Translational modulator ISRIB alleviates synaptic and behavioral phenotypes in Fragile X syndrome.

Fragile X syndrome (FXS) is caused by the loss of fragile X messenger ribonucleoprotein (FMRP), a translational regulator that binds the transcripts of proteins involved in synaptic function and plasticity. Dysregulated protein synthesis is a central effect of FMRP loss, however, direct translational modulation has not been leveraged in the treatment of FXS. Thus, we examined the effect of the translational modulator integrated stress response inhibitor (ISRIB) in treating synaptic and behavioral symptoms of FXS. We show that FMRP loss dysregulates synaptic protein abundance, stabilizing dendritic spines through increased PSD-95 levels while preventing spine maturation through reduced glutamate receptor accumulation, thus leading to the formation of dense, immature dendritic spines, characteristic of FXS patients and Fmr1 knockout (KO) mice. ISRIB rescues these deficits and improves social recognition in Fmr1 KO mice. These findings highlight the therapeutic potential of targeting core translational mechanisms in FXS and neurodevelopmental disorders more broadly.

The relationship between severe hypertensive diseases of pregnancy and moderate-severe bronchopulmonary dysplasia.

OBJECTIVE: Determine the association between severe hypertensive disease of pregnancy (HDP) with moderate-severe bronchopulmonary dysplasia (BPD) in preterm infants (< 31 weeks' gestation). STUDY DESIGN: Preterm birth cohort study of 693 mother-infant dyads. Severe HDP was defined as severe preeclampsia, HELLP syndrome or eclampsia. The outcome was moderate-severe BPD classified at 36 weeks corrected gestational age, per the NICHD Consensus statement. RESULTS: 225 (32%) mothers developed severe HDP and 234 (34%) infants had moderate-severe BPD. There was an interaction between severe HDP and gestational age (p = 0.03). Infants born at < 25 weeks gestation to mothers with HDP had increased odds for moderate-severe BPD compared to infants of normotensive mothers delivering at the same gestational age. Infants born > 28 weeks to mothers with severe HDP had decreased odds for the outcome, though not statistically significant. CONCLUSIONS: Severe HDP has a differential effect on the development of moderate-severe BPD based on gestational age.

The Relationship Between Severe Hypertensive Diseases of Pregnancy and Moderate-Severe Bronchopulmonary Dysplasia.

Objective: Determine the association between severe hypertensive disease of pregnancy (HDP) with moderate-severe bronchopulmonary dysplasia (BPD) in preterm infants (< 31 weeks' gestation). Study Design: Preterm birth cohort study of 693 mother-infant dyads. Severe HDPwas defined as severe preeclampsia, HELLP syndrome or eclampsia. The outcome was moderate-severe BPD classified at 36 weeks corrected gestational age, based on the NICHD Consensusstatement. Results: 225 (32%) mothers developed severe HDP and 234 (34%) infants hadmoderate-severe BPD. There was an interaction between severe HDP and gestational age (p=0.03). Infants born at earlier gestational ages to mothers with HDP had increased odds for moderate-severe BPD compared to infants of normotensive mothers delivering at the same gestational age. Infants born at later gestational ages to mothers with severe HDP had decreased odds for the outcome. Conclusions: Severe HDP has a differential effect on the development of moderate-severe BPD based on gestational age.

The role of the maternal and fetal inflammatory response in retinopathy of prematurity.

