Diffusion magnetic resonance imaging assessment of regional white matter maturation in preterm neonates

PURPOSE: Diffusion magnetic resonance imaging (dMRI) studies report altered white matter (WM) development in preterm infants. Neurite orientation dispersion and density imaging (NODDI) metrics provide more realistic estimations of neurite architecture in vivo compared with standard diffusion tensor imaging (DTI) metrics. This study investigated microstructural maturation of WM in preterm neonates scanned between 25 and 45 weeks postmenstrual age (PMA) with normal neurodevelopmental outcomes at 2 years using DTI and NODDI metrics. METHODS: Thirty-one neonates (n = 17 male) with median (range) gestational age (GA) 32+1 weeks (24+2-36+4) underwent 3 T brain MRI at median (range) post menstrual age (PMA) 35+2 weeks (25+3-43+1). WM tracts (cingulum, fornix, corticospinal tract (CST), inferior longitudinal fasciculus (ILF), optic radiations) were delineated using constrained spherical deconvolution and probabilistic tractography in MRtrix3. DTI and NODDI metrics were extracted for the whole tract and cross-sections along each tract to assess regional development. RESULTS: PMA at scan positively correlated with fractional anisotropy (FA) in the CST, fornix and optic radiations and neurite density index (NDI) in the cingulum, CST and fornix and negatively correlated with mean diffusivity (MD) in all tracts. A multilinear regression model demonstrated PMA at scan influenced all diffusion measures, GA and GAxPMA at scan influenced FA, MD and NDI and gender affected NDI. Cross-sectional analyses revealed asynchronous WM maturation within and between WM tracts.). CONCLUSION: We describe normal WM maturation in preterm neonates with normal neurodevelopmental outcomes. NODDI can enhance our understanding of WM maturation compared with standard DTI metrics alone

Exploring the multiple-hit hypothesis of preterm white matter damage using diffusion MRI.

Background: Preterm infants are at high risk of diffuse white matter injury and adverse neurodevelopmental outcome. The multiple hit hypothesis suggests that the risk of white matter injury increases with cumulative exposure to multiple perinatal risk factors. Our aim was to test this hypothesis in a large cohort of preterm infants using diffusion weighted magnetic resonance imaging (dMRI). Methods: We studied 491 infants (52% male) without focal destructive brain lesions born at < 34 weeks, who underwent structural and dMRI at a specialist Neonatal Imaging Centre. The median (range) gestational age (GA) at birth was 30+ 1 (23+ 2-33+ 5) weeks and median postmenstrual age at scan was 42+ 1 (38-45) weeks. dMRI data were analyzed using tract based spatial statistics and the relationship between dMRI measures in white matter and individual perinatal risk factors was assessed. We tested the hypothesis that increased exposure to perinatal risk factors was associated with lower fractional anisotropy (FA), and higher radial, axial and mean diffusivity (RD, AD, MD) in white matter. Neurodevelopmental performance was investigated using the Bayley Scales of Infant and Toddler Development, Third Edition (BSITD-III) in a subset of 381 infants at 20 months corrected age. We tested the hypothesis that lower FA and higher RD, AD and MD in white matter were associated with poorer neurodevelopmental performance. Results: Identified risk factors for diffuse white matter injury were lower GA at birth, fetal growth restriction, increased number of days requiring ventilation and parenteral nutrition, necrotizing enterocolitis and male sex. Clinical chorioamnionitis and patent ductus arteriosus were not associated with white matter injury. Multivariate analysis demonstrated that fetal growth restriction, increased number of days requiring ventilation and parenteral nutrition were independently associated with lower FA values. Exposure to cumulative risk factors was associated with reduced white matter FA and FA values at term equivalent age were associated with subsequent neurodevelopmental performance. Conclusion: This study suggests multiple perinatal risk factors have an independent association with diffuse white matter injury at term equivalent age and exposure to multiple perinatal risk factors exacerbates dMRI defined, clinically significant white matter injury. Our findings support the multiple hit hypothesis for preterm white matter injury

