[Correlation Between the Diversity Characteristics of Groundwater Bacterial Community and Environmental Factors in Typical Industrial Areas].

In order to investigate the composition and diversity of groundwater bacterial communities in typical industrial areas in Shanghai, the Illumina MiSeq high-throughput technology was adopted to explore the correlation and response mechanism of groundwater bacterial communities and environmental factors in typical industries, combined with the analysis of groundwater tri-nitrogen, heavy metals, organic matter, and other indicators. The results showed that the ammonia nitrogen in the groundwater of the petrochemical industry was 64.49%, 32.46%, and 113.91% higher than that of the textile industry, metal products industry, and other industries (P<0.05), respectively. The main detectable indicators of organic matter were total petroleum hydrocarbons (TPH) and volatile phenol. The mass concentration of volatile phenol in groundwater of the petrochemical industry was significantly higher than that of the textile industry, metal products industry, and other industries (P<0.05). The mass concentration of arsenic in the metal products industry was 49.26% and 50.59% higher than that in the petrochemical industry and other industries (P<0.05), respectively. Chloride, manganese, sulfate, etc., were significantly different in different industries (P<0.05). The Shannon index of groundwater in the textile industry was the highest at 3.14, whereas the Shannon index and Ace index of the groundwater in the metal products industry were as low as at 2.42 and 960.46, respectively. The dominant bacterial phylum in groundwater in the industrial area was Proteobacteria, accounting for 80.05%-86.18%. Arsenic, mercury, TPH, etc. were the main influencing factors in groundwater in industrial areas, whereas the nitrifying bacteria, denitrifying bacteria, and organic matter-degrading bacteria were mostly related to groundwater environmental factors. The results of this study can provide theoretical support for groundwater pollution risk management and microbial remediation in petrochemical and metal product industrial areas.

A Preliminary Study on Sympathetic Skin Response in Acute Ischemic Cerebrovascular Disease.

Purpose: The study aims to identify the characteristics of SSR in patients with AICVD and their correlation with clinical presentations. Methods: SSR of the upper limbs, the National Institute of Health stroke scale (NIHSS), the Barthel index (BI), the Essen stroke risk score (ESRS), and imaging examinations, was evaluated in 30 healthy subjects and 66 patients with AICVD. All results were recorded and analyzed via Statistical Package for the Social Sciences (SPSS 22.0) software. t-test and Spearman rank correlation were used. Results: Compared to the control group, SSR of upper limbs in patients with AICVD showed prolonged latency, reduced amplitude, and disappeared waveform (p=0.000, p=0.015, p=0.004), No statistically significant difference was observed between the affected side and the healthy side (p=0.068, p=0.661). In the case group, the higher the abnormal rate of SSR, the more severe the neurological impairment (NIHSS and ADL scores) and the worse the long-term prognosis. Specific results are as follows: Firstly, the total abnormality rate of SSR, prolonged SSR latency were positively related to the NIHSS, also the ESRS (r=0.347, p=0.004; r=0.437, p<0.001), (r=0.371, p=0.005; r=0.433, p=0.001), the reduced amplitude was positively related to the NIHSS (r=0.341, p=0.012) while the disappeared waveform was positively related to the ESRS (r=0.299, p=0.015); Secondly, the total abnormality rate of SSR, prolonged SSR latency and reduced amplitude were negatively related to the BI (r=-0.346, p=0.004) (r=-0.426, p=0.001) (r=-0.316, p=0.020). Conclusion: There may be inhibition of sympathetic reflex activity in patients with AICVD, SSR abnormality rate in patients with AICVD may be correlated with the degree of neurological impairment and long-term prognosis.

Discovery of small molecule agonists of the Relaxin Family Peptide Receptor 2.

The relaxin/insulin-like family peptide receptor 2 (RXFP2) belongs to the family of class A G-protein coupled receptors (GPCRs) and it is the only known target for the insulin-like factor 3 peptide (INSL3). The importance of this ligand-receptor pair in the development of the gubernacular ligament during the transabdominal phase of testicular descent is well established. More recently, RXFP2 has been implicated in maintaining healthy bone formation. In this report, we describe the discovery of a small molecule series of RXFP2 agonists. These compounds are highly potent, efficacious, and selective RXFP2 allosteric agonists that induce gubernacular invagination in mouse embryos, increase mineralization activity in human osteoblasts in vitro, and improve bone trabecular parameters in adult mice. The described RXFP2 agonists are orally bioavailable and display favorable pharmacokinetic properties, which allow for future evaluation of the therapeutic benefits of modulating RXFP2 activation in disease models.

