Structural basis for DNA strand separation by a hexameric replicative helicase

Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5′ ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted ‘steric exclusion’ model for dsDNA unwinding, the active 3′ ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5′ passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases

The function of STRADα during corticogenesis: Insights into the neuropathogenesis of focal cortical dysplasias

The mTORC1 signaling cascade provides regulatory control of protein translation, cell differentiation, and cell growth. Mutations in the mTORC1 inhibitory proteins TSC1 and TSC2 cause Tuberous Sclerosis Complex, an autosomal dominant disorder characterized by epilepsy and cognitive disability. Mutations in STRADα, an upstream activator of TSC2, give rise to Pretzel Syndrome, a neurodevelopmental disorder characterized by epilepsy and psychomotor retardation. We show that loss of STRADα function in vitro and in vivo is associated with enhanced cell size, aberrant nuclear localization of the kinase LKB1, and abnormal neuronal lamination as a consequence of aberrant mTORC1 signaling. We further show in human brain specimens and cultured cells in vitro that sporadic and syndromic type II Focal Cortical Dysplasias exhibit aberrant mTORC1 activation in association with persistent expression of stem cell markers. Therefore, mTORC1 hyperactivation may be a unifying feature of neurodevelopmental disorders characterized by epilepsy, autism, and cognitive disability and pharmacological inhibitors of mTOR may prove to be therapeutically beneficial in the treatment of these disorders

Investigation of drop geometry at free-falling process depending on volume of dozing

Present work performs experimental investigation of the changes in the geometric shape of the drop in terms of its free-falling, depending on the conditions of drop formation on dispenser. It was found that volume and velocity of drop formation on dispenser are the factors affecting the geometric shape of the drop in terms of its free-falling

Power plant ash and slag waste management technological direction when Kansk-Achinsk brown coal is burned

Today resource efficiency technology development in all industries where conventional raw material is being replaced by local natural resources and industrial waste is an essential matter. Along with that most producing operations are overload with wide range of waste produced during technological process. Thermal power stations are real world evidence. Their main waste is ash and slag which accumulated in great amounts in often overfull ash dumps. The goal of present work is to find perspective ash dump waste utilization methods. The study will be based on experimentally obtained data: elementary compound and properties of Kansk-Achinsk brown coal. Research methods: experimental, chemical silicate analysis, mineralogical forms identification within samples by using ASM X-ray diffraction analysis. Experiments resulted with the following conclusions: silica is ash main component, and ash has the form of ore concentrate analogy in a number of elements. We think that ASM main properties which make it useful for utilization are: high content of calcium oxide; high ash sorption properties; ASM radiation safety class which makes them safe to be used in materials, goods, and structures production for residence and public buildings construction and reconstruction; sufficiently high content of individual elements

Applied Technological Direction of Power Plant Ash and Slag Waste Management when Kuznetsk Bituminous Coal is Burned

Currently a lot of power plants have a problem with storage of coal combustion solid by-products (ash and slag). Holding capacity of existing power plants available ash dumps were enlarged and modernized repeatedly. Many plants have two or even three of them. Today new ash dump construction is economically inconvenient due to need to assign new plots of land and their inconveniently big distance from a plant, which increase ash and slag transportation expenses. The goal of our research work is to find promising directions for ash and slag waste mass utilization based on Kuznetsk bituminous coals experimental data on ultimate composition and properties. The experimental research of ash, slag and their mixture samples from ash dumps brought us to conclusion that the most promising direction for these materials application in large quantities is construction industry including road construction. Be-sides, we lined up some other directions for ash, slag, and ash and slag mixture possible application. These directions might not provide mass utilization but they are promising from a point of view of the researched waste properties

Power plant ash and slag waste management technological direction when Kansk-Achinsk brown coal is burned

Today resource efficiency technology development in all industries where conventional raw material is being replaced by local natural resources and industrial waste is an essential matter. Along with that most producing operations are overload with wide range of waste produced during technological process. Thermal power stations are real world evidence. Their main waste is ash and slag which accumulated in great amounts in often overfull ash dumps. The goal of present work is to find perspective ash dump waste utilization methods. The study will be based on experimentally obtained data: elementary compound and properties of Kansk-Achinsk brown coal. Research methods: experimental, chemical silicate analysis, mineralogical forms identification within samples by using ASM X-ray diffraction analysis. Experiments resulted with the following conclusions: silica is ash main component, and ash has the form of ore concentrate analogy in a number of elements. We think that ASM main properties which make it useful for utilization are: high content of calcium oxide; high ash sorption properties; ASM radiation safety class which makes them safe to be used in materials, goods, and structures production for residence and public buildings construction and reconstruction; sufficiently high content of individual elements

