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1.  The miR9863 Family Regulates Distinct Mla Alleles in Barley to Attenuate NLR Receptor-Triggered Disease Resistance and Cell-Death Signaling 
PLoS Genetics  2014;10(12):e1004755.
Barley (Hordeum vulgare L.) Mla alleles encode coiled-coil (CC), nucleotide binding, leucine-rich repeat (NB-LRR) receptors that trigger isolate-specific immune responses against the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). How Mla or NB-LRR genes in grass species are regulated at post-transcriptional level is not clear. The microRNA family, miR9863, comprises four members that differentially regulate distinct Mla alleles in barley. We show that miR9863 members guide the cleavage of Mla1 transcripts in barley, and block or reduce the accumulation of MLA1 protein in the heterologous Nicotiana benthamiana expression system. Regulation specificity is determined by variation in a unique single-nucleotide-polymorphism (SNP) in mature miR9863 family members and two SNPs in the Mla miR9863-binding site that separates these alleles into three groups. Further, we demonstrate that 22-nt miR9863s trigger the biogenesis of 21-nt phased siRNAs (phasiRNAs) and together these sRNAs form a feed-forward regulation network for repressing the expression of group I Mla alleles. Overexpression of miR9863 members specifically attenuates MLA1, but not MLA10-triggered disease resistance and cell-death signaling. We propose a key role of the miR9863 family in dampening immune response signaling triggered by a group of MLA immune receptors in barley.
Author Summary
Plants rely on cell-surface and intracellular immune receptors to sense pathogen invasion and to mediate defense responses. However, uncontrolled activation of immune responses is harmful to plant growth and development. Small RNAs have recently been shown to fine-tune the expression of intracellular immune receptors and contribute to the regulation of defense signaling in dicot plants, while similar processes have not been well documented in monocot grain crops, such as barley and wheat. Here, we show that, in barley, some members of the miR9863 family target a subset of Mla alleles that confer race-specific disease resistance to the powdery mildew fungus. These miRNAs act on Mla transcripts by cleavage and translational repression. Production of a type of trans-acting small RNAs, designated as phasiRNAs, enhances the effects of miRNA regulation on Mla targets. We propose that Mla-mediated immune signaling is fine-tuned by the miRNAs at later stage of MLA activation to avoid overloading of immune responses in barley cells.
PMCID: PMC4263374  PMID: 25502438
2.  Engineered RNase P Ribozymes Effectively Inhibit Human Cytomegalovirus Gene Expression and Replication 
Viruses  2014;6(6):2376-2391.
RNase P ribozyme can be engineered to be a sequence-specific gene-targeting agent with promising application in both basic research and clinical settings. By using an in vitro selection system, we have previously generated RNase P ribozyme variants that have better catalytic activity in cleaving an mRNA sequence than the wild type ribozyme. In this study, one of the variants was used to target the mRNA encoding human cytomegalovirus (HCMV) essential transcription factor immediate-early protein 2 (IE2). The variant was able to cleave IE2 mRNA in vitro 50-fold better than the wild type ribozyme. A reduction of about 98% in IE2 expression and a reduction of 3500-fold in viral production was observed in HCMV-infected cells expressing the variant compared to a 75% reduction in IE2 expression and a 100-fold reduction in viral production in cells expressing the ribozyme derived from the wild type sequence. These results suggest that ribozyme variants that are selected to be highly active in vitro are also more effective in inhibiting the expression of their targets in cultured cells. Our study demonstrates that RNase P ribozyme variants are efficient in reducing HCMV gene expression and growth and are potentially useful for anti-viral therapeutic application.
PMCID: PMC4074932  PMID: 24932966
ribozyme; RNase P; gene targeting; cytomegalovirus
3.  Upregulated PD-1 Expression Is Associated with the Development of Systemic Lupus Erythematosus, but Not the PD-1.1 Allele of the PDCD1 Gene 
Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease with complicated genetic inheritance. Programmed death 1 (PD-1), a negative T cell regulator to maintain peripheral tolerance, induces negative signals to T cells during interaction with its ligands and is therefore a candidate gene in the development of SLE. In order to examine whether expression levels of PD-1 contribute to the pathogenesis of SLE, 30 patients with SLE and 30 controls were recruited and their PD-1 expression levels in peripheral blood mononuclear cells (PBMCs) were measured via flow cytometry and quantitative real-time-reverse transcription polymerase chain reaction (RT-PCR). Also, whether PD-1 expression levels are associated with the variant of the SNP rs36084323 and the SLE Disease Activity Index (SLEDAI) was studied in this work. The PD-1 expression levels of SLE patients were significantly increased compared with those of the healthy controls. The upregulated PD-1 expression levels in SLE patients were greatly associated with SLEDAI scores. No significant difference was found between PD-1 expression levels and SNP rs36084323. The results suggest that increased expression of PD-1 may correlate with the pathogenesis of SLE, upregulated PD-1 expression may be a biomarker for SLE diagnosis, and PD-1 inhibitor may be useful to SLE treatment.
