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1.  Genetic and phenotypic diversity of NHE6 mutations in Christianson syndrome 
Annals of neurology  2014;76(4):581-593.
Recently, Christianson syndrome (CS) has been determined to be caused by mutations in the X-linked Na+/H+ Exchanger 6 (NHE6). We aimed to determine the diagnostic criteria and mutational spectrum for CS.
Twelve independent pedigrees (14 boys, ages 4 to 19) with mutations in NHE6 were administered standardized research assessments and mutations were characterized.
The mutational spectrum was composed of 9 single nucleotide variants (SNVs), 2 indels and 1 CNV deletion. All mutations were protein-truncating or splicing mutations. We identified two recurrent mutations (c.1498 c>t, p.R500X; and c.1710 g>a, p.W570X). Otherwise, all mutations were unique. In our study, seven of 12 mutations (58%) were de novo, in contrast to prior literature wherein mutations were largely inherited. We also report prominent neurological, medical and behavioral symptoms. All CS participants were non-verbal and had intellectual disability, epilepsy and ataxia. Many had prior diagnoses of autism and/or Angelman syndrome. Other neurologic symptoms included eye movement abnormalities (79%), postnatal microcephaly (92%) and MRI evidence of cerebellar atrophy (33%). Regression was noted in 50%, with recurrent presentations involving loss of words and/or the ability to walk. Medical symptoms, particularly gastrointestinal symptoms, were common. Height and body mass index measures were below normal ranges in most participants. Behavioral symptoms included hyperkinetic behavior (100%) and a majority exhibited high pain threshold.
This is the largest cohort of independent CS pedigrees reported. We propose diagnostic criteria for CS. CS represents a novel neurogenetic disorder with general relevance to autism, intellectual disability, Angelman syndrome, epilepsy and regression.
PMCID: PMC4304796  PMID: 25044251
2.  Unanchored K48-linked poly-ubiquitin synthesized by the E3-ubiquitin ligase TRIM6 stimulates the interferon-IKKε kinase mediated antiviral response 
Immunity  2014;40(6):880-895.
Type-I interferons (IFN-I) are essential antiviral cytokines produced upon microbial infection. IFN-I elicits this activity through the upregulation of hundreds of IFN-I stimulated genes (ISGs). The full breadth of ISG induction demands activation of a number of cellular factors including the IκB kinase epsilon (IKKε). However, the mechanism of IKKε activation upon IFN receptor signaling has remained elusive. Here we show that TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family of proteins, interacts with IKKε and promotes induction of IKKε-dependent ISGs. TRIM6 and the E2-ubiquitin conjugase UbE2K cooperate in the synthesis of unanchored K48-linked poly-ubiquitin chains, which activate IKKε for subsequent STAT1 phosphorylation. Our work attributes a previously unrecognized activating role of K48-linked unanchored poly-ubiquitin chains in kinase activation and identifies the UbE2K-TRIM6-ubiquitin axis as critical for IFN signaling and antiviral response.
PMCID: PMC4114019  PMID: 24882218
3.  Inhibition of Dengue Virus Replication by a Class of Small-Molecule Compounds That Antagonize Dopamine Receptor D4 and Downstream Mitogen-Activated Protein Kinase Signaling 
Journal of Virology  2014;88(10):5533-5542.
Dengue viruses (DENV) are endemic pathogens of tropical and subtropical regions that cause significant morbidity and mortality worldwide. To date, no vaccines or antiviral therapeutics have been approved for combating DENV-associated disease. In this paper, we describe a class of tricyclic small-molecule compounds—dihydrodibenzothiepines (DHBTs), identified through high-throughput screening—with potent inhibitory activity against DENV serotype 2. SKI-417616, a highly active representative of this class, displayed activity against all four serotypes of DENV, as well as against a related flavivirus, West Nile virus (WNV), and an alphavirus, Sindbis virus (SINV). This compound was characterized to determine its mechanism of antiviral activity. Investigation of the stage of the viral life cycle affected revealed that an early event in the life cycle is inhibited. Due to the structural similarity of the DHBTs to known antagonists of the dopamine and serotonin receptors, we explored the roles of two of these receptors, serotonin receptor 2A (5HTR2A) and the D4 dopamine receptor (DRD4), in DENV infection. Antagonism of DRD4 and subsequent downstream phosphorylation of epidermal growth factor receptor (EGFR)-related kinase (ERK) were found to impact DENV infection negatively, and blockade of signaling through this network was confirmed as the mechanism of anti-DENV activity for this class of compounds.