PROBLEM: There is a paucity of research on the contribution of placental inflammation to severe retinopathy of prematurity (ROP). METHOD OF STUDY: A retrospective cohort study (n = 1217) was conducted of infants screened for ROP (2006-2016). The outcomes of the study were severe ROP (type 1 or type 2 ROP) and low grade ROP. We categorized the placental pathology as the presence of (i) maternal plus fetal inflammatory response, (ii) maternal inflammatory response only, (iii) fetal inflammatory response only and, (iv) no evidence of a maternal or fetal inflammatory response. The data were analyzed using univariate and multivariate logistic regression analyses (P < .05). RESULTS: In this cohort, the number of infants with the maternal plus fetal inflammatory response, the maternal inflammatory response only, the fetal inflammatory response only, and no maternal or fetal inflammatory response was 305 (25%), 82 (7%), 8 (1%), and 822 (67%), respectively. Adjusted for covariates, the maternal plus fetal inflammatory response was a significant risk factor for severe ROP (AOR = 2.6, 95% CI 1.1-5.9, P = .03). None of the categories of placental inflammation were significantly associated with low grade ROP. CONCLUSION: Placental pathology distinguished by the maternal plus fetal inflammatory response was a significant risk factor for severe ROP. Our study supports a link between intrauterine inflammatory events and the subsequent development of severe ROP.

Human trophoblast-derived hydrogen sulfide stimulates placental artery endothelial cell angiogenesis.

Endogenous hydrogen sulfide (H2S), mainly synthesized by cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CTH), has been implicated in regulating placental angiogenesis; however, the underlying mechanisms are unknown. This study was to test a hypothesis that trophoblasts synthesize H2S to promote placental angiogenesis. Human choriocarcinoma-derived BeWo cells expressed both CBS and CTH proteins, while the first trimester villous trophoblast-originated HTR-8/SVneo cells expressed CTH protein only. The H2S producing ability of BeWo cells was significantly inhibited by either inhibitors of CBS (carboxymethyl hydroxylamine hemihydrochloride, CHH) or CTH (ß-cyano-L-alanine, BCA) and that in HTR-8/SVneo cells was inhibited by CHH only. H2S donors stimulated cell proliferation, migration, and tube formation in ovine placental artery endothelial cells (oFPAECs) as effectively as vascular endothelial growth factor. Co-culture with BeWo and HTR-8/SVneo cells stimulated oFPAEC migration, which was inhibited by CHH or BCA in BeWo but CHH only in HTR-8/SVneo cells. Primary human villous trophoblasts (HVT) were more potent than trophoblast cell lines in stimulating oFPAEC migration that was inhibited by CHH and CHH/BCA combination in accordance with its H2S synthesizing activity linked to CBS and CTH expression patterns. H2S donors activated endothelial nitric oxide synthase (NOS3), v-AKT murine thymoma viral oncogene homolog 1 (AKT1), and extracellular signal-activated kinase 1/2 (mitogen-activated protein kinase 3/1, MAPK3/1) in oFPAECs. H2S donor-induced NOS3 activation was blocked by AKT1 but not MAPK3/1 inhibition. In keeping with our previous studies showing a crucial role of AKT1, MAPK3/1, and NOS3/NO in placental angiogenesis, these data show that trophoblast-derived endogenous H2S stimulates placental angiogenesis, involving activation of AKT1, NOS3/NO, and MAPK3/1.

The relationship of the subtypes of preterm birth with retinopathy of prematurity.