[Distribution Characteristics and Risk Assessment of Antimony in Typical Urban Soil].

In order to study the distribution characteristics and potential risk of antimony (Sb) in urban soil, the concentrations of soil Sb in four different land use types were analyzed based on the data of 1670 soil samples with different vertical profiles in 102 plots in Shanghai. The risks were evaluated using the potential ecological risk index method and health risk assessment model. The results showed that the average ω(Sb) in the study area was 0.52 mg·kg-1, and the content of soil Sb gradually declined with the rise in soil profile depth. Sb was enriched in surface soil, which indicated that human activities had caused disturbance to the distribution of Sb in the soil. The content of Sb in the surface soil of industrial land was higher than that of residential land and commercial land, and the content of Sb in agricultural land was the lowest. The single-factor pollution index of industrial land was the highest, reaching a slight pollution level, whereas the residential land, commercial land, and agricultural land were at even-clean or clean levels, respectively. The whole region showed slight ecological risk, with the potential ecological risk index ranging from 4.23 to 7.61. The potential ecological risk level of industrial land was moderate, which needs to be addressed. The results of health risk assessment showed that the non-carcinogenic risk of Sb in the soil was low; however, it is of great concern to residents, especially children, when on residential land.

Multidimensional analysis and therapeutic development using patient iPSC-derived disease models of Wolfram syndrome.

Wolfram syndrome is a rare genetic disorder largely caused by pathogenic variants in the WFS1 gene and manifested by diabetes mellitus, optic nerve atrophy, and progressive neurodegeneration. Recent genetic and clinical findings have revealed Wolfram syndrome as a spectrum disorder. Therefore, a genotype-phenotype correlation analysis is needed for diagnosis and therapeutic development. Here, we focus on the WFS1 c.1672C>T, p.R558C variant, which is highly prevalent in the Ashkenazi Jewish population. Clinical investigation indicated that patients carrying the homozygous WFS1 c.1672C>T, p.R558C variant showed mild forms of Wolfram syndrome phenotypes. Expression of WFS1 p.R558C was more stable compared with the other known recessive pathogenic variants associated with Wolfram syndrome. Human induced pluripotent stem cell-derived (iPSC-derived) islets (SC-islets) homozygous for WFS1 c.1672C>T variant recapitulated genotype-related Wolfram syndrome phenotypes. Enhancing residual WFS1 function through a combination treatment of chemical chaperones mitigated detrimental effects caused by the WFS1 c.1672C>T, p.R558C variant and increased insulin secretion in SC-islets. Thus, the WFS1 c.1672C>T, p.R558C variant causes a mild form of Wolfram syndrome phenotypes, which can be remitted with a combination treatment of chemical chaperones. We demonstrate that our patient iPSC-derived disease model provides a valuable platform for further genotype-phenotype analysis and therapeutic development for Wolfram syndrome.

The influence of sea animals on selenium distribution in tundra soils and lake sediments in maritime Antarctica.