IMPORTANCE The dengue viruses are mosquito-borne, reemerging human pathogens that are the etiological agents of a spectrum of febrile diseases. Currently, there are no approved therapeutic treatments for dengue-associated disease, nor is there a vaccine. This study identifies a small molecule, SKI-417616, with potent anti-dengue virus activity. Further analysis revealed that SKI-417616 acts through antagonism of the host cell dopamine D4 receptor and subsequent repression of the ERK phosphorylation pathway. These results suggest that SKI-417616, or other compounds targeting the same cellular pathways, may have therapeutic potential for the treatment of dengue virus infections.
PMCID: PMC4019099  PMID: 24599995
4.  Gene Editing of CCR5 in Autologous CD4 T Cells of Persons Infected with HIV 
The New England journal of medicine  2014;370(10):901-910.
CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene (“gene editing”) — in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN) — is safe.
We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance.
One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (−1.81 cells per day) was significantly less than the decline in unmodified cells (−7.25 cells per day) (P = 0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients.
CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National Institute of Allergy and Infectious Diseases and others; number, NCT00842634.)
PMCID: PMC4084652  PMID: 24597865
5.  Localized serine protease activity and the establishment of Drosophila embryonic dorsoventral polarity 
Fly  2013;7(3):161-167.
Drosophila embryo dorsoventral polarity is established by a maternally encoded signal transduction pathway in which three sequentially acting serine proteases, Gastrulation Defective, Snake and Easter, generate the ligand that activates the Toll receptor on the ventral side of the embryo. The spatial regulation of this pathway depends upon ventrally restricted expression of the Pipe sulfotransferase in the ovarian follicle during egg formation. Several recent observations have advanced our understanding of the mechanism regulating the spatially restricted activation of Toll. First, several protein components of the vitelline membrane layer of the eggshell have been determined to be targets of Pipe-mediated sulfation. Second, the processing of Easter by Snake has been identified as the first Pipe-dependent, ventrally-restricted processing event in the pathway. Finally, Gastrulation Defective has been shown to undergo Pipe-dependent, ventral localization within the perivitelline space and to facilitate Snake-mediated processing of Easter. Together, these observations suggest that Gastrulation Defective, localized on the interior ventral surface of the eggshell in association with Pipe-sulfated eggshell proteins, recruits and mediates an interaction between Snake and Easter. This event leads to ventrally-restricted processing and activation of Easter and consequently, localized formation of the Toll ligand, and Toll activation.
PMCID: PMC4049848  PMID: 24047959
Drosophila; Easter; Gastrulation Defective; Pipe; Snake; Spätzle; Toll; dorsal-ventral; follicle; perivitelline; sulfation; sulfonation
6.  Developmental Regression, Depression, and Psychosocial Stress in an Adolescent with Down Syndrome 
Journal of developmental and behavioral pediatrics : JDBP  2013;34(3):10.1097/DBP.0b013e31828b2b42.
CASE: Kristen is a 13-year-old girl with Down syndrome (DS) who was seen urgently with concerns of cognitive and developmental regression including loss of language, social, and toileting skills. The evaluation in the DS clinic focused on potential medical diagnoses including atlantoaxial joint instability, vitamin deficiency, obstructive sleep apnea (OSA), and seizures. A comprehensive medical evaluation yielded only a finding of moderate OSA. A reactive depression was considered in association with several psychosocial factors including moving homes, entering puberty/onset of menses, and classroom change from an integrated setting to a self-contained classroom comprising unfamiliar peers with behavior challenges.
Urgent referrals for psychological and psychiatric evaluations were initiated. Neuropsychological testing did not suggest true regression in cognitive, language, and academic skills, although decreases in motivation and performance were noted with a reaction to stress and multiple environmental changes as a potential causative factor. Psychiatry consultation supported this finding in that psychosocial stress temporally correlated with Kristen’s regression in skills.
Working collaboratively, the team determined that Kristen’s presentation was consistent with a reactive form of depression (DSM-IV-TR: depressive disorder, not otherwise specified). Kristen’s presentation was exacerbated by salient environmental stress and sleep apnea, rather than a cognitive regression associated with a medical cause. Treatment consisted of an antidepressant medication, continuous positive airway pressure for OSA, and increased psychosocial supports. Her school initiated a change in classroom placement. With this multimodal approach to evaluation and intervention, Kristen steadily improved and she returned to her baseline function.
PMCID: PMC3884753  PMID: 23572173
Down syndrome; developmental regression; depression; obstructive sleep apnea
7.  Flaviviruses Are Sensitive to Inhibition of Thymidine Synthesis Pathways 
Journal of Virology  2013;87(17):9411-9419.
