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Mutations in POLG, the gene encoding the catalytic subunit of DNA polymerase gamma, result in clinical syndromes characterized by mitochondrial DNA (mtDNA) depletion in affected tissues with variable organ involvement. The brain is one of the most affected organs, and symptoms include intractable seizures, developmental delay, dementia, and ataxia. Patient-derived induced pluripotent stem cells (iPSCs) provide opportunities to explore mechanisms in affected cell types and potential therapeutic strategies. Fibroblasts from two patients were reprogrammed to create new iPSC models of POLG-related mitochondrial diseases. Compared with control iPSC-derived neurons, mtDNA depletion was observed upon differentiation of the POLG-mutated lines to cortical neurons. POLG-mutated neurons exhibited neurite simplification with decreased mitochondrial content, abnormal mitochondrial structure and function, and increased cell death. Expression of the mitochondrial kinase PTEN-induced kinase 1 (PINK1) mRNA was decreased in patient neurons. Overexpression of PINK1 increased mitochondrial content and ATP:ADP ratios in neurites, decreasing cell death and rescuing neuritic complexity. These data indicate an intersection of polymerase gamma and PINK1 pathways that may offer a novel therapeutic option for patients affected by this spectrum of disorders.

Encephalitis, the most significant of the central nervous system (CNS) diseases caused by Herpes simplex virus 1 (HSV-1), may have long-term sequelae in survivors treated with acyclovir, the cause of which is unclear. HSV-1 exhibits a tropism toward neurogenic niches in CNS enriched with neural precursor cells (NPCs), which play a pivotal role in neurogenesis. NPCs are susceptible to HSV-1. There is a paucity of information regarding the influence of HSV-1 on neurogenesis in humans. We investigated HSV-1 infection of NPCs from two individuals. Our results show (i) HSV-1 impairs, to different extents, the proliferation, self-renewing, and, to an even greater extent, migration of NPCs from these two subjects; (ii) The protective effect of the gold-standard antiherpetic drug acyclovir (ACV) varies with viral dose and is incomplete. It is also subject to differences in terms of efficacy of the NPCs derived from these two individuals. These results suggest that the effects of HSV-1 may have on aspects of NPC neurogenesis may vary among individuals, even in the presence of acyclovir, and this may contribute to the heterogeneity of cognitive sequelae across encephalitis survivors. Further analysis of NPC cell lines from a larger number of individuals is warranted.

Introduction: Ultrasound-targeted microbubble cavitation ( UTMC ) facilitates delivery of cell-impermeant drugs across the endothelial barrier, such as the blood brain barrier ( BBB ) . Using cultured endothelial cell ( EC ) monolayers, we reported that UTMC causes sonoporation and inter-endothelial gaps, which may mediate vascular hyperpermeability, although mechanisms are incompletely understood. Further, the effects of UTMC on extravascular cells, and extent of payload penetration are unknown, as the monolayer does not recapitulate the 3D multicellular environment. Here, we established a 3D multicellular human brain spheroid model to study UTMC bioeffects and underlying mechanisms. Methods: Spheroids were generated using neurons and astrocytes derived from human iPSCs, primary human brain- microglia, ECs and pericytes, and placed in a water tank with lipid microbubbles ( MBs ). Ultrasound (1 MHz frequency, 250-500 kPa peak negative pressures, 10 μs pulse duration, 10 ms pulse interval) was delivered for 20 s and spheroids were immediately collected. Immunostaining was used to localize cell types; propidium iodide (PI) uptake to mark sonoporation; calcein-AM and sytox to detect cell viability; and Texas red dextran (TRD) (10 kDa) to quantify permeability, using confocal microscopy. Results: Immunostaining showed ECs and pericytes at the spheroid periphery, with high expression of membranous ZO-1. BBB functionality was confirmed with histamine treatment, which increased permeability to TRD (28.8% increase in TRD mean intensity, p =0.0015). Interestingly, PI uptake (sonoporation) occurred in cells beyond the BBB. TRD penetrated up to ~100 μm beyond the BBB upon UTMC (12.2% increase). Inhibition of endothelial nitric oxide synthase with L-NAME reduced UTMC-induced TRD penetration (19% decrease, p =0.0458). Conclusions: In this novel 3D human brain spheroid model with intact BBB, UTMC caused nitric-oxide dependent BBB breach. Moreover, this is the first report of UTMC causing “remote” sonoporation of cells not in direct contact with MBs, which bears further study. Our model allows the study of mechanisms mediating UTMC efficacy for targeted delivery of drugs across the endothelial barrier, which should facilitate its clinical translation.