PMCID: PMC4016872  PMID: 24860805
4.  Phase I study on the pharmacokinetics and tolerance of ZT-1, a prodrug of huperzine A, for the treatment of Alzheimer's disease 
Acta Pharmacologica Sinica  2013;34(7):976-982.
Huperzine A isolated from the Chinese herb Huperzia serrata (Thunb) Trev is a novel reversible and selective AChE inhibitor. The aim of this study was to evaluate the pharmacokinetics and tolerance of single and multiple doses of ZT-1, a novel analogue of huperzine A, in healthy Chinese subjects.
This was a double-blinded, placebo-controlled, randomized, single- and multiple-dose study. For the single-dose study, 9 subjects were randomly divided into 3 groups receiving ZT-1 (0.5, 0.75 or 1 mg, po) according to a Three-way Latin Square Design. For the multiple-dose study, 9 subjects receiving ZT-1 (0.75 mg/d, po) for 8 consecutive days. In the tolerance study, 40 subjects were randomly divided into 5 groups receiving a single dose of ZT-1 (0.5, 0.75, 1, 1.25 or 1.5 mg, po). Plasma and urine concentrations of ZT-1 and Hup A were determined using LC-MS/MS. Pharmacokinetic parameters, including Cmax, AUC0–72 h and AUC0–∞ were calculated. Tolerance assessments were conducted throughout the study.
ZT-1 was rapidly absorbed and converted into huperzine A, thus the plasma and urine concentrations of ZT-1 were below the limit of quantification (<0.05 ng/mL). After single-dose administration of ZT-1, the mean tmax of huperzine A was 0.76–0.82 h; the AUC0–72 h and Cmax of huperzine A showed approximately dose-proportional increase over the dose range of 0.5–1 mg. After the multiple-dose administration of ZT-1, a steady-state level of huperzine A was achieved within 2 d. No serious adverse events were observed.
ZT-1 is a pro-drug that is rapidly absorbed and converted into huperzine A, and ZT-1 is well tolerated in healthy Chinese volunteers.
PMCID: PMC4002618  PMID: 23624756
ZT-1; huperzine A; prodrug; AChE inhibitor; drug tolerance; pharmacokinetics; Alzheimer's disease
5.  Partitioning, repressing and derepressing: dynamic regulations in MLA immune receptor triggered defense signaling 
Plants and animals have evolved intracellular nucleotide-binding domain and leucine-rich repeat-containing immune receptors (NLRs) to perceive non-self and trigger immune responses. Plant NLRs detect strain-specific pathogen effectors and activate immune signaling leading to extensive transcriptional reprogramming and termination of pathogen infection. Here we review the recent findings in barley MLA immune receptor mediated immune responses against the barley powdery mildew fungus. We focus on nucleocytoplasmic partitioning of immune receptor, bifurcation of immune signaling, transcriptional repression and derepression connecting receptor activation to immune responses. We also discuss similar findings from other plant NLRs where appropriate.
PMCID: PMC3792363  PMID: 24115952
plant NLRs; MLA; barley; cell death; immune signaling; transcription factors; transcription regulation
6.  Memory and Executive Screening (MES): a brief cognitive test for detecting mild cognitive impairment 
BMC Neurology  2012;12:119.
Mild cognitive impairment (MCI), defined as a transitional zone between normal cognition and dementia, requires a battery of formal neuropsychological tests administered by a trained rater for its diagnosis. The objective of this study was to develop a screening tool for MCI.
One hundred ninety seven cognitively normal controls (NC), one hundred sixteen patients with amnestic MCI –single domain (aMCI-sd), one hundred ninety five patients with amnestic MCI-multiple domain (aMCI-md), and two hundred twenty eight patients with mild Alzheimer’s disease (AD) were evaluated by comprehensive neuropsychological tests and by the Memory and Executive Screening (MES).