Dengue virus has emerged as a global health threat to over one-third of humankind. As a positive-strand RNA virus, dengue virus relies on the host cell metabolism for its translation, replication, and egress. Therefore, a better understanding of the host cell metabolic pathways required for dengue virus infection offers the opportunity to develop new approaches for therapeutic intervention. In a recently described screen of known drugs and bioactive molecules, we observed that methotrexate and floxuridine inhibited dengue virus infections at low micromolar concentrations. Here, we demonstrate that all serotypes of dengue virus, as well as West Nile virus, are highly sensitive to both methotrexate and floxuridine, whereas other RNA viruses (Sindbis virus and vesicular stomatitis virus) are not. Interestingly, flavivirus replication was restored by folinic acid, a thymidine precursor, in the presence of methotrexate and by thymidine in the presence of floxuridine, suggesting an unexpected role for thymidine in flavivirus replication. Since thymidine is not incorporated into RNA genomes, it is likely that increased thymidine production is indirectly involved in flavivirus replication. A possible mechanism is suggested by the finding that p53 inhibition restored dengue virus replication in the presence of floxuridine, consistent with thymidine-less stress triggering p53-mediated antiflavivirus effects in infected cells. Our data reveal thymidine synthesis pathways as new and unexpected therapeutic targets for antiflaviviral drug development.
PMCID: PMC3754125  PMID: 23824813
8.  Inaugural Christianson Syndrome Association conference: families meeting for the first time 
Christianson syndrome (CS) is an X-linked neurodevelopmental disorder caused by deleterious mutations in SLC9A6. Affected families organized the inaugural Christianson Syndrome Association conference to advance CS knowledge and develop questions that may be prioritized in future research.
PMCID: PMC4038054  PMID: 25273398
Christianson syndrome; Intellectual disability; NHE6; SLC9A6; X-linked developmental disorder
9.  A ventrally localized protease in the Drosophila egg controls embryo dorsoventral polarity 
Current Biology  2012;22(11):1013-1018.
Drosophila embryo dorsoventral (DV) polarity is defined by serine protease activity in the perivitelline space (PVS) between the embryonic membrane and the inner layer of the eggshell [1, 2, 3, 4, 5]. Gastrulation Defective (GD) cleaves and activates Snake (Snk). Activated Snk cleaves and activates Easter (Ea), exclusively on the ventral side of the embryo [6, 7, 8]. Activated Ea then processes Spätzle (Spz) into the activating ligand for Toll, a transmembrane receptor that is distributed throughout the embryonic plasma membrane [9]. Ventral activation of Toll depends upon the activity of the Pipe sulfotransferase in the ventral region of the follicular epithelium that surrounds the developing oocyte [10]. Pipe transfers sulfate residues to several protein components of the inner vitelline membrane layer of the eggshell [11]. Here we show that GD protein becomes localized in the ventral PVS in a Pipe-dependent process. Moreover, ventrally concentrated GD acts to promote the cleavage of Ea by Snk through an extracatalytic mechanism that is distinct from GD's proteolytic activation of Snk. Together, these observations illuminate the mechanism through which spatially restricted sulfotransferase activity in the developing egg chamber leads to localization of serine protease activity and ultimately to spatially specific activation of the Toll receptor in the Drosophila embryo.
PMCID: PMC3371173  PMID: 22578419
10.  Cognitive Stimulation and Cognitive and Functional Decline in Alzheimer's Disease: The Cache County Dementia Progression Study 
To examine the association of engagement in cognitively stimulating activities with cognitive and functional decline in a population-based sample of incident Alzheimer's disease (AD).
After diagnosis, 187 participants (65% females) were followed semiannually for a mean 2.7 (SD = 0.4) years. Mean age and education were 84.6 (SD = 5.8) and 13.2 (SD = 2.9) years. Caregivers enumerated cognitively stimulating leisure activities via the Lifestyle Activities Questionnaire. Cognition was assessed using the Mini-Mental State Examination and functional ability via the Clinical Dementia Rating sum of boxes. Linear mixed models tested the association between stimulating activities and change over time in each outcome. Covariates were demographic factors, estimated premorbid IQ, presence/absence of the APOE ϵ4 allele, duration of dementia, level of physical activity, and general health.
At initial assessment, 87% of participants were engaged in one or more stimulating activities, with mean (SD) activities = 4.0 (3.0). This number declined to 2.4 (2.0) at the final visit. There was a statistical interaction between dementia duration and number of activities in predicting rate of cognitive decline (p = .02) and overall functional ability (p = .006).
Active involvement in cognitively stimulating pursuits may be beneficial for persons with AD.
PMCID: PMC3132266  PMID: 21441386
Alzheimer's disease; Cognitive activity; Cognitive decline; Dementia
11.  A Rare Case of Myeloid Sarcoma Presenting as an Anorectal Ulcer 
Case Reports in Medicine  2012;2012:537278.