Background Drug repurposing is a cost-effective strategy to identify drugs with novel effects. We searched for drugs exhibiting inhibitory activity to Herpes Simplex virus 1 (HSV-1). Our strategy utilized gene expression data generated from HSV-1-infected cell cultures which was paired with drug effects on gene expression. Gene expression data from HSV-1 infected and uninfected neurons were analyzed using BaseSpace Correlation Engine (Illumina®). Based on the general Signature Reversing Principle (SRP), we hypothesized that the effects of candidate antiviral drugs on gene expression would be diametrically opposite (negatively correlated) to those effects induced by HSV-1 infection. Results We initially identified compounds capable of inducing changes in gene expression opposite to those which were consequent to HSV-1 infection. The most promising negatively correlated drugs (Valproic acid, Vorinostat) did not significantly inhibit HSV-1 infection further in African green monkey kidney epithelial cells (Vero cells). Next, we tested Sulforaphane and Menadione which showed effects similar to those caused by viral infections (positively correlated). Intriguingly, Sulforaphane caused a modest but significant inhibition of HSV-1 infection in Vero cells (IC50 = 180.4 µM, p = 0.008), but exhibited toxicity when further explored in human neuronal progenitor cells (NPCs) derived from induced pluripotent stem cells. Conclusions These results reveal the limits of the commonly used SRP strategy when applied to the identification of novel antiviral drugs and highlight the necessity to refine the SRP strategy to increase its utility.

The pathogen hypothesis of Alzheimer’s disease (AD) proposes that brain HSV-1 infection is an initial source of amyloid beta (Aβ) peptide-containing amyloid plaque development. Aβ accumulation was reported in HSV-1-infected 2D neuronal cultures and neural stem cell cultures as well as in HSV-1-infected 3D neuronal culture models., Alzheimer's disease is a progressive neurodegenerative disease characterized neuropathologically by the presence of extracellular amyloid plaques composed of fibrillar amyloid beta (Aβ) peptides and intracellular neurofibrillary tangles. Postmortem and in vivo studies implicate herpes simplex virus 1 (HSV-1) infection in the brain as a precipitating factor in disease/pathology initiation. HSV-1 infection of two-dimensional (2D) neuronal cultures causes intracellular accumulation of Aβ42 peptide, but these 2D models do not recapitulate the three-dimensional (3D) architecture of brain tissue. We employed human induced pluripotent stem cells (hiPSCs) to compare patterns of Aβ42 accumulation in HSV-1-infected 2D (neuronal monolayers) and 3D neuronal cultures (brain organoids). Akin to prior studies, HSV-1-infected 2D cultures showed Aβ42 immunoreactivity in cells expressing the HSV-1 antigen ICP4 (ICP4+). Conversely, accumulation of Aβ42 in ICP4+ cells in infected organoids was rarely observed. These results highlight the importance of considering 3D cultures to model host-pathogen interaction. IMPORTANCE The pathogen hypothesis of Alzheimer’s disease (AD) proposes that brain HSV-1 infection is an initial source of amyloid beta (Aβ) peptide-containing amyloid plaque development. Aβ accumulation was reported in HSV-1-infected 2D neuronal cultures and neural stem cell cultures as well as in HSV-1-infected 3D neuronal culture models. The current study extends these findings by showing different patterns of Aβ42 accumulation following HSV-1 infection of 2D compared to 3D neuronal cultures (brain organoids). Specifically, 2D neuronal cultures showed Aβ42 immunoreactivity mainly in HSV-1-infected cells and only rarely in uninfected cells or infected cells exposed to antivirals. Conversely, 3D brain organoids showed accumulation of Aβ42 mainly in noninfected cells surrounding HSV-1-infected cells. We suggest that because brain organoids better recapitulate architectural features of a developing brain than 2D cultures, they are a more suitable model to investigate the involvement of HSV-1 in the onset of AD pathology.

Herpes simplex virus 1 (HSV-1) can induce damage in brain regions that include the hippocampus and associated limbic structures. These neurogenic niches are important because they are associated with memory formation and are highly enriched with neural progenitor cells (NPCs). The susceptibility and fate of HSV-1-infected NPCs are largely unexplored. We differentiated human induced pluripotent stem cells (hiPSCs) into NPCs to generate two-dimensional (2D) and three-dimensional (3D) culture models to examine the interaction of HSV-1 with NPCs. Here, we show that (i) NPCs can be efficiently infected by HSV-1, but infection does not result in cell death of most NPCs, even at high multiplicities of infection (MOIs); (ii) limited HSV-1 replication and gene expression can be detected in the infected NPCs; (iii) a viral silencing mechanism is established in NPCs exposed to the antivirals (E)-5-(2-bromovinyl)-2'-deoxyuridine (5BVdU) and alpha interferon (IFN-α) and when the antivirals are removed, spontaneous reactivation can occur at low frequency; (iv) HSV-1 impairs the ability of NPCs to migrate in a dose-dependent fashion in the presence of 5BVdU plus IFN-α; and (v) 3D cultures of NPCs are less susceptible to HSV-1 infection than 2D cultures. These results suggest that NPC pools could be sites of HSV-1 reactivation in the central nervous system (CNS). Finally, our results highlight the potential value of hiPSC-derived 3D cultures to model HSV-1-NPC interaction.