The biogeochemical behavior of selenium (Se) has been extensively studied in Se-enriched or Se contaminated soils at low and middle latitudes. However, the Se distribution patterns have not been studied in tundra ecosystems of remote Antarctica. Here, the soils/sediments were collected from penguin and seal colonies, their adjacent tundra and lakes, tundra marsh, human-activity areas, normal tundra and the periglacial in maritime Antarctica, and total Se and seven operationally defined Se fractions were analyzed. Overall the regional distribution of Se levels showed high spatial heterogeneity (coefficient of variation, CV = 114%) in tundra soils, with the highest levels in penguin (mean 6.12 ± 2.66 µg g-1) and seal (mean 2.29 ± 1.43 µg g-1) colony soils, and the lowest in normal tundra soils and periglacial sediments (<0.5 µg g-1). The contribution rates of penguins and seals to tundra soil Se levels amounted to 91.7% and 78.0%. The lake sediment Se levels (mean 2.15 ± 0.87 µg g-1) close to penguin colonies were one order of magnitude higher than those (mean 0.49 ± 0.87 µg g-1) around normal tundra. Strong positive correlations (p < 0.01) of Se concentrations between lake sediments and adjacent tundra soils, and lower Se: P (<0.001) and S: P (<1) ratios in the lake sediments close to penguin colonies, indicated the infiltration or leaching of penguin guano as the predominant Se source in lake sediment. The Se species in penguin and seal guano were dominated by SeCys2 (76.6%) and SeMet (73.5%), respectively. The evidence from the predominant proportions of total organic matter-bound Se (Seom, 67%-70% of total Se) in penguin or seal colony soils further supported penguin or seal guano had a great influence on the distribution patterns of Se fractions in the tundra. This study confirmed that sea animal activities transported substantial amount Se from ocean to land, and significantly altered the biogeochemical cycle of Se in maritime Antarctica.

Identification of Small-Molecule Inhibitors of Human Inositol Hexakisphosphate Kinases by High-Throughput Screening.

Inositol hexakisphosphate kinases (IP6Ks) catalyze pyrophosphorylation of inositol hexakisphosphate (IP6) into inositol 5-diphospho-1,2,3,4,6-pentakisphosphate (IP7), which is involved in numerous areas of cell physiology including glucose homeostasis, blood coagulation, and neurological development. Inhibition of IP6Ks may be effective for the treatment of Type II diabetes, obesity, metabolic complications, thrombosis, and psychiatric disorders. We performed a high-throughput screen (HTS) of 158 410 compounds for IP6K1 inhibitors using a previously developed ADP-Glo Max assay. Of these, 1206 compounds were found to inhibit IP6K1 kinase activity by more than 25%, representing a 0.8% hit rate. Structural clustering analysis of HTS-active compounds, which were confirmed in the dose-response testing using the same kinase assay, revealed diverse clusters that were feasible for future structure-activity relationship (SAR) optimization to potent IP6K inhibitors. Medicinal chemistry SAR efforts in three chemical series identified potent IP6K1 inhibitors which were further validated in an orthogonal LC-MS IP7 analysis. The effects of IP6K1 inhibitors on cellular IP7 levels were further confirmed and were found to correlate with cellular IP6K1 binding measured by a high-throughput cellular thermal shift assay (CETSA).

A high-throughput screening to identify small molecules that suppress huntingtin promoter activity or activate huntingtin-antisense promoter activity.

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of huntingtin (HTT). While there are currently no disease-modifying treatments for HD, recent efforts have focused on the development of nucleotide-based therapeutics to lower HTT expression. As an alternative to siRNA or oligonucleotide methods, we hypothesized that suppression of HTT expression might be accomplished by small molecules that either (1) directly decrease HTT expression by suppressing HTT promoter activity or (2) indirectly decrease HTT expression by increasing the promoter activity of HTT-AS, the gene antisense to HTT that appears to inhibit expression of HTT. We developed and employed a high-throughput screen for modifiers of HTT and HTT-AS promoter activity using luminescent reporter HEK293 cells; of the 52,041 compounds tested, we identified 898 replicable hits. We used a rigorous stepwise approach to assess compound toxicity and the capacity of the compounds to specifically lower huntingtin protein in 5 different cell lines, including HEK293 cells, HD lymphoblastoid cells, mouse primary neurons, HD iPSCs differentiated into cortical-like neurons, and HD hESCs. We found no compounds which were able to lower huntingtin without lowering cell viability in all assays, though the potential efficacy of a few compounds at non-toxic doses could not be excluded. Our results suggest that more specific targets may facilitate a small molecule approach to HTT suppression.

Selenium volatilization from tundra soils in maritime Antarctica.