Myeloid Sarcoma is a rare tumor composed of myeloblasts occurring at an extramedullary site like bones, or various soft tissues. Myeloid sarcoma may involve the gastrointestinal tract very rarely either solitarily, or occurring simultaneously with acute myeloid leukemia. Its diagnosis is challenging and needs biopsy and immunohistochemical staining. We are describing a case of myeloid sarcoma which presented as a painful anal ulcer mimicking an atypical fissure. Its appearance resembled crohn's disease on sigmoidoscopy. A biopsy of the ulcer along with histochemical staining led to the diagnosis of myeloid sarcoma. Our case demonstrates the need for aggressive evaluation of any common gastrointestinal complaint with an atypical presentation.
PMCID: PMC3364052  PMID: 22666268
12.  Development of Peptide-Conjugated Morpholino Oligomers as Pan-Arenavirus Inhibitors▿† 
Antimicrobial Agents and Chemotherapy  2011;55(10):4631-4638.
Members of the Arenaviridae family are a threat to public health and can cause meningitis and hemorrhagic fever, and yet treatment options remain limited by a lack of effective antivirals. In this study, we found that peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) complementary to viral genomic RNA were effective in reducing arenavirus replication in cell cultures and in vivo. PPMO complementary to the Junín virus genome were designed to interfere with viral RNA synthesis or translation or both. However, only PPMO designed to potentially interfere with translation were effective in reducing virus replication. PPMO complementary to sequences that are highly conserved across the arenaviruses and located at the 5′ termini of both genomic segments were effective against Junín virus, Tacaribe virus, Pichinde virus, and lymphocytic choriomeningitis virus (LCMV)-infected cell cultures and suppressed viral titers in the livers of LCMV-infected mice. These results suggest that arenavirus 5′ genomic termini represent promising targets for pan-arenavirus antiviral therapeutic development.
PMCID: PMC3186971  PMID: 21825302
13.  Inhibition of Dengue Virus Infections in Cell Cultures and in AG129 Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence ▿  
Journal of Virology  2011;85(19):10154-10166.
The dengue viruses (DENVs) exist as numerous genetic strains that are grouped into four antigenically distinct serotypes. DENV strains from each serotype can cause severe disease and threaten public health in tropical and subtropical regions worldwide. No licensed antiviral agent to treat DENV infections is currently available, and there is an acute need for the development of novel therapeutics. We found that a synthetic small interfering RNA (siRNA) (DC-3) targeting the highly conserved 5′ cyclization sequence (5′CS) region of the DENV genome reduced, by more than 100-fold, the titers of representative strains from each DENV serotype in vitro. To determine if DC-3 siRNA could inhibit DENV in vivo, an “in vivo-ready” version of DC-3 was synthesized and tested against DENV-2 by using a mouse model of antibody-dependent enhancement of infection (ADE)-induced disease. Compared with the rapid weight loss and 5-day average survival time of the control groups, mice receiving the DC-3 siRNA had an average survival time of 15 days and showed little weight loss for approximately 12 days. DC-3-treated mice also contained significantly less virus than control groups in several tissues at various time points postinfection. These results suggest that exogenously introduced siRNA combined with the endogenous RNA interference processing machinery has the capacity to prevent severe dengue disease. Overall, the data indicate that DC-3 siRNA represents a useful research reagent and has potential as a novel approach to therapeutic intervention against the genetically diverse dengue viruses.
PMCID: PMC3196423  PMID: 21795337
14.  Co-selection of West Nile virus nucleotides that confer resistance to an antisense oligomer while maintaining long-distance RNA/RNA base-pairings 
Virology  2008;382(1):98-106.
West Nile virus (WNV) genome cyclization is mediated by two pairs of long-distance RNA/RNA interactions: the 5′CS/3′CSI (conserved sequence) and the 5′UAR/3′UAR (upstream AUG region) base pairings. Antisense peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), designed to interfere with the 5′CS/3′CSI or 5′UAR/3′UAR base pairings, were previously shown to inhibit WNV. In this study, we selected and characterized WNVs resistant to a PPMO targeting the 3′UAR (3′UAR-PPMO). All resistant viruses accumulated one-nucleotide mutations within the 3′UAR, leading to a single-nucleotide mismatch or a weakened base-pairing interaction with the 3′UAR-PPMO. Remarkably, a one-nucleotide mutation within the 5′UAR was correspondingly co-selected; the 5′UAR mutation restored the base-pairing with the 3′UAR mutation. Mutagenesis of WNV demonstrated that the single-nucleotide change within the 3′UAR-PPMO-target site conferred the resistance. RNA binding analysis indicated that the single-nucleotide change reduced the ability of 3′UAR-PPMO to block the RNA/RNA interaction required for genome cyclization. The results suggest a mechanism by which WNV develops resistance to 3′UAR-PPMO, through co-selection of the 5′UAR and 3′UAR, to create a mismatch or a weakened base-pairing interaction with the PPMO, while maintaining the 5′UAR/3′UAR base pairings.
