The neurointensive nursery: concept, development, and insights gained.

PURPOSE OF REVIEW: With the advent of therapeutic hypothermia for treatment of hypoxic ischemic encephalopathy, and improvements in neuroimaging and bedside neuromonitoring, a new era of neonatal brain-focused care has emerged in recent years. We describe the development of the first neurointensive care nursery (NICN) as a model for comanagement of neonates with identified neurologic risk factors by a multidisciplinary team constituted of neurologists, neonatologists, specialized nurses, and others with the goal of optimizing management, preventing secondary injury and maximizing long-term outcomes. RECENT FINDINGS: Optimizing brain metabolic environment and perfusion and preventing secondary brain injury are key to neurocritical care. This includes close management of temperature, blood pressure, oxygenation, carbon dioxide, and glucose levels. Early developmental interventions and involvement of physical and occupational therapy provide additional assessment information. Finally, long-term follow-up is essential for any neurocritical care program. SUMMARY: The NICN model aims to optimize evidence-based care of infants at risk for neurologic injury. Results from ongoing hypothermia and neuroprotective trials are likely to yield additional treatments. New technologies, such as functional MRI, continuous neurophysiological assessment, and whole genomic approaches to rapid diagnosis may further enhance clinical protocols and neonatal precision medicine. Importantly, advances in neurocritical care improve our ability to provide comprehensive information when counseling families. Long-term follow-up data will determine if the NICN/Neuro-NICU provides enduring benefit to infants at risk for neurologic injury

Characterization of Pax-2 Regulatory Sequences That Direct Transgene Expression in the Wolffian Duct and Its Derivatives

AbstractThe Pax family of transcription factors plays important roles in vertebrate organogenesis. Pax-2 is a critical factor in the development of the mammalian urogenital system. Pax-2 is expressed in the epithelia of the ureter, the Müllerian duct, and the Wolffian duct and in the nephrogenic mesenchyme. Gene targeting in the mouse as well as natural mutations in mouse and man have demonstrated the requirement of Pax-2 in the development of these structures. Little is known about the molecular mechanisms regulating Pax-2 expression in the developing urogenital system. As a first step to reveal these mechanisms and to search for the elements and factors controlling Pax-2 expression we have characterized regulatory sequences of the Pax-2 gene in an in vivo reporter assay in the mouse. An 8.5-kb genomic region upstream of the Pax-2 transcription start site directed reporter gene activity in the epithelium of the pronephric duct at 8.25 days postcoitum (dpc) and in the Wolffian duct starting from 9.0 dpc. Expression in the Wolffian duct and its derivatives, the ureter, the collecting duct system, the seminal vesicles, the vas deferens, and the epididymis, was maintained at least until 18.5 dpc. Hence, an element(s) in the 8.5-kb upstream region is sufficient to initiate and maintain Pax-2 expression in the Wolffian duct and its derivatives. In order to more precisely map the Wolffian duct regulatory sequences, a deletion analysis of the 8.5-kb upstream region was performed in a transient in vivo reporter assay. A 0.4-kb subfragment was required for marker gene expression in the Wolffian duct. Misexpression of fgf8 under the control of the 8.5-kb upstream region resulted in polycystic kidneys, demonstrating the general usefulness of Pax-2 regulatory sequences in misexpression of foreign genes in the ureter and collecting duct system of the kidney in transgenic approaches in mice

Variable electrostatic interaction between DNA and coat protein in filamentous bacteriophage assembly

A restriction fragment carrying the major coat protein gene (gene VIII) was excised from the DNA of the class I filamentous bacteriophage fd, which infects Escherichia coli. This fragment was cloned into the expression plasmid pKK223-3, where it came under the control of the tac promoter, generating plasmid pKf8P. Bacteriophage fd gene VIII was similarly cloned into the plasmid pEMBL9 +, enabling it to be subjected to site-directed mutagenesis. By this means the positively charged lysine residue at position 48, one of four positively charged residues near the C terminus of the protein, was turned into a negatively charged glutamic acid residue. The mutated fd gene VIII was cloned back from the pEMBL plasmid into the expression plasmid pKK223-3, creating plasmid pKE48. In the presence of the inducer isopropyl-P-o-thiogalactoside, the wild-type and mutated coat protein genes were strongly expressed in E. coli TG 1 cells transformed with plasmids pKf8P and pKE48, respectively, and the product procoat proteins underwent processing and insertion into the E. coli cell inner membrane. A net positive charge of only 2 on the side-chains in the C-terminal region is evidently sufficient for this initial stage of the virus assembly process. However, the mutated coat protein could not encapsidate the DNA of bacteriophage R252, an fd bacteriophage carrying an amber mutation in its own gene VIII, when tested on non-suppressor strains of E. coli. On the other hand, elongated hybrid bacteriophage particles could be generated whose capsids contained mixtures of wild-type (K48) and mutant (E48) subunits. This suggests that the defect in assembly may occur at the initiation rather than the elongation step(s) in virus assembly. Other mutations of lysine-48 that removed or reversed the positive charge at this position in the C-terminal region of the coat protein were also found to lead to the production of commensurately longer bacteriophage particles. Taken together, these results indicate direct electrostatic interaction between the DNA and the coat protein in the capsid and support a model of non-specific binding between DNA and coat protein subunits with a stoicheiometry that can be varied during assembly.peer-reviewe