Ultrasound (US)-targeted microbubble (MB) cavitation (UTMC) facilitates delivery of cell-impermeant drugs across the blood brain barrier (BBB) . The effects of UTMC on extravascular cells, and the extent of payload penetration, are unknown. Spheroids were generated using neurons and astrocytes derived from human iPSCs, primary human brain- microglia, endothelial cells (ECs) and pericytes, and placed in a water tank with lipid MBs. US (1 MHz frequency, 250 kPa peak negative pressure, 10 μs pulse duration, 10 ms pulse interval) was delivered for 10 s. Immunostaining showed ECs and pericytes at the spheroid periphery, with high expression of membranous ZO-1. BBB functionality was confirmed with histamine treatment, which increased permeability to Texas red dextran (TRD) (28.8% increase, p = 0.0015). TRD penetrated up to 100 μm beyond the BBB upon UTMC (12% increase compared to 0 kPa control). Inhibition of endothelial nitric oxide synthase with L-NAME reduced UTMC-induced TRD penetration (19% decrease, p

This study employed human induced pluripotent stem cells (hiPSCs) to model the interaction of HSV-1 with NPCs, which reside in the neurogenic niches of the CNS and play a fundamental role in adult neurogenesis. Herein, we provide evidence that in NPCs infected at an MOI as low as 0.001, HSV-1 can establish a latent state, suggesting that (i) a variant of classical HSV-1 latency can be established during earlier stages of neuronal differentiation and (ii) neurogenic niches in the brain may constitute additional sites of viral reactivation. Lytic HSV-1 infections impaired NPC migration, which represents a critical step in neurogenesis. A difference in susceptibility to HSV-1 infection between two-dimensional (2D) and three-dimensional (3D) NPC cultures was observed, highlighting the potential value of 3D cultures for modeling host-pathogen interactions. Together, our results are relevant in light of observations relating HSV-1 infection to postencephalitic cognitive dysfunction., Herpes simplex virus 1 (HSV-1) can induce damage in brain regions that include the hippocampus and associated limbic structures. These neurogenic niches are important because they are associated with memory formation and are highly enriched with neural progenitor cells (NPCs). The susceptibility and fate of HSV-1-infected NPCs are largely unexplored. We differentiated human induced pluripotent stem cells (hiPSCs) into NPCs to generate two-dimensional (2D) and three-dimensional (3D) culture models to examine the interaction of HSV-1 with NPCs. Here, we show that (i) NPCs can be efficiently infected by HSV-1, but infection does not result in cell death of most NPCs, even at high multiplicities of infection (MOIs); (ii) limited HSV-1 replication and gene expression can be detected in the infected NPCs; (iii) a viral silencing mechanism is established in NPCs exposed to the antivirals (E)-5-(2-bromovinyl)-2′-deoxyuridine (5BVdU) and alpha interferon (IFN-α) and when the antivirals are removed, spontaneous reactivation can occur at low frequency; (iv) HSV-1 impairs the ability of NPCs to migrate in a dose-dependent fashion in the presence of 5BVdU plus IFN-α; and (v) 3D cultures of NPCs are less susceptible to HSV-1 infection than 2D cultures. These results suggest that NPC pools could be sites of HSV-1 reactivation in the central nervous system (CNS). Finally, our results highlight the potential value of hiPSC-derived 3D cultures to model HSV-1–NPC interaction. IMPORTANCE This study employed human induced pluripotent stem cells (hiPSCs) to model the interaction of HSV-1 with NPCs, which reside in the neurogenic niches of the CNS and play a fundamental role in adult neurogenesis. Herein, we provide evidence that in NPCs infected at an MOI as low as 0.001, HSV-1 can establish a latent state, suggesting that (i) a variant of classical HSV-1 latency can be established during earlier stages of neuronal differentiation and (ii) neurogenic niches in the brain may constitute additional sites of viral reactivation. Lytic HSV-1 infections impaired NPC migration, which represents a critical step in neurogenesis. A difference in susceptibility to HSV-1 infection between two-dimensional (2D) and three-dimensional (3D) NPC cultures was observed, highlighting the potential value of 3D cultures for modeling host-pathogen interactions. Together, our results are relevant in light of observations relating HSV-1 infection to postencephalitic cognitive dysfunction.