BACKGROUND: Retinopathy of prematurity is an adverse outcome of preterm birth and is a leading cause of childhood blindness. The relationship between the subtypes of preterm birth with retinopathy of prematurity is understudied. OBJECTIVE: To investigate whether there is a difference in the incidence of type 1 or type 2 retinopathy of prematurity in infants with preterm birth resulting from spontaneous preterm labor, a medical indication of preterm birth, or preterm premature rupture of the membranes. STUDY DESIGN: A retrospective cohort study was conducted of 827 infants screened for retinopathy of prematurity who were delivered at a single tertiary care center in Colorado. All infants fulfilled the American Academy of Pediatrics 2013 screening criteria for retinopathy of prematurity defined as "infants with a birth weight of ≤1500 g or gestational age of 30 weeks or less (as defined by the attending neonatologist) and selected infants with a birth weight between 1500 and 2000 g or gestational age of >30 weeks with an unstable clinical course, including those requiring cardiorespiratory support and who are believed by their attending pediatrician or neonatologist to be at high risk for retinopathy of prematurity." Two independent reviewers masked to retinopathy of prematurity outcomes determined whether preterm birth resulted from spontaneous preterm labor, medical indication of preterm birth, or preterm premature rupture of the membranes. Discrepancies were resolved by a third reviewer. Data were analyzed with univariate and multivariable logistic regression. RESULTS: In our cohort, the frequency of preterm birth resulting from spontaneous preterm labor, medical indication of preterm birth, or preterm premature rupture of the membranes was 34%, 40%, and 26%, respectively. The mean gestational age (weeks, days) ± SD (range) in the cohort and across the preterm birth subtypes was as follows: entire cohort, 28 weeks, 6 days ± 2 weeks, 3 days (23 weeks, 3 days - 36 weeks, 4 days); spontaneous preterm labor, 28 weeks 1 day ± 2 weeks, 3 days (23 weeks, 3 days - 33 weeks, 4 days); medical indication of preterm birth, 29 weeks, 1 day ± 2 weeks, 2 days (24-36 weeks, 4 days); preterm premature rupture of the membranes, 28 weeks, 4 days ± 2 weeks, 1 day (24-33 weeks, 1 day). Among infants with type 1, type 2, or no retinopathy of prematurity, the incidence of type 1 or type 2 retinopathy of prematurity in births from spontaneous preterm labor, medical indication of preterm birth, and preterm premature rupture of the membranes was 37 of 218 (17%), 27 of 272 (10%), and 10 of 164 (6%), respectively. Adjusted for gestational age, birth weight, and multiparity and compared with the preterm premature rupture of the membranes group, the odds ratios of spontaneous preterm labor and medical indication of preterm birth for type 1 or type 2 retinopathy of prematurity were 6.1 (95% confidence interval, 1.8 to 20, P = .003) and 5.5 (95% confidence interval, 1.4 to 21, P = .01), respectively. Among neonates born after preterm premature rupture of the membranes, the probability of developing type 1 or type 2 retinopathy of prematurity was greatest in infants with rupture of membrane duration of up to 24 hours. After 24 hours, the probability of developing type 1 or type 2 retinopathy of prematurity declined. The odds of developing type 1 or type 2 retinopathy of prematurity was 9.0 (95% confidence interval 2.3 to 34, P = .002) in infants who had preterm premature rupture of the membranes ≤ 24 hours compared with infants who had preterm premature rupture of the membranes > 24 hours. CONCLUSION: Type 1 or type 2 retinopathy of prematurity are adverse ocular outcomes linked with not only lower gestational age and birth weight at delivery but also with events in the intrauterine environment that trigger a preterm birth. The reduced incidence of type 1 or type 2 retinopathy of prematurity in the preterm premature rupture of the membranes group compared with other causes of preterm birth may be related to the perinatal therapies associated with preterm premature rupture of the membranes (such as corticosteroids, antibiotics, maternal-fetal surveillance), which may have an inhibitory effect on the development of retinopathy of prematurity. We suggest that the physiologic events that predispose infants to type 1 or type 2 retinopathy of prematurity begin before delivery.

Astrocytic Contributions to Synaptic and Learning Abnormalities in a Mouse Model of Fragile X Syndrome.

BACKGROUND: Fragile X syndrome (FXS) is the most common type of mental retardation attributable to a single-gene mutation. It is caused by FMR1 gene silencing and the consequent loss of its protein product, fragile X mental retardation protein. Fmr1 global knockout (KO) mice recapitulate many behavioral and synaptic phenotypes associated with FXS. Abundant evidence suggests that astrocytes are important contributors to neurological diseases. This study investigates astrocytic contributions to the progression of synaptic abnormalities and learning impairments associated with FXS. METHODS: Taking advantage of the Cre-lox system, we generated and characterized mice in which fragile X mental retardation protein is selectively deleted or exclusively expressed in astrocytes. We performed in vivo two-photon imaging to track spine dynamics/morphology along dendrites of neurons in the motor cortex and examined associated behavioral defects. RESULTS: We found that adult astrocyte-specific Fmr1 KO mice displayed increased spine density in the motor cortex and impaired motor-skill learning. The learning defect coincided with a lack of enhanced spine dynamics in the motor cortex that normally occurs in response to motor skill acquisition. Although spine density was normal at 1 month of age in astrocyte-specific Fmr1 KO mice, new spines formed at an elevated rate. Furthermore, fragile X mental retardation protein expression in only astrocytes was insufficient to rescue most spine or behavioral defects. CONCLUSIONS: Our work suggests a joint astrocytic-neuronal contribution to FXS pathogenesis and reveals that heightened spine formation during adolescence precedes the overabundance of spines and behavioral defects found in adult Fmr1 KO mice.