PMCID: PMC3202013  PMID: 18842280
West Nile virus; Flavivirus replication; Antiviral therapy; genome cyclization; RNA cis elements
15.  High-Content Assay to Identify Inhibitors of Dengue Virus Infection 
Dengue virus (DENV) infections are vectored by mosquitoes and constitute one of the most prevalent infectious diseases in many parts of the world, affecting millions of people annually. Current treatments for DENV infections are nonspecific and largely ineffective. In this study, we describe the adaptation of a high-content cell-based assay for screening against DENV-infected cells to identify inhibitors and modulators of DENV infection. Using this high-content approach, we monitored the inhibition of test compounds on DENV protein production by means of immunofluorescence staining of DENV glycoprotein envelope, simultaneously evaluating cytotoxicity in HEK293 cells. The adapted 384-well microtiter-based assay was validated using a small panel of compounds previously reported as having inhibitory activity against DENV infections of cell cultures, including compounds with antiviral activity (ribavirin), inhibitors of cellular signaling pathways (U0126), and polysaccharides that are presumed to interfere with virus attachment (carrageenan). A screen was performed against a collection of 5,632 well-characterized bioactives, including U.S. Food and Drug Administration–approved drugs. Assay control statistics show an average Z' of 0.63, indicative of a robust assay in this cell-based format. Using a threshold of >80% DENV inhibition with <20% cellular cytotoxicity, 79 compounds were initially scored as positive hits. A follow-up screen confirmed 73 compounds with IC50 potencies ranging from 60 nM to 9 μM and yielding a hit rate of 1.3%. Over half of the confirmed hits are known to target transporters, receptors, and protein kinases, providing potential opportunity for drug repurposing to treat DENV infections. In summary, this assay offers the opportunity to screen libraries of chemical compounds, in an effort to identify and develop novel drug candidates against DENV infections.
PMCID: PMC2962577  PMID: 20973722
16.  Inhibition of Influenza Virus Infection in Human Airway Cell Cultures by an Antisense Peptide-Conjugated Morpholino Oligomer Targeting the Hemagglutinin-Activating Protease TMPRSS2 ▿  
Journal of Virology  2010;85(4):1554-1562.
Influenza A viruses constitute a major and ongoing global public health concern. Current antiviral strategies target viral gene products; however, the emergence of drug-resistant viruses highlights the need for novel antiviral approaches. Cleavage of the influenza virus hemagglutinin (HA) by host cell proteases is crucial for viral infectivity and therefore presents a potential drug target. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded-DNA-like antisense agents that readily enter cells and can act as antisense agents by sterically blocking cRNA. Here, we evaluated the effect of PPMO targeted to regions of the pre-mRNA or mRNA of the HA-cleaving protease TMPRSS2 on proteolytic activation and spread of influenza viruses in human Calu-3 airway epithelial cells. We found that treatment of cells with a PPMO (T-ex5) designed to interfere with TMPRSS2 pre-mRNA splicing resulted in TMPRSS2 mRNA lacking exon 5 and consequently the expression of a truncated and enzymatically inactive form of TMPRSS2. Altered splicing of TMPRSS2 mRNA by the T-ex5 PPMO prevented HA cleavage in different human seasonal and pandemic influenza A viruses and suppressed viral titers by 2 to 3 log10 units, strongly suggesting that TMPRSS2 is responsible for HA cleavage in Calu-3 airway cells. The data indicate that PPMO provide a useful reagent for investigating HA-activating proteases and may represent a promising strategy for the development of novel therapeutics to address influenza infections.
PMCID: PMC3028871  PMID: 21123387
17.  Pipe-dependent ventral processing of Easter by Snake is the defining step in Drosophila embryo DV axis formation 
Current biology : CB  2010;20(12):1133-1137.