Mystery Solved: The Identification of the Two Missing Romanov Children Using DNA Analysis

One of the greatest mysteries for most of the twentieth century was the fate of the Romanov family, the last Russian monarchy. Following the abdication of Tsar Nicholas II, he and his wife, Alexandra, and their five children were eventually exiled to the city of Yekaterinburg. The family, along with four loyal members of their staff, was held captive by members of the Ural Soviet. According to historical reports, in the early morning hours of July 17, 1918 the entire family along with four loyal members of their staff was executed by a firing squad. After a failed attempt to dispose of the remains in an abandoned mine shaft, the bodies were transported to an open field only a few kilometers from the mine shaft. Nine members of the group were buried in one mass grave while two of the children were buried in a separate grave. With the official discovery of the larger mass grave in 1991, and subsequent DNA testing to confirm the identities of the Tsar, the Tsarina, and three of their daughters – doubt persisted that these remains were in fact those of the Romanov family. In the summer of 2007, a group of amateur archeologists discovered a collection of remains from the second grave approximately 70 meters from the larger grave. We report forensic DNA testing on the remains discovered in 2007 using mitochondrial DNA (mtDNA), autosomal STR, and Y- STR testing. Combined with additional DNA testing of material from the 1991 grave, we have virtually irrefutable evidence that the two individuals recovered from the 2007 grave are the two missing children of the Romanov family: the Tsarevich Alexei and one of his sisters

Synchronized Retrovirus Fusion in Cells Expressing Alternative Receptor Isoforms Releases the Viral Core into Distinct Sub-cellular Compartments

Disparate enveloped viruses initiate infection by fusing with endosomes. However, the highly diverse and dynamic nature of endosomes impairs mechanistic studies of fusion and identification of sub-cellular sites supporting the nucleocapsid release. We took advantage of the extreme stability of avian retrovirus-receptor complexes at neutral pH and of acid-dependence of virus-endosome fusion to isolate the latter step from preceding asynchronous internalization/trafficking steps. Viruses were trapped within endosomes in the presence of NH4Cl. Removal of NH4Cl resulted in a quick and uniform acidification of all subcellular compartments, thereby initiating synchronous viral fusion. Single virus imaging demonstrated that fusion was initiated within seconds after acidification and often culminated in the release of the viral core from an endosome. Comparative studies of cells expressing either the transmembrane or GPI-anchored receptor isoform revealed that the transmembrane receptor delivered the virus to more fusion-permissive compartments. Thus the identity of endosomal compartments, in addition to their acidity, appears to modulate viral fusion. A more striking manifestation of the virus delivery to distinct compartments in the presence of NH4Cl was the viral core release into the cytosol of cells expressing the transmembrane receptor and into endosomes of cells expressing the GPI-anchored isoform. In the latter cells, the newly released cores exhibited restricted mobility and were exposed to a more acidic environment than the cytoplasm. These cores appear to enter into the cytosol after an additional slow temperature-dependent step. We conclude that the NH4Cl block traps the virus within intralumenal vesicles of late endosomes in cells expressing the GPI-anchored receptor. Viruses surrounded by more than one endosomal membrane release their core into the cytoplasm in two steps – fusion with an intralumenal vesicle followed by a yet unknown temperature-dependent step that liberates the core from late endosomes

Complementation of diverse HIV-1 Env defects through cooperative subunit interactions: a general property of the functional trimer