Interactions between DNA and coat protein in the structure and assembly of filamentous bacteriophage fd

Bacteriophage fd is a class I filamentous virus (others are M13 and fl) that comprises a circular, single-stranded DNA molecule enclosed in a cylindrical protein sheath to form a flexible particle -890 nm long and 7 nm in diameter (for reviews, see refs 1 and 2). The viral DNA contains 6,408 nucleotidesJ.-5 incorporating 10 genes, and the protein sheath is composed of about 2,700 major coat protein subunits6 in a shingled helical array, the symmetry of which is defined by a fivefold rotational axis combined with a twofold screw axis of pitch 3.2 nm (refs 7-9). The DNA extends throughout the length of the particle but is not base-paired and has a symmetry different from that of the protein helix. How the DNA is packed remains unclear but the number (2.4) of nucleotides packaged per major coat protein subunit is certainly not integral, in contrast with, say, the packaging of RNA in tobacco mosaic virus. The coat protein subunit is SO amino-acid residues in length and, in the virus particle, adopts a largely a-helical conformation, with the long axis of the helix aligned close to the long axis of the filament'-9·n. This protein is arranged with its negatively charged N-terminal region on the outside of the filament and its positively charged C-terminal region on the inside abutting the DNA7. We report here that positive charge on one of the four lysine side chains in the latter region has a direct effect on DNA packaging, because when this charge is absent, elongated particles are produced with lengths that can be correlated with the residual positive charge in the C-terminal region of the coat protein subunit.peer-reviewe

Lineage-Restricted OLIG2-RTK Signaling Governs the Molecular Subtype of Glioma Stem-like Cells

SummaryThe basic helix-loop-helix (bHLH) transcription factor OLIG2 is a master regulator of oligodendroglial fate decisions and tumorigenic competence of glioma stem-like cells (GSCs). However, the molecular mechanisms underlying dysregulation of OLIG2 function during gliomagenesis remains poorly understood. Here, we show that OLIG2 modulates growth factor signaling in two distinct populations of GSCs, characterized by expression of either the epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor alpha (PDGFRα). Biochemical analyses of OLIG2 function in normal and malignant neural progenitors reveal a positive feedforward loop between OLIG2 and EGFR to sustain co-expression. Furthermore, loss of OLIG2 function results in mesenchymal transformation in PDGFRαHIGH GSCs, a phenomenon that appears to be circumscribed in EGFRHIGH GSCs. Exploitation of OLIG2′s dual and antithetical, pro-mitotic (EGFR-driven), and lineage-specifying (PDGFRα-driven) functions by glioma cells appears to be critical for sustaining growth factor signaling and GSC molecular subtype

Ferret brain possesses young interneuron collections equivalent to human postnatal migratory streams.

The human early postnatal brain contains late migratory streams of immature interneurons that are directed to cortex and other focal brain regions. However, such migration is not observed in rodent brain, and whether other small animal models capture this aspect of human brain development is unclear. Here, we investigated whether the gyrencephalic ferret cortex possesses human-equivalent postnatal streams of doublecortin positive (DCX+) young neurons. We mapped DCX+ cells in the brains of ferrets at P20 (analogous to human term gestation), P40, P65, and P90. In addition to the rostral migratory stream, we identified three populations of young neurons with migratory morphology at P20 oriented toward: (a) prefrontal cortex, (b) dorsal posterior sigmoid gyrus, and (c) occipital lobe. These three neuronal collections were all present at P20 and became extinguished by P90 (equivalent to human postnatal age 2 years). DCX+ cells in such collections all expressed GAD67, identifying them as interneurons, and they variously expressed the subtype markers SP8 and secretagogin (SCGN). SCGN+ interneurons appeared in thick sections to be oriented from white matter toward multiple cortical regions, and persistent SCGN-expressing cells were observed in cortex. These findings indicate that ferret is a suitable animal model to study the human-relevant process of late postnatal cortical interneuron integration into multiple regions of cortex

An ‘oligarchy’ rules neural development.

Review Oligodendrocytes engage in complex interactions with nerve cell bodies and axons in the CNS, notably in the formation of myelin sheaths Transcription factors as arbiters of oligodendroglial cell fate The roles of transcription factors in neuronal cell fate specification in the CNS have been intensively studied over the past decade (reviewed in Ref

Axin2 as regulatory and therapeutic target in newborn brain injury and remyelination.

Permanent damage to white matter tracts, comprising axons and myelinating oligodendrocytes, is an important component of brain injuries of the newborn that cause cerebral palsy and cognitive disabilities, as well as multiple sclerosis in adults. However, regulatory factors relevant in human developmental myelin disorders and in myelin regeneration are unclear. We found that AXIN2 was expressed in immature oligodendrocyte progenitor cells (OLPs) in white matter lesions of human newborns with neonatal hypoxic-ischemic and gliotic brain damage, as well as in active multiple sclerosis lesions in adults. Axin2 is a target of Wnt transcriptional activation that negatively feeds back on the pathway, promoting β-catenin degradation. We found that Axin2 function was essential for normal kinetics of remyelination. The small molecule inhibitor XAV939, which targets the enzymatic activity of tankyrase, acted to stabilize Axin2 levels in OLPs from brain and spinal cord and accelerated their differentiation and myelination after hypoxic and demyelinating injury. Together, these findings indicate that Axin2 is an essential regulator of remyelination and that it might serve as a pharmacological checkpoint in this process