The establishment of Drosophila embryonic dorsal-ventral (DV) polarity relies on serine proteolytic activity in the perivitelline space between the embryonic membrane and the eggshell [1]. Gastrulation Defective cleaves and activates Snake, which processes and activates Easter, which cleaves Spätzle to form the activating ligand for the Toll receptor. Ventral restriction of ligand formation depends on the Pipe sulfotransferase, which is expressed in ventral cells of the follicular epithelium surrounding the developing oocyte [2]. Pipe modifies components of the developing eggshell to produce a ventral cue embedded in the vitelline membrane [3]. This ventral cue is believed to promote one or more of the proteolysis steps in the perivitelline space. By examining the processing of transgenic, tagged versions of the perivitelline proteins during DV patterning we find that the proteolysis of Easter by Snake is the first Pipe-dependent step and therefore the key ventrally-restricted event in the protease cascade. We also find that Snake and Easter associate together in a complex in both wild-type and pipe mutant-derived embryos. This observation suggests a mechanism in which the sulfated target of Pipe promotes a productive interaction between Snake and Easter, perhaps by facilitating conformational changes in a complex containing the two proteins.
PMCID: PMC2902586  PMID: 20605458
18.  Coxsackievirus B3 Infection Activates the Unfolded Protein Response and Induces Apoptosis through Downregulation of p58IPK and Activation of CHOP and SREBP1▿  
Journal of Virology  2010;84(17):8446-8459.
Cardiomyocyte apoptosis is a hallmark of coxsackievirus B3 (CVB3)-induced myocarditis. We used cardiomyocytes and HeLa cells to explore the cellular response to CVB3 infection, with a focus on pathways leading to apoptosis. CVB3 infection triggered endoplasmic reticulum (ER) stress and differentially regulated the three arms of the unfolded protein response (UPR) initiated by the proximal ER stress sensors ATF6a (activating transcription factor 6a), IRE1-XBP1 (X box binding protein 1), and PERK (PKR-like ER protein kinase). Upon CVB3 infection, glucose-regulated protein 78 expression was upregulated, and in turn ATF6a and XBP1 were activated via protein cleavage and mRNA splicing, respectively. UPR activity was further confirmed by the enhanced expression of UPR target genes ERdj4 and EDEM1. Surprisingly, another UPR-associated gene, p58IPK, which often is upregulated during infections with other types of viruses, was downregulated at both mRNA and protein levels after CVB3 infection. These findings were observed similarly for uninfected Tet-On HeLa cells induced to overexpress ATF6a or XBP1. In exploring potential connections between the three UPR pathways, we found that the ATF6a-induced downregulation of p58IPK was associated with the activation of PKR (PERK) and the phosphorylation of eIF2α, suggesting that p58IPK, a negative regulator of PERK and PKR, mediates cross-talk between the ATF6a/IRE1-XBP1 and PERK arms. Finally, we found that CVB3 infection eventually produced the induction of the proapoptoic transcription factor CHOP and the activation of SREBP1 and caspase-12. Taken together, these data suggest that CVB3 infection activates UPR pathways and induces ER stress-mediated apoptosis through the suppression of P58IPK and induction/activation of CHOP, SREBP1, and caspase-12.
PMCID: PMC2918999  PMID: 20554776
19.  No requirement for localized Nudel protein expression in Drosophila embryonic axis determination 
Fly  2008;2(4):220-228.
Drosophila embryonic dorsal-ventral polarity is defined by a maternally encoded signal transduction pathway. Gastrulation Defective, Snake and Easter comprise a serine protease cascade that operates in the perivitelline space to generate active ligand for the Toll receptor, which resides in the embryonic membrane. Toll is activated only on the ventral side of the embryo. Spatial regulation of this pathway is initiated by the ventrally restricted expression of the sulfotransferase Pipe in the follicular epithelium that surrounds the developing oocyte. Pipe is thought to modify a target molecule that is secreted and localized within the ventral region of the egg and future embryo, where it influences the activity of the pathway such that active Toll ligand is produced only ventrally. A potential substrate for Pipe is encoded by nudel, which is expressed throughout the follicle cell layer and encodes a large, multi-functional secreted protein that contains a serine protease domain as well as other structural features characteristic of extracellular matrix proteins. A previous mosaic analysis suggested that the protease domain of Nudel is not a target for Pipe activity as its expression is not required in pipe-expressing cells, but failed to rule out such a role for other functional domains of the protein. To investigate this possibility, we carried out a mosaic analysis of additional nudel alleles, including some that affect the entire protein. Our analysis demonstrated that proteolytically processed segments of Nudel are secreted into the perivitelline space and stably localized, as would be expected for the target of Pipe, However, we found no requirement for nudel to be expressed in ventral, pipe-expressing follicle cells, thereby eliminating Nudel as an essential substrate of Pipe sulfotransferase activity.
PMCID: PMC2941771  PMID: 18776742
Drosophila; dorsoventral; dorsal-ventral; dorsal group; pipe; nudel; oogenesis; follicle
20.  Sulfation of Eggshell Proteins by Pipe Defines Dorsal-Ventral Polarity in the Drosophila embryo 
Current biology : CB  2009;19(14):1200-1205.