<p>Abstract</p> <p>Background</p> <p>The HIV-1 Env glycoprotein mediates virus entry by catalyzing direct fusion between the virion membrane and the target cell plasma membrane. Env is composed of two subunits: gp120, which binds to CD4 and the coreceptor, and gp41, which is triggered upon coreceptor binding to promote the membrane fusion reaction. Env on the surface of infected cells is a trimer consisting of three gp120/gp41 homo-dimeric protomers. An emerging question concerns cooperative interactions between the protomers in the trimer, and possible implications for Env function.</p> <p>Results</p> <p>We extended studies on cooperative subunit interactions within the HIV-1 Env trimer, using analysis of functional complementation between coexpressed inactive variants harboring different functional deficiencies. In assays of Env-mediated cell fusion, complementation was observed between variants with a wide range of defects in both the gp120 and gp41 subunits. The former included gp120 subunits mutated in the CD4 binding site or incapable of coreceptor interaction due either to mismatched specificity or V3 loop mutation. Defective gp41 variants included point mutations at different residues within the fusion peptide or heptad repeat regions, as well as constructs with modifications or deletions of the membrane proximal tryptophan-rich region or the transmembrane domain. Complementation required the defective variants to be coexpressed in the same cell. The observed complementation activities were highly dependent on the assay system. The most robust activities were obtained with a vaccinia virus-based expression and reporter gene activation assay for cell fusion. In an alternative system involving Env expression from integrated provirus, complementation was detected in cell fusion assays, but not in virus particle entry assays.</p> <p>Conclusion</p> <p>Our results indicate that Env function does not require every subunit in the trimer to be competent for all essential activities. Through cross-talk between subunits, the functional determinants on one defective protomer can cooperatively interact to trigger the functional determinants on an adjacent protomer(s) harboring a different defect, leading to fusion. Cooperative subunit interaction is a general feature of the Env trimer, based on complementation activities observed for a highly diverse range of functional defects.</p

Protection of Stem Cell-Derived Lymphocytes in a Primate AIDS Gene Therapy Model after In Vivo Selection

Background: There is currently no effective AIDS vaccine, emphasizing the importance of developing alternative therapies. Recently, a patient was successfully transplanted with allogeneic, naturally resistant CCR5-negative (CCR5 delta 32) cells, setting the stage for transplantation of naturally resistant, or genetically modified stem cells as a viable therapy for AIDS. Hematopoietic stem cell (HSC) gene therapy using vectors that express various anti-HIV transgenes has also been attempted in clinical trials, but inefficient gene transfer in these studies has severely limited the potential of this approach. Here we evaluated HSC gene transfer of an anti-HIV vector in the pigtailed macaque (Macaca nemestrina) model, which closely models human transplantation. Methods and Findings: We used lentiviral vectors that inhibited both HIV-1 and simian immunodeficiency virus (SIV)/HIV-1 (SHIV) chimera virus infection, and also expressed a P140K mutant methylguanine methyltransferase (MGMT) transgene to select gene-modified cells by adding chemotherapy drugs. Following transplantation and MGMT-mediated selection we demonstrated transgene expression in over 7% of stem-cell derived lymphocytes. The high marking levels allowed us to demonstrate protection from SHIV in lymphocytes derived from gene-modified macaque long-term repopulating cells that expressed an HIV-1 fusion inhibitor. We observed a statistically significant 4-fold increase of gene-modified cells after challenge of lymphocytes from one macaque that received stem cells transduced with an anti-HIV vector (p<0.02, Student's t-test), but not in lymphocytes from a macaque that received a control vector. We also established a competitive repopulation assay in a second macaque for preclinical testing of promising anti-HIV vectors. The vectors we used were HIV-based and thus efficiently transduce human cells, and the transgenes we used target HIV-1 genes that are also in SHIV, so our findings can be rapidly translated to the clinic. Conclusions: Here we demonstrate the ability to select protected HSC-derived lymphocytes in vivo in a clinically relevant nonhuman primate model of HIV/SHIV infection. This approach can now be evaluated in human clinical trials in AIDS lymphoma patients. In this patient setting, chemotherapy would not only kill malignant cells, but would also increase the number of MGMTP140K-expressing HIV-resistant cells. This approach should allow for high levels of HIV-protected cells in AIDS patients to evaluate AIDS gene therapy

Imaging Single Retrovirus Entry through Alternative Receptor Isoforms and Intermediates of Virus-Endosome Fusion

A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes

APOBEC3G-Augmented Stem Cell Therapy to Modulate HIV Replication: A Computational Study

