24% (12/50) of the families. A further 22% (11/50) of families carried sequence variants in IPN genes in which the significance remains unclear. An additional 12% (6/50) of families had variants in novel IPN candidate genes, 3 of which have been published thus far as novel discoveries (KIF1A, TBCK, and MCM3AP). This study highlights the use of WES in the molecular diagnostic approach of highly heterogeneous disorders, such as IPNs, places it in context of other published neuropathy cohorts, while further highlighting associated benefits for discovery.

Debunking Occam's razor: Diagnosing multiple genetic diseases in families by whole-exome sequencing.

BACKGROUND: Recent clinical whole exome sequencing (WES) cohorts have identified unanticipated multiple genetic diagnoses in single patients. However, the frequency of multiple genetic diagnoses in families is largely unknown. AIMS: We set out to identify the rate of multiple genetic diagnoses in probands and their families referred for analysis in two national research programs in Canada. MATERIALS & METHODS: We retrospectively analyzed WES results for 802 undiagnosed probands referred over the past 5 years in either the FORGE or Care4Rare Canada WES initiatives. RESULTS: Of the 802 probands, 226 (28.2%) were diagnosed based on mutations in known disease genes. Eight (3.5%) had two or more genetic diagnoses explaining their clinical phenotype, a rate in keeping with the large published studies (average 4.3%; 1.4 - 7.2%). Seven of the 8 probands had family members with one or more of the molecularly diagnosed diseases. Consanguinity and multisystem disease appeared to increase the likelihood of multiple genetic diagnoses in a family. CONCLUSION: Our findings highlight the importance of comprehensive clinical phenotyping of family members to ultimately provide accurate genetic counseling.

Loss of the arginine methyltranserase PRMT7 causes syndromic intellectual disability with microcephaly and brachydactyly.

Post-translational protein modifications exponentially expand the functional complement of proteins encoded by the human genome. One such modification is the covalent addition of a methyl group to arginine or lysine residues, which is used to regulate a substantial proportion of the proteome. Arginine and lysine methylation are catalyzed by protein arginine methyltransferase (PRMTs) and protein lysine methyltransferase proteins (PKMTs), respectively; each methyltransferase has a specific set of target substrates. Here, we report a male with severe intellectual disability, facial dysmorphism, microcephaly, short stature, brachydactyly, cryptorchidism and seizures who was found to have a homozygous 15,309 bp deletion encompassing the transcription start site of PRMT7, which we confirmed is functionally a null allele. We show that the patient's cells have decreased levels of protein arginine methylation, and that affected proteins include the essential histones, H2B and H4. Finally, we demonstrate that patient cells have altered Wnt signaling, which may have contributed to the skeletal abnormalities. Our findings confirm the recent disease association of PRMT7, expand the phenotypic manifestations of this disorder and provide insight into the molecular pathogenesis of this new condition.

Comparison of the mineral and fatty acid profiles of ostrich, turkey and broiler chicken livers.

The aim of this study was to determine the mineral and lipid profiles in ostrich, turkey and broiler chicken livers. The highest concentrations of phosphorus, potassium and magnesium were found in turkey livers, at 3751.50, 2153.05 and 363.98 mg/kg fresh weight (FW), respectively, whereas ostrich livers were the richest in calcium (82.77 mg/kg FW). Ostrich liver was the richest source of iron, calcium, zinc and silicon, at 947.8, 82.77, 47.50, 36.69 mg/kg FW, respectively. Ostrich livers proved to be the richest source of long-chain fatty acids (5.18% C20:4n+6, 0.48% EPA and 1.08% DHA).

Utility of whole-exome sequencing for those near the end of the diagnostic odyssey: time to address gaps in care.

An accurate diagnosis is an integral component of patient care for children with rare genetic disease. Recent advances in sequencing, in particular whole-exome sequencing (WES), are identifying the genetic basis of disease for 25-40% of patients. The diagnostic rate is probably influenced by when in the diagnostic process WES is used. The Finding Of Rare Disease GEnes (FORGE) Canada project was a nation-wide effort to identify mutations for childhood-onset disorders using WES. Most children enrolled in the FORGE project were toward the end of the diagnostic odyssey. The two primary outcomes of FORGE were novel gene discovery and the identification of mutations in genes known to cause disease. In the latter instance, WES identified mutations in known disease genes for 105 of 362 families studied (29%), thereby informing the impact of WES in the setting of the diagnostic odyssey. Our analysis of this dataset showed that these known disease genes were not identified prior to WES enrollment for two key reasons: genetic heterogeneity associated with a clinical diagnosis and atypical presentation of known, clinically recognized diseases. What is becoming increasingly clear is that WES will be paradigm altering for patients and families with rare genetic diseases.