Drosophila embryonic dorsal-ventral (DV) polarity is controlled by a group of sequentially acting serine proteases located in the fluid-filled perivitelline space between the embryonic membrane and the eggshell, which generate the ligand for the Toll receptor on the ventral side of the embryo [1, 2, 3]. Spatial control of the protease cascade relies on the Pipe sulfotransferase, a fly homologue of vertebrate glycosaminoglycan modifying enzymes [4, 5, 6], which is expressed in ventral cells of the follicular epithelium surrounding the developing oocyte. The identification of the Pipe enzymatic target has remained a major gap in our understanding of the mechanism controlling the perivitelline protease cascade, and hence embryonic DV patterning. Here we show that the protein Vitelline Membrane-Like (VML) [7] undergoes Pipe-dependent sulfation and, consistent with a role in conveying positional information from the egg chamber to the embryo, becomes incorporated into the eggshell at a position corresponding to the location of the follicle cells from which it was secreted. Although VML influences embryonic DV pattern in a sensitized genetic background, VML is not essential for DV axis formation, suggesting that there is redundancy in the composition of the Pipe enzymatic target. Correspondingly, we find that additional structural components of the vitelline membrane undergo Pipe-dependent sulfation. In identifying the elusive targets of Pipe, this ork points to the vitelline membrane as the source of signals that generate the Drosophila DV axis and provides a framework for understanding the mechanism controlling spatially-specific activation of serine protease activity during embryonic pattern formation.
PMCID: PMC2733793  PMID: 19540119
Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in infants, young children, and high-risk adults. Currently, there is no vaccine for the prevention of RSV infection, and available therapeutics are of limited utility. Peptide conjugated phosphorodiamidate morpholino oligomers (PPMO) are a class of antisense agents that can enter cells readily and interfere with viral protein expression through steric blocking of complementary RNA. Two antisense PPMO, designed to target sequence that includes the 5′ terminal- and translation start site-regions of RSV L mRNA, were tested for anti-RSV activity in cultures of two human airway cell lines. Both PPMO showed minimal cytotoxicity, and one of them (AUG-2), reduced viral titers by more than 2.0 log10. Intranasal treatment of BALB/c mice with AUG-2 PPMO prior to RSV inoculation produced a reduction in viral titer of 1.2 log10 in lung tissue at day 5 post-infection, and attenuated pulmonary inflammation at day 7 post-infection. These data show that the AUG-2 PPMO possessed potent anti-RSV activity and is worthy of further investigation as a candidate for therapeutic development.
PMCID: PMC2782410  PMID: 18443602
RSV; morpholino oligomers; antisense; PPMO; antiviral agents
22.  Mis-specified cells die by an active gene-directed process, and inhibition of this death results in cell fate transformation in Drosophila 
Development (Cambridge, England)  2005;132(24):5343-5352.
Incorrectly specified or mis-specified cells often undergo cell death or are transformed to adopt a different cell fate during development. The underlying cause for this distinction is largely unknown. In many developmental mutants in Drosophila, large numbers of mis-specified cells die synchronously, providing a convenient model for analysis of this phenomenon. The maternal mutant bicoid is particularly useful model with which to address this issue because its mutant phenotype is a combination of both transformation of tissue (acron to telson) and cell death in the presumptive head and thorax regions. We show that a subset of these mis-specified cells die through an active gene-directed process involving transcriptional upregulation of the cell death inducer hid. Upregulation of hid also occurs in oskar mutants and other segmentation mutants. In hid bicoid double mutants, mis-specified cells in the presumptive head and thorax survive and continue to develop, but they are transformed to adopt a different cell fate. We provide evidence that the terminal torso signaling pathway protects the mis-specified telson tissue in bicoid mutants from hid-induced cell death, whereas mis-specified cells in the head and thorax die, presumably because equivalent survival signals are lacking. These data support a model whereby mis-specification can be tolerated if a survival pathway is provided, resulting in cellular transformation.
PMCID: PMC2760325  PMID: 16280349
Mis-specification; Cell death; Transformation; Bicoid; Oskar; Hid; Drosophila
23.  Severe Acute Respiratory Syndrome Coronavirus Triggers Apoptosis via Protein Kinase R but Is Resistant to Its Antiviral Activity▿  
Journal of Virology  2008;83(5):2298-2309.