Maritime Antarctica harbors a large number of penguins and seals that provide considerable input of selenium (Se) originating as guano into terrestrial ecosystems. Subsequent Se emissions via biomethylation and volatilization from these sources of Se have not been studied. Here, penguin colony soils (PCS) and adjacent tundra marsh soils (TMS), seal colony soils (SCS) and adjacent tundra soils (STS), and normal upland tundra soils (NTS) were collected in maritime Antarctica. For the first time, Se volatilization and speciation were investigated in these soils through incubation experiments using chemo-trapping method. The Se contents in PCS, SCS, STS and TMS were highly enriched compared with NTS, with organic matter-bound Se accounting for 70%-80%. Laboratory incubations yielded the greatest Se volatilization rates (VRSe) in PCS (0.20 ± 0.01 µg kg-1 d-1), followed by SCS (0.14 ± 0.01 µg kg-1 d-1) at low temperature (4 °C). Soil frozen-thawing induced 1-4 fold increase in VRSe, and the VRSe continuously increased until the soils fully thawed. The VRSe showed a significant positive correlation (R2 = 0.96, p < 0.01) with soil temperature. Methylated Se species were dominated by dimethylselenide (DMSe) in PCS and dimethyldiselenide (DMDSe) in SCS. Our results imply that the combination of climate warming, frozen-thawing processes, and high-Se inputs from sea animals will significantly increase tundra soil Se volatilization in maritime Antarctica. High VRSe from penguin colony soils, and significantly elevated Se levels in the mosses close to penguin colony, suggest that volatilization of Se from penguin colony soils play an important role in the mobilization and regional biogeochemical cycling of Se in maritime Antarctica.

Validation and Characterization of Five Distinct Novel Inhibitors of Human Cytomegalovirus.

The critical consequences of human cytomegalovirus (HCMV) infection in the transplant population and in congenitally infected infants, the limited treatment options for HCMV, and the rise of resistant mutants toward existing therapies has fueled the search for new anti-HCMV agents. A pp28-luciferase recombinant HCMV was used as a reporter system for high-throughput screening of HCMV inhibitors. Approximately 400 000 compounds from existing libraries were screened. Subsequent validation assays using resynthesized compounds, several virus strains, and detailed virology assays resulted in the identification of five structurally unique and selective HCMV inhibitors, active at sub to low micromolar concentrations. Further characterization revealed that each compound inhibited a specific stage of HCMV replication. One compound was also active against herpes simplex virus (HSV1 and HSV2), and another compound was active against Epstein-Barr virus (EBV). Drug combination studies revealed that all five compounds were additive with ganciclovir or letermovir. Future studies will focus on optimization of these new anti-HCMV compounds along with mechanistic studies.

Effects of multiple environmental variables on tundra ecosystem respiration in maritime Antarctica.

Summertime ecosystem respiration (ER) rates through seven sites were measured at an upland tundra on Fildes Peninsula in maritime Antarctica to investigate effects of topographic gradient, vegetation types and climatic factors on tundra ER rates. Overall the highest ER rates occurred at the tundra top, followed at the middle slope, and the lowest rates at the lower slope. The daily highest ER rates occurred at noon whereas the lowest at 6 am. There was a significant positive correlation (P < 0.05) between tundra ER and 0-10 cm soil temperature, but a significant negative correlation (P < 0.01) between ER and soil moisture. A high Q10 value of 2.69 was obtained when all the data were combined, indicating soil temperature sensitivity of tundra ER. The mean ER at the tundra sites with moss coverage (72.2 ± 4.4 mg CO2 m-2 h-1) was significantly higher (P < 0.01) than that at the sites with lichen coverage (46.8 ± 8.7 mg CO2 m-2 h-1). The tundra sites without snow coverage experienced significant CO2 release, whereas the emission through ER was very low at the tundra sites with snow coverage. Our results indicated that topographic gradient, soil temperature, soil moisture, vegetation types and snow coverage might affect tundra ER in maritime Antarctica.

[Expression of COX-1 and COX-2 in the Platelet of Iron Deficiency Anemia Women at Childbearing Age and Its Clinical Significance].