X-ray reflectivity and grazing incidence diffraction studies of interaction between human adhesion/growth-regulatory galectin-1 and DPPE-GM1 lipid monolayer at an air/water interface.

The specific interaction of ganglioside GM1 with the homodimeric (prototype) endogenous lectin galectin-1 triggers growth regulation in tumor and activated effector T cells. This proven biorelevance directed interest to studying association of the lectin to a model surface, i.e. a 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine/ganglioside GM1 (80 : 20 mol%) monolayer, at a bioeffective concentration. Surface expansion by the lectin insertion was detected at a surface pressure of 20 mN/m. On combining the methods of grazing incidence X-ray diffraction and X-ray reflectivity, a transient decrease in lipid-ordered phase of the monolayer was observed. The measured electron density distribution indicated that galectin-1 is oriented with its long axis in the surface plane, ideal for cis-crosslinking. The data reveal a conspicuous difference to the way the pentameric lectin part of the cholera toxin, another GM1-specific lectin, is bound to the monolayer. They also encourage further efforts to monitor effects of structurally different members of the galectin family such as the functionally antagonistic chimera-type galectin-3.

LIMS2 mutations are associated with a novel muscular dystrophy, severe cardiomyopathy and triangular tongues.

Limb girdle muscular dystrophy (LGMD) is a heterogeneous group of genetic disorders leading to progressive muscle degeneration and often associated with cardiac complications. We present two adult siblings with childhood-onset of weakness progressing to a severe quadriparesis with the additional features of triangular tongues and biventricular cardiac dysfunction. Whole exome sequencing identified compound heterozygous missense mutations that are predicted to be pathogenic in LIMS2. Biopsy of skeletal muscle demonstrated disrupted immunostaining of LIMS2. This is the first report of mutations in LIMS2 and resulting disruption of the integrin linked kinase (ILK)-LIMS-parvin complex associated with LGMD.

Mutations in PNPLA6 are linked to photoreceptor degeneration and various forms of childhood blindness.

Blindness due to retinal degeneration affects millions of people worldwide, but many disease-causing mutations remain unknown. PNPLA6 encodes the patatin-like phospholipase domain containing protein 6, also known as neuropathy target esterase (NTE), which is the target of toxic organophosphates that induce human paralysis due to severe axonopathy of large neurons. Mutations in PNPLA6 also cause human spastic paraplegia characterized by motor neuron degeneration. Here we identify PNPLA6 mutations in childhood blindness in seven families with retinal degeneration, including Leber congenital amaurosis and Oliver McFarlane syndrome. PNPLA6 localizes mostly at the inner segment plasma membrane in photoreceptors and mutations in Drosophila PNPLA6 lead to photoreceptor cell death. We also report that lysophosphatidylcholine and lysophosphatidic acid levels are elevated in mutant Drosophila. These findings show a role for PNPLA6 in photoreceptor survival and identify phospholipid metabolism as a potential therapeutic target for some forms of blindness.

Whole-exome sequencing broadens the phenotypic spectrum of rare pediatric epilepsy: a retrospective study.

Whole-exome sequencing (WES) has transformed our ability to detect mutations causing rare diseases. FORGE (Finding Of Rare disease GEnes) and Care4Rare Canada are nation-wide projects focused on identifying disease genes using WES and translating this technology to patient care. Rare forms of epilepsy are well-suited for WES and we retrospectively selected FORGE and Care4Rare families with clinical descriptions that included childhood-onset epilepsy or seizures not part of a recognizable syndrome or an early-onset encephalopathy where standard-of-care investigations were unrevealing. Nine families met these criteria and a diagnosis was made in seven, and potentially eight, of the families. In the eight families we identified mutations in genes associated with known neurological and epilepsy disorders: ASAH1, FOLR1, GRIN2A (two families), SCN8A, SYNGAP1 and SYNJ1. A novel and rare mutation was identified in KCNQ2 and was likely responsible for the benign seizures segregating in the family though additional evidence would be required to be definitive. In retrospect, the clinical presentation of four of the patients was considered atypical, thereby broadening the phenotypic spectrum of these conditions. Given the extensive clinical and genetic heterogeneity associated with epilepsy, our findings suggest that WES may be considered when a specific gene is not immediately suspected as causal.