Correlation analysis showed that the three indicators of the MES were significantly negatively related with age (P<0.05), yet not related with education (P>0.05). There was no ceiling or floor effect. Test completion averaged seven minutes (421.14±168.31 seconds). The receiver operating characteristics (ROC) analyses performed on the aMCI-sd group yielded 0.89 for the area under the curve (AUC) (95% CI, 0.85–0.92) for the MES-total score, with sensitivity of 0.795 and specificity of 0.828. There was 81% correct classification rate when the cut-off was set at less than 75. Meanwhile, the aMCI-md group yielded 0.95 for the AUC (95% CI, 0.93–0.97) for the MES-total score, with sensitivity of 0.87 and specificity of 0.91, and 90% correct classification rate when the cut-off was set at less than 72.
The MES, minimally time-consuming, may be a valid and easily administered cognitive screening tool with high sensitivity and specificity for aMCI, with single or multiple domain impairment.
PMCID: PMC3492138  PMID: 23050770
Mild cognitive impairment (MCI); Amnestic MCI-single domain (aMCI-sd); Amnestic MCI-multiple domain (aMCI-md); Alzheimer’s disease (AD); Memory and Executive Screening (MES); Mini-Mental State Examination (MMSE)
7.  Structure-Function Analysis of Barley NLR Immune Receptor MLA10 Reveals Its Cell Compartment Specific Activity in Cell Death and Disease Resistance 
PLoS Pathogens  2012;8(6):e1002752.
Plant intracellular immune receptors comprise a large number of multi-domain proteins resembling animal NOD-like receptors (NLRs). Plant NLRs typically recognize isolate-specific pathogen-derived effectors, encoded by avirulence (AVR) genes, and trigger defense responses often associated with localized host cell death. The barley MLA gene is polymorphic in nature and encodes NLRs of the coiled-coil (CC)-NB-LRR type that each detects a cognate isolate-specific effector of the barley powdery mildew fungus. We report the systematic analyses of MLA10 activity in disease resistance and cell death signaling in barley and Nicotiana benthamiana. MLA10 CC domain-triggered cell death is regulated by highly conserved motifs in the CC and the NB-ARC domains and by the C-terminal LRR of the receptor. Enforced MLA10 subcellular localization, by tagging with a nuclear localization sequence (NLS) or a nuclear export sequence (NES), shows that MLA10 activity in cell death signaling is suppressed in the nucleus but enhanced in the cytoplasm. By contrast, nuclear localized MLA10 is sufficient to mediate disease resistance against powdery mildew fungus. MLA10 retention in the cytoplasm was achieved through attachment of a glucocorticoid receptor hormone-binding domain (GR), by which we reinforced the role of cytoplasmic MLA10 in cell death signaling. Together with our data showing an essential and sufficient nuclear MLA10 activity in disease resistance, this suggests a bifurcation of MLA10-triggered cell death and disease resistance signaling in a compartment-dependent manner.
Author Summary
Plants utilize a multilayered immune system to protect themselves against pathogens. One layer of innate immunity is controlled by intracellular immune receptors called disease resistance (R) proteins. Plant R proteins are powerful molecules capable of triggering host cell suicide thereby restricting pathogen growth. Therefore, it is crucial for plants to control R protein activity in signaling cell death to avoid harmful autoimmune responses. The Barley MLA locus encodes a number of immune receptors that each recognizes a specific powdery mildew fungal strain. Upon pathogen recognition MLAs trigger host defenses concomitant with a rapid cell death response. We here show that MLA10 cell death-inducing activity is tightly regulated by conserved motifs located in two of its domains and by specific cellular chaperone components. Furthermore, we show distinct functions for the nuclear and cytoplasmic MLA10 pools in disease resistance and cell death signaling and provide evidence for a model uncoupling MLA10 cell death signaling from its disease resistance activity. Our results suggest that plant immune receptors integrate signals from multiple sub-cellular compartments to coordinate effective immune responses against pathogen attack.
PMCID: PMC3369952  PMID: 22685408
8.  Occupational Solvent Exposure and Brain Function: An fMRI Study 
Environmental Health Perspectives  2011;119(7):908-913.
Background: Deficits in cognitive function have been demonstrated among workers chronically exposed to solvents, but the neural basis for these deficits has not been shown.
Objectives: We used functional magnetic resonance imaging (fMRI) to compare pathophysiological changes in brain function between solvent-exposed and control workers.
Methods: Painters, drywall tapers, and carpenters were recruited from the International Union of Painters and Allied Trades, District Council 9 in New York City and District Council 21 in Philadelphia, Pennsylvania, and from the Carpenters Union in New Jersey. Twenty-seven solvent-exposed and 27 control subjects of similar age, education, and occupational status completed the N-Back working memory test during fMRI. After controlling for confounders (age; lifetime marijuana, cocaine, and alcohol use; blood lead; symptoms of depression; verbal intelligence), voxelwise group analysis and regional activation levels were compared and then correlated with an index of lifetime solvent exposure.