Acyclovir (ACV) is an effective antiviral agent for treating lytic Herpes Simplex virus, type 1 (HSV-1) infections, and it has dramatically reduced the mortality rate of herpes simplex encephalitis. However, HSV-1 resistance to ACV and its derivatives is being increasingly documented, particularly among immunocompromised individuals. The burgeoning drug resistance compels the search for a new generation of more efficacious anti-herpetic drugs. We have previously shown that trans-dihydrolycoricidine (R430), a lycorane-type alkaloid derivative, effectively inhibits HSV-1 infections in cultured cells. We now report that R430 also inhibits ACV-resistant HSV-1 strains, accompanied by global inhibition of viral gene transcription and enrichment of H3K27me3 methylation on viral gene promoters. Furthermore, we demonstrate that R430 prevents HSV-1 reactivation from latency in an ex vivo rodent model. Finally, among a panel of DNA viruses and RNA viruses, R430 inhibited Zika virus with high therapeutic index. Its therapeutic index is comparable to standard antiviral drugs, though it has greater toxicity in non-neuronal cells than in neuronal cells. Synthesis of additional derivatives could enable more efficacious antivirals and the identification of active pharmacophores.

Development of a chiral pool-based synthesis of 10b-aza-analogues of biologically active Amaryllidaceae alkaloids is described, involving a concise reductive amination and condensation sequence, leading to ring-B/C-modified, fully functionalized ring-C derivatives. Differentiated anticancer and antiviral activities of these analogues are presented. Despite complete conformational and functional group overlap, the 10b-aza-analogues have diminished anticancer activity and no antiviral activity. These unprecedented electronic effects suggest a possible role for π-type secondary orbital interactions with the biological target.

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented possibility to investigate defects occurring during neuronal differentiation in neuropsychiatric and neurodevelopmental disorders, but the density and intricacy of intercellular connections in neuronal cultures challenge currently available analytic methods. Low-density neuronal cultures facilitate the morphometric and functional analysis of neurons. We describe a differentiation protocol to generate low-density neuronal cultures (∼2,500 neurons/cm2) from human iPSC-derived neural stem cells/early neural progenitor cells. We generated low-density cultures using cells from 3 individuals. We also evaluated the morphometric features of neurons derived from 2 of these individuals, one harboring a microdeletion on chromosome 15q11.2 and the other without the microdeletion. An approximately 7.5-fold increase in the density of dendritic filopodia was observed in the neurons with the microdeletion, consistent with previous reports. Low-density neuronal cultures enable facile and unbiased comparisons of iPSC-derived neurons from different individuals or clones.

A recent report suggested Complement 4 (C4A) gene copy numbers (GCN) as risk factors for schizophrenia. Rodent model showed association of C4 with synaptic pruning suggesting its pathophysiological significance (Sekar, A. et al. (2016)). We, therefore, predicted that C4A GCN would be positively correlated with neuropil contraction in the human brain among schizophrenia patients showing more prominent correlations in ventral regions among young adults and dorsal regions among adolescents since neuromaturation progresses dorsoventrally. Whole-brain, multi-voxel, in vivo phosphorus magnetic resonance spectroscopy (31P MRS) assessed neuropil changes by estimating levels of membrane phospholipid (MPL) precursors and catabolites. Increased MPL catabolites and/or decreased MPL precursors indexed neuropil contraction. Digital droplet PCR-based assay was used to estimate C4A and C4B GCN. We evaluated two independent cohorts (young adult-onset early-course schizophrenia (YASZ = 15) and adolescent-onset schizophrenia (AOSZ = 12) patients), and controls matched for each group, n = 22 and 15, respectively. Separate forward stepwise linear regression models with Akaike information Criterion were built for MPL catabolites and precursors. YASZ cohort: Consistent with the rodent model (Sekar, A. et al. 2016)), C4A GCN positively correlated with neuropil contraction (increased pruning/decreased formation) in the inferior frontal cortex and inferior parietal lobule. AOSZ cohort: C4A GCN positively correlated with neuropil contraction in the dorsolateral prefrontal cortex and thalamus. Exploratory analysis of C4B GCN showed positive correlation with neuropil contraction in the cerebellum and superior temporal gyrus among YASZ while AOSZ showed neuropil contraction in the prefrontal and subcortical structures. Thus, C4A and C4B GCN are associated with neuropil contraction in regions often associated with schizophrenia, and may be neuromaturationally dependent.