PMC3661658The interplay between the innate immune system restriction factor APOBEC3G and the HIV protein Vif is a key host-retrovirus interaction. APOBEC3G can counteract HIV infection in at least two ways: by inducing lethal mutations on the viral cDNA; and by blocking steps in reverse transcription and viral integration into the host genome. HIV-Vif blocks these antiviral functions of APOBEC3G by impeding its encapsulation. Nonetheless, it has been shown that overexpression of APOBEC3G, or interfering with APOBEC3G-Vif binding, can efficiently block in vitro HIV replication. Some clinical studies have also suggested that high levels of APOBEC3G expression in HIV patients are correlated with increased CD4+ T cell count and low levels of viral load; however, other studies have reported contradictory results and challenged this observation. Stem cell therapy to replace a patient's immune cells with cells that are more HIV-resistant is a promising approach. Pre-implantation gene transfection of these stem cells can augment the HIV-resistance of progeny CD4+ T cells. As a protein, APOBEC3G has the advantage that it can be genetically encoded, while small molecules cannot. We have developed a mathematical model to quantitatively study the effects on in vivo HIV replication of therapeutic delivery of CD34+ stem cells transfected to overexpress APOBEC3G. Our model suggests that stem cell therapy resulting in a high fraction of APOBEC3G-overexpressing CD4+ T cells can effectively inhibit in vivo HIV replication. We extended our model to simulate the combination of APOBEC3G therapy with other biological activities, to estimate the likelihood of improved outcomes.JH Libraries Open Access Fun

Measuring the efficacy of insecticide treated bednets: the use of DNA fingerprinting to increase the accuracy of personal protection estimates in Tanzania.

Summary Insecticide-treated nets have proved successful in the prevention of malaria as a result of both the personal protection with which they provide the sleeper and also the 'mass effect' on the local mosquito population when they are used on a community-wide basis. Personal protection estimates are normally based on comparisons of the numbers of bloodfed mosquitoes found in rooms with and without nets, however it seemed possible that a number of those mosquitoes may not have fed on the occupants of the rooms in which they were found but had entered after feeding elsewhere. To address this possible source of error, we used an 8-locus microsatellite system to identify the source of bloodmeals of Anopheles gambiaes.l. and A. funestus mosquitoes collected in rooms and window traps in Tanzanian villages with and without nets treated with alphacypermethrin. DNA fingerprints were produced from blood samples taken from people who had slept in these rooms and were matched to fingerprints obtained from the mosquito bloodmeals. We were able to type successfully over 90% of the bloodmeals collected and found that proportions of bloodfed mosquitoes that had fed on occupants of the rooms in which they were found were high and only slightly greater in villages without treated nets than those with them (95% and 88%, respectively). When these percentages were used to adjust estimates of personal protection, it was found that the error due to mosquitoes not feeding in the rooms in which they were collected is negligible

Pronounced impact of atmospheric conditions on Ba4Nb2O9 and Ba4Ta2O9

Ba(4)Nb(2)O(9) and Ba(4)Ta(2)O(9) decompose upon heating in a CO(2)-rich atmosphere. Here, we report further in situ synchrotron X-ray diffraction studies under CO(2)-free atmospheres of variable humidity and under vacuum and show that this strongly influences the reconstructive phase transitions normally seen. Under normal atmospheric conditions, alpha-Ba(4)Nb(2)O(9) transforms to gamma-Ba(4)Nb(2)O(9) at 1493 K and alpha-Ba(4)Ta(2)O(9) transforms to 6H-Ba(4)Ta(2)O(9) at 1513 K. Under wet argon, these transitions shift to lower temperatures of 1453 K and 1443 K for Ba(4)Nb(2)O(9) and Ba(4)Ta(2)O(9) respectively. Upon heating both the alpha samples under vacuum, no phase transition is observed at all up to 1723 K. Heating 6H-Ba(4)Ta(2)O(9) under vacuum saw the monoclinic -> hexagonal symmetry-transition temperature increase from 1050 K under air to 1700K. The significance of these observations in terms of possible practical applications of their mixed oxide ionic, protonic and electronic conduction properties are discussed. © 2012, Elsevier Ltd