Results: Solvent-exposed workers’ performance on the N-Back was significantly worse than that of controls. Activation of the anterior cingulate, prefrontal, and parietal cortices—areas serving working memory function and attention—was also significantly lower for solvent-exposed workers relative to controls. After controlling for confounders, we observed a negative correlation between lifetime solvent exposure and activation in these same regions among the solvent-exposed workers.
Conclusions: This study is one of the few to document neural structures affected by exposure to solvents. Our findings provide a biological mechanism for the neurobehavioral deficits in working memory and attention that have previously been reported by other groups studying the effects of chronic exposure to solvents. These imaging markers, which are consistent with the neurobehavioral measures in our subject population, are consistent with altered brain pathology caused by prolonged exposure to solvent mixtures during construction work.
PMCID: PMC3222975  PMID: 21296712
brain function; fMRI; solvent exposure
9.  Occupational Exposure to Organic Solvents during Bridge Painting 
Annals of Occupational Hygiene  2010;54(4):417-426.
Exposure to volatile organic compounds (VOCs) from bridge painting was measured in New York City and New Jersey during the summer and fall seasons from 2005 to 2007. The effect of painting activities (paint coating layer, confinement setup, and application method) and meteorological conditions (temperature, humidity, and wind speed) on solvent exposure to aromatic, ketone, ester, and alkane compounds were individually evaluated. Mixed-effect models were used to examine the combination effects of these factors on the air concentration of total VOCs as the individual compound groups were not present in all samples. Air concentration associated with spraying was not affected by meteorological conditions since spraying was done in a confined space, thus reducing their impact on solvent air concentration. The mixed models for brushing and rolling samples included two fixed factors, i.e. application method and temperature, and one random factor, i.e. sampling day. An independent dataset (daily air samples) was used to validate the mixed model constructed for brushing and rolling samples. The regression line of the predicted values and actual measurements had a slope of 1.32 ± 0.15 for daily brushing and rolling samples, with almost all points being within the 95% confidence bands. The constructed model provides practical approaches for estimating the solvent exposure from brushing and rolling activities among construction painters. An adjusted mean air concentration derived from the activity-specific spray samples was the best estimate for that painting application.
PMCID: PMC2902363  PMID: 20354053
construction; occupational exposure; oil-based paints; painting; solvents
10.  Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys 
Nature medicine  2005;11(12):1295-1298.
We transplanted kidneys from α1,3-galactosyltransferase knockout (GalT-KO) pigs into six baboons using two different immunosuppressive regimens, but most of the baboons died from severe acute humoral xenograft rejection. Circulating induced antibodies to non-Gal antigens were markedly elevated at rejection, which mediated strong complement-dependent cytotoxicity against GalT-KO porcine target cells. These data suggest that antibodies to non-Gal antigens will present an additional barrier to transplantation of organs from GalT-KO pigs to humans.
PMCID: PMC3018862  PMID: 16311604
11.  European solvent industry group generic exposure scenario risk and exposure tool 
The European Solvents Industry Group (ESIG) Generic Exposure Scenario (GES) Risk and Exposure Tool (EGRET) was developed to facilitate the safety evaluation of consumer uses of solvents, as required by the European Union Registration, Evaluation and Authorization of Chemicals (REACH) Regulation. This exposure-based risk assessment tool provides estimates of both exposure and risk characterization ratios for consumer uses. It builds upon the consumer portion of the European Center for Ecotoxicology and Toxicology of Chemicals (ECETOC) Targeted Risk Assessment (TRA) tool by implementing refinements described in ECETOC TR107. Technical enhancements included the use of additional data to refine scenario defaults and the ability to include additional parameters in exposure calculations. Scenarios were also added to cover all frequently encountered consumer uses of solvents. The TRA tool structure was modified to automatically determine conditions necessary for safe use. EGRET reports results using specific standard phrases in a format consistent with REACH exposure scenario guidance, in order that the outputs can be readily assimilated within safety data sheets and similar information technology systems. Evaluation of tool predictions for a range of commonly encountered consumer uses of solvents found it provides reasonable yet still conservative exposure estimates.
PMCID: PMC3941027  PMID: 23361440
consumer; exposure model; exposure scenario; REACH; safety data sheet; ECETOC-TRA

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