Background A variety of neurological disorders including neurodegenerative diseases and infection by neurotropic viruses can cause structural and functional changes in the central nervous system (CNS), resulting in long-term neurological sequelae. An improved understanding of the pathogenesis of these disorders is important for developing efficacious interventions. Human induced pluripotent stem cells (hiPSCs) offer an extraordinary window for modeling pathogen-CNS interactions, and other cellular interactions, in three-dimensional (3D) neuronal cultures that can recapitulate several aspects of in vivo brain tissue. Methods Herein, we describe a prototype of scaffold-free hiPSC-based adherent 3D (A-3D) human neuronal cultures in 96-well plates. To test their suitability for drug screening, A-3D neuronal cultures were infected with herpes simplex virus type 1 (HSV-1) with or without acyclovir. Results The half maximal inhibitory concentration (IC50) of acyclovir was 3.14 μM and 3.12 μM determined using flow cytometry and the CX7 High Content Screening platform, respectively. Conclusions Our A-3D neuronal cultures provide an unprecedented opportunity for high-content drug screening programs to treat human CNS infections. Electronic supplementary material The online version of this article (10.1186/s13287-018-0881-6) contains supplementary material, which is available to authorized users.

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.

Background Herpes simplex virus, type 1 (HSV-1) commonly produces lytic mucosal lesions. It invariably initiates latent infection in sensory ganglia enabling persistent, lifelong infection. Acute HSV-1 encephalitis is rare and definitive evidence of latent infection in the brain is lacking. However, exposure untraceable to encephalitis has been repeatedly associated with impaired working memory and executive functions, particularly among schizophrenia patients. Methods Patterns of HSV-1 infection and gene expression changes were examined in human induced pluripotent stem cell (iPSC)-derived neurons. Separately, differences in blood oxygenation level-dependent (BOLD) responses to working memory challenges using letter n-back tests were investigated using functional magnetic resonance imaging (fMRI) among schizophrenia cases/controls. Results HSV-1 induced lytic changes in iPSC-derived glutamatergic neurons and neuroprogenitor cells. In neurons, HSV-1 also entered a quiescent state following coincubation with antiviral drugs, with distinctive changes in gene expression related to functions such as glutamatergic signaling. In the fMRI studies, main effects of schizophrenia (P = .001) and HSV-1 exposure (1-back, P = 1.76 × 10(-4); 2-back, P = 1.39 × 10(-5)) on BOLD responses were observed. We also noted increased BOLD responses in the frontoparietal, thalamus, and midbrain regions among HSV-1 exposed schizophrenia cases and controls, compared with unexposed persons. Conclusions The lytic/quiescent cycles in iPSC-derived neurons indicate that persistent neuronal infection can occur, altering cellular function. The fMRI studies affirm the associations between nonencephalitic HSV-1 infection and functional brain changes linked with working memory impairment. The fMRI and iPSC studies together provide putative mechanisms for the cognitive impairments linked to HSV-1 exposure.

High levels of DNA methyltransferase 1 (DNMT1), hypermethylation, and downregulation of GAD(67) and reelin have been described in GABAergic interneurons of patients with schizophrenia (SZ) and bipolar (BP) disorders. However, overexpression of DNMT1 is lethal, making it difficult to assess the direct effect of high levels of DNMT1 on neuronal development in vivo. We therefore used Dnmt1(tet/tet) mouse ES cells that overexpress DNMT1 as an in vitro model to investigate the impact of high levels of DNMT1 on neuronal differentiation. Although there is down-regulation of DNMT1 during early stages of differentiation in wild type and Dnmt1(tet/tet) ES cell lines, neurons derived from Dnmt1(tet/tet) cells showed abnormal dendritic arborization and branching. The Dnmt1(tet/tet) neuronal cells also showed elevated levels of functional N-methyl d-aspartate receptor (NMDAR), a feature also reported in some neurological and neurodegenerative disorders. Considering the roles of reelin and GAD(67) in neuronal networking and excitatory/inhibitory balance, respectively, we studied methylation of these genes' promoters in Dnmt1(tet/tet) ES cells and neurons. Both reelin and GAD(67) promoters were not hypermethylated in the Dnmt1(tet/tet) ES cells and neurons, suggesting that overexpression of DNMT1 may not directly result in methylation-mediated repression of these two genes. Taken together, our results suggest that overexpression of DNMT1 in ES cells results in an epigenetic change prior to the onset of differentiation. This epigenetic change in turn results in abnormal neuronal differentiation and upregulation of functional NMDA receptor.