A regulatory motif in nonmuscle myosin II-B regulates its role in migratory front-back polarity.

In this study, we show that the role of nonmuscle myosin II (NMII)-B in front-back migratory cell polarity is controlled by a short stretch of amino acids containing five serines (1935-1941). This motif resides near the junction between the C terminus helical and nonhelical tail domains. Removal of this motif inhibited NMII-B assembly, whereas its insertion into NMII-A endowed an NMII-B-like ability to generate large actomyosin bundles that determine the rear of the cell. Phosphomimetic mutation of the five serines also inhibited NMII-B assembly, rendering it unable to support front-back polarization. Mass spectrometric analysis showed that several of these serines are phosphorylated in live cells. Single-site mutagenesis showed that serine 1935 is a major regulatory site of NMII-B function. These data reveal a novel regulatory mechanism of NMII in polarized migrating cells by identifying a key molecular determinant that confers NMII isoform functional specificity.

α-Actinin-2 mediates spine morphology and assembly of the post-synaptic density in hippocampal neurons.

Dendritic spines are micron-sized protrusions that constitute the primary post-synaptic sites of excitatory neurotransmission in the brain. Spines mature from a filopodia-like protrusion into a mushroom-shaped morphology with a post-synaptic density (PSD) at its tip. Modulation of the actin cytoskeleton drives these morphological changes as well as the spine dynamics that underlie learning and memory. Several PSD molecules respond to glutamate receptor activation and relay signals to the underlying actin cytoskeleton to regulate the structural changes in spine and PSD morphology. α-Actinin-2 is an actin filament cross-linker, which localizes to dendritic spines, enriched within the post-synaptic density, and implicated in actin organization. We show that loss of α-actinin-2 in rat hippocampal neurons creates an increased density of immature, filopodia-like protrusions that fail to mature into a mushroom-shaped spine during development. α-Actinin-2 knockdown also prevents the recruitment and stabilization of the PSD in the spine, resulting in failure of synapse formation, and an inability to structurally respond to chemical stimulation of the N-methyl-D-aspartate (NMDA)-type glutamate receptor. The Ca2+-insensitive EF-hand motif in α-actinin-2 is necessary for the molecule's function in regulating spine morphology and PSD assembly, since exchanging it for the similar but Ca2+-sensitive domain from α-actinin-4, another α-actinin isoform, inhibits its function. Furthermore, when the Ca2+-insensitive domain from α-actinin-2 is inserted into α-actinin-4 and expressed in neurons, it creates mature spines. These observations support a model whereby α-actinin-2, partially through its Ca2+-insensitive EF-hand motif, nucleates PSD formation via F-actin organization and modulates spine maturation to mediate synaptogenesis.

Human placental expression of SLIT/ROBO signaling cues: effects of preeclampsia and hypoxia.