Whole exome sequencing unravels disease-causing genes in consanguineous families in Qatar.

Whole exome sequencing (WES) has greatly facilitated the identification of causal mutations for diverse human genetic disorders. We applied WES as a molecular diagnostic tool to identify disease-causing genes in consanguineous families in Qatar. Seventeen consanguineous families with diverse disorders were recruited. Initial mutation screening of known genes related to the clinical diagnoses did not reveal the causative mutations. Using WES approach, we identified the definitive disease-causing mutations in four families: (i) a novel nonsense homozygous (c.1034C>G) in PHKG2 causing glycogen storage disease type 9C (GSD9C) in a male with initial diagnosis of GSD3; (ii) a novel homozygous 1-bp deletion (c.915del) in NSUN2 in a male proband with Noonan-like syndrome; (iii) a homozygous SNV (c.1598C>G) in exon 11 of IDUA causing Hurler syndrome in a female proband with unknown clinical diagnosis; (iv) a de novo known splicing mutation (c.1645+1G>A) in PHEX in a female proband with initial diagnosis of autosomal recessive hypophosphatemic rickets. Applying WES as a diagnostic tool led to the unambiguous identification of disease-causing mutations in phenotypically complex disorders or correction of the initial clinical diagnosis in ˜25% of our cases.

Evidence for clinical, genetic and biochemical variability in spinal muscular atrophy with progressive myoclonic epilepsy.

Spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) is a recently delineated, autosomal recessive condition caused by rare mutations in the N-acylsphingosine amidohydrolase 1 (acid ceramidase) ASAH1 gene. It is characterized by motor neuron disease followed by progressive myoclonic seizures and eventual death due to respiratory insufficiency. Here we report an adolescent female who presented with atonic and absence seizures and myoclonic jerks and was later diagnosed as having myoclonic-absence seizures. An extensive genetic and metabolic work-up was unable to arrive at a molecular diagnosis. Whole exome sequencing (WES) identified two rare, deleterious mutations in the ASAH1 gene: c.850G>T;p.Gly284X and c.456A>C;p.Lys152Asn. These mutations were confirmed by Sanger sequencing in the patient and her parents. Functional studies in cultured fibroblasts showed that acid ceramidase was reduced in both overall amount and enzymatic activity. Ceramide level was doubled in the patient's fibroblasts as compared to control cells. The results of the WES and the functional studies prompted an electromyography (EMG) study that showed evidence of motor neuron disease despite only mild proximal muscle weakness. These findings expand the phenotypic spectrum of SMA-PME caused by novel mutations in ASAH1 and highlight the clinical utility of WES for rare, intractable forms of epilepsy.

Whole-exome sequencing expands the phenotype of Hunter syndrome.

Whole-exome sequencing (WES) has proven its utility in finding novel genes associated with rare conditions and its usefulness is being further demonstrated in expanding the phenotypes of well known diseases. We present here a family with a previously undiagnosed X-linked condition characterized by progressive restriction of joint range of motion, prominence of the supraorbital ridge, audiology issues and hernias. They had an average stature, normal occipitofrontal circumference and intelligence, absence of dysostosis multiplex and otherwise good health. A diagnosis of Hunter syndrome was determined using WES and further supported by biochemical investigations. The phenotype of this family does not correspond to either the severe or attenuated clinical subtypes of Hunter syndrome. As further atypical families are reported, this classification will need to be modified. Our findings highlight the utility of WES in expanding the recognized phenotypic spectrum of known syndromes.

WDR19: an ancient, retrograde, intraflagellar ciliary protein is mutated in autosomal recessive retinitis pigmentosa and in Senior-Loken syndrome.