STRADα deficiency results in aberrant mTORC1 signaling during corticogenesis in humans and mice

Polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome (PMSE) is a rare human autosomalrecessive disorder characterized by abnormal brain development, cognitive disability, and intractable epilepsy. It is caused by homozygous deletions of STE20-related kinase adaptor α (STRADA). The underlying pathogenic mechanisms of PMSE and the role of STRADA in cortical development remain unknown. Here, we found that a human PMSE brain exhibits cytomegaly, neuronal heterotopia, and aberrant activation of mammalian target of rapamycin complex 1 (mTORC1) signaling. STRADα normally binds and exports the protein kinase LKB1 out of the nucleus, leading to suppression of the mTORC1 pathway. We found that neurons in human PMSE cortex exhibited abnormal nuclear localization of LKB1. To investigate this further, we modeled PMSE in mouse neural progenitor cells (mNPCs) in vitro and in developing mouse cortex in vivo by knocking down STRADα expression. STRADα-deficient mNPCs were cytomegalic and showed aberrant rapamycin-dependent activation of mTORC1 in association with abnormal nuclear localization of LKB1. Consistent with the observations in human PMSE brain, knockdown of STRADα in vivo resulted in cortical malformation, enhanced mTORC1 activation, and abnormal nuclear localization of LKB1. Thus, we suggest that the aberrant nuclear accumulation of LKB1 caused by STRADα deficiency contributes to hyperactivation of mTORC1 signaling and disruption of neuronal lamination during corticogenesis, and thereby the neurological features associated with PMSE

Early Progenitor Cell Marker Expression Distinguishes Type II From Type I Focal Cortical Dysplasias

Type I and type II focal cortical dysplasias (FCDs) exhibit distinct histopathologic features that suggest different pathogenic mechanisms. Type I FCDs are characterized by mild laminar disorganization and hypertrophic neurons, whereas type II FCDs exhibit dramatic laminar disorganization and cytomegalic cells (balloon cells). Both FCD types are associated with intractable epilepsy; therefore, identifying cellular or molecular differences between these lesion types that explains the histologic differences could provide new diagnostic and therapeutic insights. Type II FCDs express nestin, a neuroglial progenitor protein that is modulated in vitro by the stem cell proteins c-Myc, sex-determining region Y-box 2 (SOX2), and Octamer-4 (Oct-4) after activation of mammalian target of rapamycin complex 1 (mTORC1). Because mTORC1 activation has been demonstrated in type II FCDs, we hypothesized that c-Myc, SOX2, and Oct-4 expression would distinguish type II from type I FCDs. In addition, we assayed the expression of progenitor cell proteins forkhead box G1 (FOXG1), Kruppel-like factor 4 (KLF4), Nanog, and SOX3. Differential expression of 7 stem cellproteins and aberrant phosphorylation of 2 mTORC1 substrates, S6 and S6 kinase 1 proteins, clearly distinguished type II from type I FCDs (n = 10 each). Our results demonstrate new potential pathogenic pathways in type II FCDs and suggest biomarkers for diagnostic pathology in resected epilepsy specimen

Rapamycin Prevents Seizures After Depletion of STRADA in a Rare Neurodevelopmental Disorder

A rare neurodevelopmental disorder in the Old Order Mennonite population called PMSE (polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome; also called Pretzel syndrome) is characterized by infantile-onset epilepsy, neurocognitive delay, craniofacial dysmorphism, and histopathological evidence of heterotopic neurons in subcortical white matter and subependymal regions. PMSE is caused by a homozygous deletion of exons 9 to 13 of the LYK5/STRADA gene, which encodes the pseudokinase STRADA, an upstream inhibitor of mammalian target of rapamycin complex 1 (mTORC1). We show that disrupted pathfinding in migrating mouse neural progenitor cells in vitro caused by STRADA depletion is prevented by mTORC1 inhibition with rapamycin or inhibition of its downstream effector p70 S6 kinase (p70S6K) with the drug PF-4708671 (p70S6Ki). We demonstrate that rapamycin can rescue aberrant cortical lamination and heterotopia associated with STRADA depletion in the mouse cerebral cortex. Constitutive mTORC1 signaling and a migration defect observed in fibroblasts from patients with PMSE were also prevented by mTORC1 inhibition. On the basis of these preclinical findings, we treated five PMSE patients with sirolimus (rapamycin) without complication and observed a reduction in seizure frequency and an improvement in receptive language. Our findings demonstrate a mechanistic link between STRADA loss and mTORC1 hyperactivity in PMSE, and suggest that mTORC1 inhibition may be a potential treatment for PMSE as well as other mTOR-associated neurodevelopmental disorders