In this study, infection of 293/ACE2 cells with severe acute respiratory syndrome coronavirus (SARS-CoV) activated several apoptosis-associated events, namely, cleavage of caspase-3, caspase-8, and poly(ADP-ribose) polymerase 1 (PARP), and chromatin condensation and the phosphorylation and hence inactivation of the eukaryotic translation initiation factor 2α (eIF2α). In addition, two of the three cellular eIF2α kinases known to be virus induced, protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK), were activated by SARS-CoV. The third kinase, general control nonderepressible-2 kinase (GCN2), was not activated, but late in infection the level of GCN2 protein was significantly reduced. Reverse transcription-PCR analyses revealed that the reduction of GCN2 protein was not due to decreased transcription or stability of GCN2 mRNA. The specific reduction of PKR protein expression by antisense peptide-conjugated phosphorodiamidate morpholino oligomers strongly reduced cleavage of PARP in infected cells. Surprisingly, the knockdown of PKR neither enhanced SARS-CoV replication nor abrogated SARS-CoV-induced eIF2α phosphorylation. Pretreatment of cells with beta interferon prior to SARS-CoV infection led to a significant decrease in PERK activation, eIF2α phosphorylation, and SARS-CoV replication. The various effects of beta interferon treatment were found to function independently on the expression of PKR. Our results show that SARS-CoV infection activates PKR and PERK, leading to sustained eIF2α phosphorylation. However, virus replication was not impaired by these events, suggesting that SARS-CoV possesses a mechanism to overcome the inhibitory effects of phosphorylated eIF2α on viral mRNA translation. Furthermore, our data suggest that viral activation of PKR can lead to apoptosis via a pathway that is independent of eIF2α phosphorylation.
PMCID: PMC2643707  PMID: 19109397
24.  Inhibition of alphavirus infection in cell culture and in mice with antisense morpholino oligomers 
Virology  2008;376(2):357-370.
The genus Alphavirus contains members that threaten human health, both as natural pathogens and as potential biological weapons. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) enter cells readily and can inhibit viral replication through sequence-specific steric blockade of viral RNA. Sindbis virus (SINV) has low pathogenicity in humans and is regularly utilized as a model alphavirus. PPMO targeting the 5′-terminal and AUG translation start site-regions of the SINV genome blocked the production of infectious SINV in tissue culture. PPMO designed against corresponding regions in Venezuelan equine encephalitis virus (VEEV) were likewise found to be effective in vitro against several strains of VEEV. Mice treated with PPMO before and after VEEV infection were completely protected from lethal outcome while mice receiving only post-infection PPMO treatment were partially protected. Levels of virus in tissue samples correlated with animal survival. Uninfected mice suffered no apparent ill-effects from PPMO treatment. Thus, PPMO appear promising as candidates for therapeutic development against alphaviruses.
PMCID: PMC2447162  PMID: 18468653
Venezuelan equine encephalitis virus; Sindbis virus; pathogenic alphaviruses; antiviral agents; antisense therapy; morpholino oligomers
25.  Vascular Factors and Risk for Neuropsychiatric Symptoms in Alzheimer’s Disease: The Cache County Study 
To examine, in an exploratory analysis, the association between vascular conditions and the occurrence of neuropsychiatric symptoms (NPS) in a population-based sample of incident Alzheimer’s disease (AD).
The sample consisted of 254 participants, identified through two waves of assessment. NPS were assessed using the Neuropsychiatric Inventory. Prior to the onset of AD, data regarding a history of stroke, hypertension, hyperlipidemia, heart attack or CABG, and diabetes were recorded. Logistic regression procedures were used to examine the relationship of each vascular condition to individual neuropsychiatric symptoms. Covariates considered were age, gender, education, APOE genotype, dementia severity, and overall health status.
One or more NPS were observed in 51.0% of participants. Depression was most common (25.8%), followed by apathy (18.6%), and irritability (17.7%). Least common were elation (0.8%), hallucinations (5.6%), and disinhibition (6.0%). Stroke prior to the onset of AD was associated with increased risk of delusions (OR=4.76, p=0.02), depression (OR=3.87, p=0.03), and apathy (OR=4.48, p=0.02). Hypertension was associated with increased risk of delusions (OR=2.34, p=0.02), anxiety (OR=4.10, p=0.002), and agitation/aggression (OR=2.82, p=0.01). No associations were observed between NPS and diabetes, hyperlipidemia, heart attack or CABG, or overall health.
Results suggest that a history of stroke and hypertension increase the risk of specific NPS in patients with AD. These conditions may disrupt neural circuitry in brain areas involved in NPS. Findings may provide an avenue for reduction in occurrence of NPS through the treatment or prevention of vascular risk conditions.
PMCID: PMC2692675  PMID: 18289451
dementia; Alzheimer; neuropsychiatric; disturbance; risk factors; vascular

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