OBJECTIVE: To detect the expression level of cyclooxygenase-1（COX-1） and cyclooxygenase-2（COX-2） in the platelet of iron deficiency anemia（IDA）women at childbearing age and to explore its correlation with the different indexes of anemia and platelets. METHODS: Forty female IDA patients at childbearing age and 35 healthy controls were enrolled in this study. The Flow cytometry was used to detect the expression of platelet COX-1 and COX-2，the platelet aggregation function as examined by turbidimetric method，and the levels of serum ferritin were analyzed by electrochemical luminescence method，the leval of serum iron was determined by ELISA，and the correlation of different indexes was analyzed. RESULTS: Compared with healthy controls，the levels of platelet COX-1 and COX-2 were significantly lower in female IDA patients at Childbearing age（P<0.05），but platelet count（Plt），mean platelet volume（MPV） and platelet aggregation rate（PAgT）were not statistically different between the 2 groups（P > 0.05）. The expression level of platelet COX-1 positively correlated with those of Hb（r =0.623，P<0.01），serum iron（r =0.321，P<0.05） and HCT（r=0.305，P<0.05）. but the platelet COX-2 expression did not corelate with these indexs. CONCLUSION: The expression of platelet COX-1 and COX-2 in female IDA patients at Childbearing age markedly decrease，and the expression level of platelet COX-1 closely relates with the severity of anemia，that possesses reference value for clinical diagnosis of female IDA patients at Childbearing age..

A modified gene trap approach for improved high-throughput cancer drug discovery.

While advances in laboratory automation has dramatically increased throughout of compound screening efforts, development of robust cell-based assays in relevant disease models remain resource-intensive and time-consuming, presenting a bottleneck to drug discovery campaigns. To address this issue, we present a modified gene trap approach to efficiently generate pathway-specific reporters that result in a robust "on" signal when the pathway of interest is inhibited. In this proof-of-concept study, we used vemurafenib and trametinib to identify traps that specifically detect inhibition of the mitogen-activated protein kinase (MAPK) pathway in a model of BRAFV600E driven human malignant melanoma. We demonstrate that insertion of our trap into particular loci results in remarkably specific detection of MAPK pathway inhibitors over compounds targeting any other pathway or cellular function. The accuracy of our approach was highlighted in a pilot screen of ~6000 compounds where 40 actives were detected, including 18 MEK, 10 RAF, and 3 ERK inhibitors along with a few compounds representing previously under-characterized inhibitors of the MAPK pathway. One such compound, bafetinib, a second generation BCR/ABL inhibitor, reduced phosphorylation of ERK and when combined with trametinib, both in vitro and in vivo, reduced growth of vemurafenib resistant melanoma cells. While piloted in a model of BRAF-driven melanoma, our results set the stage for using this approach to rapidly generate reporters against any transcriptionally active pathway across a wide variety of disease-relevant cell-based models to expedite drug discovery efforts.

Potential effects of ultraviolet radiation reduction on tundra nitrous oxide and methane fluxes in maritime Antarctica.

Stratospheric ozone has begun to recover in Antarctica since the implementation of the Montreal Protocol. However, the effects of ultraviolet (UV) radiation on tundra greenhouse gas fluxes are rarely reported for Polar Regions. In the present study, tundra N2O and CH4 fluxes were measured under the simulated reduction of UV radiation in maritime Antarctica over the last three-year summers. Significantly enhanced N2O and CH4 emissions occurred at tundra sites under the simulated reduction of UV radiation. Compared with the ambient normal UV level, a 20% reduction in UV radiation increased tundra emissions by an average of 8 µg N2O m-2 h-1 and 93 µg CH4 m-2 h-1, whereas a 50% reduction in UV radiation increased their emissions by an average of 17 µg N2O m-2 h-1 and 128 µg CH4 m-2 h-1. No statistically significant correlation (P > 0.05) was found between N2O and CH4 fluxes and soil temperature, soil moisture, total carbon, total nitrogen, NO3--N and NH4+-N contents. Our results confirmed that UV radiation intensity is an important factor affecting tundra N2O and CH4 fluxes in maritime Antarctica. Exclusion of the effects of reduced UV radiation might underestimate their budgets in Polar Regions with the recovery of stratospheric ozone.

Icariin modulates mitochondrial function and apoptosis in high glucose-induced glomerular podocytes through G protein-coupled estrogen receptors.