Autosomal recessive retinitis pigmentosa (arRP) is a clinically and genetically heterogeneous retinal disease that causes blindness. Our purpose was to identify the causal gene, describe the phenotype and delineate the mutation spectrum in a consanguineous Quebec arRP family. We performed Arrayed Primer Extension (APEX) technology to exclude ∼500 arRP mutations in ∼20 genes. Homozygosity mapping [single nucleotide polymorphism (SNP) genotyping] identified 10 novel significant homozygous regions. We performed next generation sequencing and whole exome capture. Sanger sequencing provided cosegregation. We screened another 150 retinitis pigmentosa (RP) and 200 patients with Senior-Løken Syndrome (SLS). We identified a novel missense mutation in WDR19, c.2129T>C which lead to a p.Leu710Ser. We found the same mutation in a second Quebec arRP family. Interestingly, two of seven affected members of the original family developed 'sub-clinical' renal cysts. We hypothesized that more severe WDR19 mutations may lead to severe ciliopathies and found seven WDR19 mutations in five SLS families. We identified a new gene for both arRP and SLS. WDR19 is a ciliary protein associated with the intraflagellar transport machinery. We are currently investigating the full extent of the mutation spectrum. Our findings are crucial in expanding the understanding of childhood blindness and identifying new genes.

Origin of bulk uniaxial anisotropy in zinc-blende dilute magnetic semiconductors.

It is demonstrated that the nearest-neighbor Mn pair on the GaAs (001) surface has a lower energy for the [110] direction compared to the [110] case. According to the group theory and Luttinger's method of invariants, this specific Mn distribution results in bulk uniaxial in-plane and out-of-plane anisotropies. The sign and magnitude of the corresponding anisotropy energies determined by a perturbation method and ab initio computations are consistent with experimental results.

Early and late indications for implantation of cardiac pacemakers in patients after heart transplantation: a single-center experience.

BACKGROUND: Data regarding the incidence and predictors of permanent pacemaker (PPM) implantation following cardiac transplantation are still limited and inconsistent. The aim of our study was to assess the incidence and indications for PPM implantation in cardiac transplant recipients in a single center. METHODS: We retrospectively analyzed the records of 508 consecutive patients including 425 men and an overall mean age of 45 y who underwent heart transplantation from 1988 to 2011 at our institution, using the biatrial surgical technique. RESULTS: Twenty-three (4.5%) subjects developed intermittent third-degree atrioventricular block (AVB) or sinus node dysfunction (SND) requiring PPM: 7 early (<3 months) and 16, late (>3 months) after transplantation. The incidence of AVB was higher than SND early (0.98% vs 0.4%; P<.05) and late (2.4% vs 0.8%; P<.01). The time from transplantation to PPM implantation was shorter among the SND group compared with the AVB group early (mean, 4.5 vs 6.5 weeks; P<.05) and late (mean, 4 vs 7 years; P<.05). CONCLUSION: The incidence of PPM after heart transplantation was 4.5%. AVB was the main indication for PPM early and late after transplantation. The time interval from transplantation to PPM implantation was shorter among SND compared with AVB patients.

Membrane texture induced by specific protein binding and receptor clustering: active roles for lipids in cellular function.

Biological membranes are complex, self-organized structures that define boundaries and compartmentalize space in living matter. Composed of a wide variety of lipid and protein molecules, these responsive surfaces mediate transmembrane signaling and material transport within the cell and with its environment. It is well known that lipid membrane properties change as a function of composition and phase state, and that protein-lipid interactions can induce changes in the membrane's properties and biochemical response. Here, molecular level changes in lipid organization induced by multivalent toxin binding were investigated using grazing incidence X-ray diffraction. Structural changes to lipid monolayers at the air-water interface and bilayers at the solid-water interface were studied before and after specific binding of cholera toxin to membrane embedded receptors. At biologically relevant surface pressures, protein binding perturbed lipid packing within monolayers and bilayers resulting in topological defects and the emergence of a new orientationally textured lipid phase. In bilayers this altered lipid order was transmitted from the receptor laden exterior membrane leaflet to the inner leaflet, representing a potential mechanism for lipid mediated outside-in signaling by multivalent protein binding. It is further hypothesized that cell-surface micro-domains exhibiting this type of lipid order may serve as nucleation sites for vesicle formation in clathrin independent endocytosis of cholera toxin.