Genomic imprinting is a conserved epigenetic phenomenon in eutherian mammals, with regards both to the genes that are imprinted and the mechanism underlying the expression of just one of the parental alleles. Epigenetic modifications of alleles of imprinted genes are established during oogenesis and spermatogenesis, and these modifications are then inherited. Differentially methylated domains (DMDs) of imprinted genes are the genomic sites of these inherited epigenetic imprints. We previously showed that CpG-rich imperfect tandem direct repeats within three different mouse DMDs (Snurf/Snrpn, Kcnq1 and Igf2r), each with a unique sequence, play a central role in maintaining the differential methylation. This finding implicates repeat-related DNA structure, not sequence, in the imprinting mechanism. To better define the important features of this signal, we compared sequences of these three DMD tandem repeats among mammalian species. All DMD repeats contain short indirect repeats, many of which are organized into larger unit repeats. Even though the larger repeat units undergo deletion and addition during evolution (most likely through unequal crossovers during meiosis), the size of DMD tandem repeated regions has remained remarkably stable during mammalian evolution. Moreover, all three DMD tandem repeats have a high-CpG content, an ordered arrangement of CpG dinucleotides, and similar predicted secondary structures. These observations suggest that a structural feature or features of these DMD tandem repeats is the conserved DMD imprinting signal.

The Amaryllidaceae alkaloid trans-dihydrolycoricidine 7 and three analogues 8-10 were produced via asymmetric chemical synthesis. Alkaloid 7 proved superior to acyclovir, the current standard for herpes simplex virus, type 1 (HSV-1) infection. Compound 7 potently inhibited lytic HSV-1 infection, significantly reduced HSV-1 reactivation, and more potently inhibited varicella zoster virus (VZV) lytic infection. A configurationally defined (3R)-secondary alcohol at C3 proved crucial for efficacious inhibition of lytic HSV-1 infection.

Background: Copy number variation on chromosome 15q11.2 (BP1-BP2) causes a deletion of CYFIP1, NIPA1, NIPA2 and TUBGCP5. Furthermore, it also affects brain structure and elevates the risk for several neurodevelopmental disorders that are associated with dendritic spine abnormalities. In rodents, altered cyfip1 expression changes dendritic spine morphology, motivating analyses of human neuronal cells derived from induced pluripotent stem cells (iPSCs; iPSC-neurons). Methods: iPSCs were generated from a mother and her offspring, both carrying the 15q11.2 (BP1-BP2) deletion, and a non-deletion control. Gene expression in the deletion region was estimated using quantitative real-time PCR assays. Neural progenitor cells (NPCs) and iPSC-neurons were characterized using immunocytochemistry. Results:CYFIP1, NIPA1, NIPA2 and TUBGCP5 gene expression was lower in iPSCs, NPCs and iPSC-neurons from the mother and her offspring in relation to control cells. CYFIP1 and PSD-95 protein levels were lower in iPSC-neurons derived from the copy number variant-bearing individuals using Western blot analysis. Ten weeks after differentiation, iPSC-neurons appeared to show dendritic spines, and qualitative analysis suggested that dendritic morphology was altered in 15q11.2-deletion subjects compared with control cells. Conclusions: The 15q11.2 (BP1-BP2) deletion is associated with a reduced expression of four genes in iPSC-derived neuronal cells; it may also be associated with altered iPSC-neuron dendritic morphology.

Herpesvirus infections cause considerable morbidity and mortality through lifelong recurrent cycles of lytic and latent infection in several tissues, including the human nervous system. Acyclovir (ACV) and its prodrug, the current antivirals of choice for herpes simplex virus (HSV) and, to some extent, varicella zoster virus (VZV) infections are nucleoside analogues that inhibit viral DNA replication. Rising viral resistance and the need for more effective second-line drugs have motivated searches for additional antiviral agents, particularly non-nucleoside based agents. We evaluated the antiviral activity of five compounds with predicted lysosomotropic activity using conventional and human induced pluripotent stem cell-derived neuronal (iPSC-neurons) cultures. Their potency and toxicity were compared with ACV and the lysosomotropic agents chloroquine and bafilomycin A1. Out of five compounds tested, micromolar concentrations of 30N12, 16F19, and 4F17 showed antiviral activity comparable to ACV (50μM) during lytic herpes simplex virus type 1 (HSV-1) infections, reduced viral DNA copy number, and reduced selected HSV-1 protein levels. These compounds also inhibited the reactivation of 'quiescent' HSV-1 infection established in iPSC-neurons, but did not inhibit viral entry into host cells. The same compounds had greater potency than ACV against lytic VZV infection; they also inhibited replication of human cytomegalovirus. The anti-herpetic effects of these non-nucleoside agents merit further evaluation in vivo.