Podocyte apoptosis in glomerular lesions has been found to have a dominant role in the progression of diabetic nephropathy. The present research aimed to explore the beneficial effect of icariin on diabetic podocytes by interfering in the process of apoptosis. Podocyte apoptosis was significantly exacerbated after high glucose treatment, with the level of reactive oxygen species (ROS) increasing simultaneously. Here, we demonstrated that icariin, which is a G protein-coupled estrogen receptor 1 (GPER) agonist, inhibited podocyte apoptosis by reducing ROS, maintaining the integrity of mitochondrial membranes. Moreover, the stabilization of mitochondria by icariin was reversed when GPER was knocked down in podocytes. Meanwhile, icariin inhibited the caspase cascade in podocyte apoptosis by promoting Bcl-2 expression and mitochondrial translocation. The above findings at least partly elucidated the mechanism by which icariin stabilized podocytes by inducing the mitochondrial Bcl-2 translocation and therefore preventing downstream apoptosis.

Cell-based high-throughput screening identifies galactocerebrosidase enhancers as potential small-molecule therapies for Krabbe's disease.

Krabbe's disease, also known as globoid cell leukodystrophy (GLD), is a lysosomal storage disease caused by the deficiency of the lysosomal enzyme ß-galactocerebrosidase (GALC), resulting in severe neurological manifestations related to demyelination secondary to elevated galactosylsphingosine (psychosine) with its subsequent cytotoxicity. The only available treatment is hematopoietic stem cell transplantation, which delays disease onset but does not prevent long-term neurological manifestations. This article describes the identification of small molecules that enhance mutant GALC activity, identified by quantitative cell-based high-throughput screening (qHTS). Using a specific neurologically relevant murine cell line (145M-Twi) modified to express common human hGALC-G270D mutant, we were able to detect GALC activity in a 1,536-well microplate format. The qHTS of approximately 46,000 compounds identified three small molecules that showed significant enhancements of residual mutant GALC activity in primary cell lines from GLD patients. These compounds were shown to increase the levels of GALC-G270D mutant in the lysosomal compartment. In kinetic assessments, these small molecules failed to disturb the GALC kinetic profile under acidic conditions, which is highly desirable for folding-assisting molecules operating in the endoplasmic reticulum and not affecting GALC catalytic properties in the lysosomal compartment. In addition, these small molecules rescued the decreased GALC activity at neutral pH and partially stabilized GALC under heat-denaturating conditions. These drug-like compounds can be used as the starting point to develop novel small-molecule agents to treat the progressive neurodegenerative course of GLD. © 2016 Wiley Periodicals, Inc.

Monitoring atmospheric nitrous oxide background concentrations at Zhongshan Station, east Antarctica.

At present, continuous observation data for atmospheric nitrous oxide (N2O) concentrations are still lacking, especially in east Antarctica. In this paper, nitrous oxide background concentrations were measured at Zhongshan Station (69°22'25″S, 76°22'14″E), east Antarctica during the period of 2008-2012, and their interannual and seasonal characteristics were analyzed and discussed. The mean N2O concentration was 321.9nL/L with the range of 320.5-324.8nL/L during the five years, and it has been increasing at a rate of 0.29% year(-1). Atmospheric N2O concentrations showed a strong seasonal fluctuation during these five years. The concentrations appeared to follow a downtrend from spring to autumn, and then increased in winter. Generally the highest concentrations occurred in spring. This trend was very similar to that observed at other global observation sites. The overall N2O concentration at the selected global sites showed an increasing annual trend, and the mean N2O concentration in the Northern Hemisphere was slightly higher than that in the Southern Hemisphere. Our result could be representative of atmospheric N2O background levels at the global scale. This study provided valuable data for atmospheric N2O concentrations in east Antarctica, which is important to study on the relationships between N2O emissions and climate change.

Efficacy and Mechanism of Action of Low Dose Emetine against Human Cytomegalovirus.