Preeclampsia is characterized by dysfunctional endothelium and impaired angiogenesis. Recent studies suggest that the neuronal guidance SLIT/ROBO system regulates tumor angiogenesis. This study investigated if SLIT and ROBO are differentially expressed in healthy term and preeclamptic placentas and if hypoxia regulates SLIT and ROBO expression in placental trophoblast and endothelial cells. Total RNA and protein were extracted from placental tissues of healthy term (n = 5) and preeclamptic (n = 6) pregnancies and used for SLIT/ROBO expression analyses with reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative-PCR, and immunoblotting. Paraffin-embedded tissues were processed to localize SLIT/ROBO proteins in placental villi by immunohistochemistry. BeWo choriocarcinoma cells and human umbilical vein endothelial cells (HUVEC) were treated with 2% or 10% oxygen or the hypoxia mimetic deferoxamine mesylate (100 µM) to test if hypoxia regulates SLIT/ROBO expression. SLIT2, SLIT3, ROBO1, and ROBO4 mRNA and proteins were detected in the placenta. SLIT2 and ROBO1 proteins localized in the syncytiotrophoblast, and SLIT3, ROBO1, and ROBO4 in capillary endothelium of the placental villi. Levels of ROBO1 and ROBO4 as well as sFLT1 (soluble fms-like tyrosine kinase-1) proteins were significantly greater in preeclamptic placentas compared to normal controls. Hypoxia significantly increased both mRNA and protein levels of SLIT2 in BeWo cells and of SLIT3, ROBO1, and ROBB4 in HUVEC. Thus, trophoblast and endothelial coexpression of SLIT/ROBO suggests an autocrine/paracrine regulatory system for regulating placental function. Differential expression of SLITs and ROBOs in healthy term and preeclamptic placentas and hypoxia regulation of their expressions in placental cells implicate a potential pathophysiological role for this system in preeclampsia.

Myosin IIb activity and phosphorylation status determines dendritic spine and post-synaptic density morphology.

Dendritic spines in hippocampal neurons mature from a filopodia-like precursor into a mushroom-shape with an enlarged post-synaptic density (PSD) and serve as the primary post-synaptic location of the excitatory neurotransmission that underlies learning and memory. Using myosin II regulatory mutants, inhibitors, and knockdowns, we show that non-muscle myosin IIB (MIIB) activity determines where spines form and whether they persist as filopodia-like spine precursors or mature into a mushroom-shape. MIIB also determines PSD size, morphology, and placement in the spine. Local inactivation of MIIB leads to the formation of filopodia-like spine protrusions from the dendritic shaft. However, di-phosphorylation of the regulatory light chain on residues Thr18 and Ser19 by Rho kinase is required for spine maturation. Inhibition of MIIB activity or a mono-phosphomimetic mutant of RLC similarly prevented maturation even in the presence of NMDA receptor activation. Expression of an actin cross-linking, non-contractile mutant, MIIB R709C, showed that maturation into a mushroom-shape requires contractile activity. Loss of MIIB also leads to an elongated PSD morphology that is no longer restricted to the spine tip; whereas increased MIIB activity, specifically through RLC-T18, S19 di-phosphorylation, increases PSD area. These observations support a model whereby myosin II inactivation forms filopodia-like protrusions that only mature once NMDA receptor activation increases RLC di-phosphorylation to stimulate MIIB contractility, resulting in mushroom-shaped spines with an enlarged PSD.

Genome remodelling in a basal-like breast cancer metastasis and xenograft.

Massively parallel DNA sequencing technologies provide an unprecedented ability to screen entire genomes for genetic changes associated with tumour progression. Here we describe the genomic analyses of four DNA samples from an African-American patient with basal-like breast cancer: peripheral blood, the primary tumour, a brain metastasis and a xenograft derived from the primary tumour. The metastasis contained two de novo mutations and a large deletion not present in the primary tumour, and was significantly enriched for 20 shared mutations. The xenograft retained all primary tumour mutations and displayed a mutation enrichment pattern that resembled the metastasis. Two overlapping large deletions, encompassing CTNNA1, were present in all three tumour samples. The differential mutation frequencies and structural variation patterns in metastasis and xenograft compared with the primary tumour indicate that secondary tumours may arise from a minority of cells within the primary tumour.

The B73 maize genome: complexity, diversity, and dynamics.