Fluorescence in situ hybridization (FISH) with probes representing sheep satellite I and satellite II DNAs shows a different distribution of the two repetitive DNA families in the centromeric region of most chromosomes. The single signal per chromosome produced by the satellite I probe suggests close proximity of this DNA family to the primary constriction. Satellite II produces two separate signals on the sister chromatids, and large blocks of satellite II DNA constitute most of the short arm of all acrocentric chromosomes. We have isolated and sequenced a phage clone containing a junction between discrete blocks of satellite I and satellite II sequences. The junction is characterized by an abrupt juxtaposition of arrays of the two satellites. The possibility that the peculiar structural features of this junction could have a functional significance is discussed.

Human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS) cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs), neural progenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate.

Roberto Di Maio1, Kommu Naga Mohan2, Brianna Heath3 and Leonardo D’Aiuto4 1 Department of Neurology, University of Pittsburgh, & Ri.Med Foundation, Palermo 2 Department of Molecular and Human Genetics, Baylor College of Medicine 3 Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, 4 Department of Psychiatry, University of Pittsburgh School of Medicine, Western Psychiatric Institute and Clinic 1Italy 2,3,4United States

Reprogramming of DNA methylation patterns during mammalian preimplantation development involves the concurrent maintenance of methylation on differentially methylated domains (DMDs) of imprinted genes and a marked reduction of global (non-DMD) genomic methylation. In the developing mammalian embryo, one allele of a DMD is unmethylated, and the opposite parental allele is methylated, having inherited this methylation from the parental gamete. The maintenance of DMDs is important for monoallelic imprinted gene expression and normal development of the embryo. Because the DNMT1 cytosine methyltransferase governs maintenance methylation in mammals, rearrangements of non-DMD, but not DMD methylation in preimplantation embryos suggest that the preimplantation DNMT1-dependent maintenance mechanism specifically targets DMD sequences. We explored this possibility using an engineered mouse ES cell line to screen for mutant DNMT1 proteins that protect against the loss of DMD and/or global (non-DMD) methylation in the absence of the wild-type endogenous DNMT1 methyltransferase. We identified DNMT1 mutants that were defective in maintenance of either DMD and/or non-DMD methylation. Among these, one mutant maintained non-DMD methylation but not imprinted DMD methylation and another mutant maintained just DMD methylation. The mutated amino acids of these mutants reside in a mammal-specific, disordered region near the amino terminus of DNMT1. These findings suggest that DNMT1 participates in epigenetic reprogramming through its ability to distinguish different categories of methylated sequences.

We have isolated a new interspersed sequence present in a high copy number in the ovine genome. This patchwork sequence, named 3.79 AS1, is part of a larger element encompassing similarities to constant region of reverse transcriptase and to art2 shared with the Bovine Dimer Driven Family (BDDF). The 3.79 AS1 sequence includes homologies to amplification promoting sequences (APS), to a potential origin of bidirectional DNA replication (OBR), to the Alu core sequence motif GGAGGC required for RNA polymerase III promoter function and to the ATGGCTGCCAT sequence that has been shown to be able to induce amplification-dependent transformation in murine cells. Fluorescent in situ hybridization experiments using probes derived from both ends of the 3.79 AS1 sequence showed a widespread signal over all sheep chromosomes, except the Y chromosome. We propose that the structural features of the 3.79 AS1 patchwork sequence, that is likely to be a subfamily of Bov B LINE that invaded the Artiodactyl genome prior to the separation of the Bovidae species, facilitated its massive amplification and dispersion in the ovine genome.

We have isolated and characterized two human alphoid DNA clones: p4n1/4 and pZ4.1. Clone p4n1/4 identifies specifically the centromeric region of chromosome 4; pZ4.1 recognizes a subset of alphoid DNA shared by chromosomes 4 and 9. The specificity was determined using fluorescence in situ hybridization experiments on metaphase spreads and Southern blotting analysis of human-hamster somatic cell hybrids. The genomic organization of both subsets was also investigated. Comparative mapping on chimpanzee and gorilla chromosomes was performed. p4n1/4 hybridizes to chimpanzee chromosomes 11 and 13, homologs of human chromosomes 9 and 2q, respectively. On gorilla metaphase spreads, p4n1/4 hybridizes exclusively to the centromeric region of chromosome 19, partially homologous to human chromosome 17. No hybridization signal was detected on chromosome 3 of both chimpanzee and gorilla, in both species homolog of human chromosome 4. Identical comparative mapping results were obtained using pZ4.1 probe, although the latter recognizes an alphoid subset distinct from the one recognized by p4n1/4. The implications of these results in the evolution of centromeric regions of primate chromosomes are discussed.