We report an improved draft nucleotide sequence of the 2.3-gigabase genome of maize, an important crop plant and model for biological research. Over 32,000 genes were predicted, of which 99.8% were placed on reference chromosomes. Nearly 85% of the genome is composed of hundreds of families of transposable elements, dispersed nonuniformly across the genome. These were responsible for the capture and amplification of numerous gene fragments and affect the composition, sizes, and positions of centromeres. We also report on the correlation of methylation-poor regions with Mu transposon insertions and recombination, and copy number variants with insertions and/or deletions, as well as how uneven gene losses between duplicated regions were involved in returning an ancient allotetraploid to a genetically diploid state. These analyses inform and set the stage for further investigations to improve our understanding of the domestication and agricultural improvements of maize.

Dendritic Spines: Similarities with Protrusions and Adhesions in Migrating Cells.

Dendritic spines are specialized, micron-sized post-synaptic compartments that support synaptic function. These actin-based protrusions push the post-synaptic membrane, establish contact with the presynaptic membrane and undergo dynamic changes in morphology during development, as well as in response to synaptic neurotransmission. These processes are propelled by active remodeling of the actin cytoskeleton, which includes polymerization, filament disassembly, and organization of the actin in supramolecular arrays, such as branched networks or bundles. Dendritic spines contain a plethora of adhesion and synaptic receptors, signaling, and cytoskeletal proteins that regulate their formation, maturation and removal. Whereas many of the molecules involved in dendritic spine formation have been identified, their actual roles in spine formation, removal and maturation are not well understood. Using parallels between migrating fibroblasts and dendritic spines, we point to potential mechanisms and approaches for understanding spine development and dynamics.

Inherited and acquired variations in the hyaluronan synthase 1 (HAS1) gene may contribute to disease progression in multiple myeloma and Waldenstrom macroglobulinemia.

To characterize genetic contributions toward aberrant splicing of the hyaluronan synthase 1 (HAS1) gene in multiple myeloma (MM) and Waldenstrom macroglobulinemia (WM), we sequenced 3616 bp in HAS1 exons and introns involved in aberrant splicing, from 17 patients. We identified a total of 197 HAS1 genetic variations (GVs), a range of 3 to 24 GVs/patient, including 87 somatic GVs acquired in splicing regions of HAS1. Nearly all newly identified inherited and somatic GVs in MM and/or WM were absent from B chronic lymphocytic leukemia, nonmalignant disease, and healthy donors. Somatic HAS1 GVs recurred in all hematopoietic cells tested, including normal CD34(+) hematopoietic progenitor cells and T cells, or as tumor-specific GVs restricted to malignant B and plasma cells. An in vitro splicing assay confirmed that HAS1 GVs direct aberrant HAS1 intronic splicing. Recurrent somatic GVs may be enriched by strong mutational selection leading to MM and/or WM.

Nuclear import of TFIIB is mediated by Kap114p, a karyopherin with multiple cargo-binding domains.

Nuclear import and export is mediated by an evolutionarily conserved family of soluble transport factors, the karyopherins (referred to as importins and exportins). The yeast karyopherin Kap114p has previously been shown to import histones H2A and H2B, Nap1p, and a component of the preinitiation complex (PIC), TBP. Using a proteomic approach, we have identified several potentially new cargoes for Kap114p. These cargoes include another PIC component, the general transcription factor IIB or Sua7p, which interacted directly with Kap114p. Consistent with its role as a Sua7p import factor, deletion of KAP114 led to specific mislocalization of Sua7p to the cytoplasm. An interaction between Sua7p and TBP was also detected in cytosol, raising the possibility that both Sua7p and TBP can be coimported by Kap114p. We have also shown that Kap114p possesses multiple overlapping binding sites for its partners, Sua7p, Nap1p, and H2A and H2B, as well as RanGTP and nucleoporins. In addition, we have assembled an in vitro complex containing Sua7p, Nap1p, and histones H2A and H2B, suggesting that this Kap may import several proteins simultaneously. The import of more than one cargo at a time would increase the efficiency of each import cycle and may allow the regulation of coimported cargoes.