Using vector-CENP-B box polymerase chain reaction (PCR) we isolated and cloned from a human chromosome 21-specific plasmid library, a 1 kb DNA sequence, named p alpha H21. In in situ hybridization experiments, p alpha H21 hybridized, under high stringency conditions, to the centromeric region of all the human, chimpanzee, gorilla and orangutan chromosomes. On human chromosomes p alpha H21 also identified non-centromeric sequences at 2q21 (locus D2F33S1) and 9q13 (locus D9F33S2). The possible derivation of these sequences from ancestral centromeres is discussed. Sequence analysis confirmed the alphoid nature of the whole p alpha H21 insert.

Deletion analysis of mouse DNMT1, the primary maintenance methyltransferase in mammals, showed that most of the N-terminal regulatory domain (amino acid residues 412-1112) is required for its enzymatic activity. Although analysis of deletion mutants helps to identify regions of a protein sequence required for a particular activity, amino acid deletions can have drastic effects on protein structure and/or stability. Alternative approaches represented by rational design and directed evolution are resource demanding, and require high-throughput selection or screening systems. We developed Regional Frame-shift Mutagenesis (RFM) as a new approach to identify portions required for the methyltransferase activity of DNMT1 within the N-terminal 89-905 amino acids. In this method, a short stretch of amino acids in the wild-type protein is converted to a different amino acid sequence. The resultant mutant protein retains the same amino acid length as the wild type, thereby reducing physical constrains on normal folding of the mutant protein. Using RFM, we identified three small regions in the amino-terminal one-third of the protein that are essential for DNMT1 function. Two of these regions (amino acids 124-160 and 341-368) border a large disordered region that regulates maintenance methylation activity. This organization of DNMT1's amino terminus suggests that the borders define the position of the disordered region within the DNMT1 protein, which in turn allows for its proper function.

Background Establishing a suitable level of exogenous gene expression in mammalian cells in general, and embryonic stem (ES) cells in particular, is an important aspect of understanding pathways of cell differentiation, signal transduction and cell physiology. Despite its importance, this process remains challenging because of the poor correlation between the presence of introduced exogenous DNA and its transcription. Consequently, many transfected cells must be screened to identify those with an appropriate level of expression. To improve the screening process, we investigated the utility of the human interleukin 12 (IL-12) p40 cDNA as a reporter gene for studies of mammalian gene expression and for high-throughput screening of engineered mouse embryonic stem cells. Results A series of expression plasmids were used to study the utility of IL-12 p40 as an accurate reporter of gene activity. These studies included a characterization of the IL-12 p40 expression system in terms of: (i) a time course of IL-12 p40 accumulation in the medium of transfected cells; (ii) the dose-response relationship between the input DNA and IL-12 p40 mRNA levels and IL-12 p40 protein secretion; (iii) the utility of IL-12 p40 as a reporter gene for analyzing the activity of cis-acting genetic elements; (iv) expression of the IL-12 p40 reporter protein driven by an IRES element in a bicistronic mRNA; (v) utility of IL-12 p40 as a reporter gene in a high-throughput screening strategy to identify successful transformed mouse embryonic stem cells; (vi) demonstration of pluripotency of IL-12 p40 expressing ES cells in vitro and in vivo; and (vii) germline transmission of the IL-12 p40 reporter gene. Conclusion IL-12 p40 showed several advantages as a reporter gene in terms of sensitivity and ease of the detection procedure. The IL-12 p40 assay was rapid and simple, in as much as the reporter protein secreted from the transfected cells was accurately measured by ELISA using a small aliquot of the culture medium. Remarkably, expression of Il-12 p40 does not affect the pluripotency of mouse ES cells. To our knowledge, human IL-12 p40 is the first secreted reporter protein suitable for high-throughput screening of mouse ES cells. In comparison to other secreted reporters, such as the widely used alkaline phosphatase (SEAP) reporter, the IL-12 p40 reporter system offers other real advantages.

The causative agent of Severe Acute Respiratory Syndrome (SARS) has been identified as a new type of coronavirus. Here we have examined the ability of adenoviral gene transfer of codon-optimized SARS-CoV strain Urbani structural antigens spike (S) protein S1 fragment, membrane protein (M), and nucleocapsid protein (N) to induce broad immunity in the non-human primate. Six rhesus macaques were immunized intramuscularly with a combination of the three Ad5-SARS-CoV vectors and boosted on day 28. The vaccinated animals all had antibody responses against S1 and T-cell responses against the antigens. Importantly, all six vaccinated animals showed strong neutralizing antibody responses to SARS-CoV infection in vitro. These results demonstrate that an adenoviral-based vaccine can induce strong SARS-CoV-specific immune responses in the monkey, and hold promise for development of a protective vaccine against the SARS etiologic agent.