Infection with human cytomegalovirus (HCMV) is a threat for pregnant women and immunocompromised hosts. Although limited drugs are available, development of new agents against HCMV is desired. Through screening of the LOPAC library, we identified emetine as HCMV inhibitor. Additional studies confirmed its anti-HCMV activities in human foreskin fibroblasts: EC50-40±1.72 nM, CC50-8±0.56 µM, and selectivity index of 200. HCMV inhibition occurred after virus entry, but before DNA replication, and resulted in decreased expression of viral proteins. Synergistic virus inhibition was achieved when emetine was combined with ganciclovir. In a mouse CMV (MCMV) model, emetine was well-tolerated, displayed long half-life, preferential distribution to tissues over plasma, and effectively suppressed MCMV. Since the in vitro anti-HCMV activity of emetine decreased significantly in low-density cells, a mechanism involving cell cycle regulation was suspected. HCMV inhibition by emetine depended on ribosomal processing S14 (RPS14) binding to MDM2, leading to disruption of HCMV-induced MDM2-p53 and MDM2-IE2 interactions. Irrespective of cell density, emetine induced RPS14 translocation into the nucleus during infection. In infected high-density cells, MDM2 was available for interaction with RPS14, resulting in disruption of MDM2-p53 interaction. However, in low-density cells the pre-existing interaction of MDM2-p53 could not be disrupted, and RPS14 could not interact with MDM2. In high-density cells the interaction of MDM2-RPS14 resulted in ubiquitination and degradation of RPS14, which was not observed in low-density cells. In infected-only or in non-infected emetine-treated cells, RPS14 failed to translocate into the nucleus, hence could not interact with MDM2, and was not ubiquitinated. HCMV replicated similarly in RPS14 knockdown or control cells, but emetine did not inhibit virus replication in the former cell line. The interaction of MDM2-p53 was maintained in infected RPS14 knockdown cells despite emetine treatment, confirming a unique mechanism by which emetine exploits RPS14 to disrupt MDM2-p53 interaction. Summarized, emetine may represent a promising candidate for HCMV therapy alone or in combination with ganciclovir through a novel host-dependent mechanism.

Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease.

The Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) cysteine protease (EC 3.4.22.-) is essential for viral replication and is involved in the cytopathic effects (CPE) of the virus. The VEEV nsP2 protease is a member of MEROPS Clan CN and characteristically contains a papain-like protease linked to an S-adenosyl-l-methionine-dependent RNA methyltransferase (SAM MTase) domain. The protease contains an alternative active site motif, (475)NVCWAK(480), which differs from papain's (CGS(25)CWAFS), and the enzyme lacks a transition state-stabilizing residue homologous to Gln-19 in papain. To understand the roles of conserved residues in catalysis, we determined the structure of the free enzyme and the first structure of an inhibitor-bound alphaviral protease. The peptide-like E64d inhibitor was found to bind beneath a ß-hairpin at the interface of the SAM MTase and protease domains. His-546 adopted a conformation that differed from that found in the free enzyme; one or both of the conformers may assist in leaving group departure of either the amine or Cys thiolate during the catalytic cycle. Interestingly, E64c (200 µM), the carboxylic acid form of the E64d ester, did not inhibit the nsP2 protease. To identify key residues involved in substrate binding, a number of mutants were analyzed. Mutation of the motif residue, N475A, led to a 24-fold reduction in kcat/Km, and the conformation of this residue did not change after inhibition. N475 forms a hydrogen bond with R662 in the SAM MTase domain, and the R662A and R662K mutations both led to 16-fold decreases in kcat/Km. N475 forms the base of the P1 binding site and likely orients the substrate for nucleophilic attack or plays a role in product release. An Asn homologous to N475 is similarly found in coronaviral papain-like proteases (PLpro) of the Severe Acute Respiratory Syndrome (SARS) virus and Middle East Respiratory Syndrome (MERS) virus. Mutation of another motif residue, K480A, led to a 9-fold decrease in kcat and kcat/Km. K480 likely enhances the nucleophilicity of the Cys. Consistent with our substrate-bound models, the SAM MTase domain K706A mutation increased Km 4.5-fold to 500 µM. Within the ß-hairpin, the N545A mutation slightly but not significantly increased kcat and Km. The structures and identified active site residues may facilitate the discovery of protease inhibitors with antiviral activity.

Part 1: Notch-sparing γ-secretase inhibitors: The identification of novel naphthyl and benzofuranyl amide analogs.

γ-Secretase is one of two proteases directly involved in the production of the amyloid ß-peptide (Aß), which is pathogenic in Alzheimer's disease. Inhibition of γ-secretase to suppress the production of Aß should not block processing of one of its alternative substrates, Notch1 receptors, as interference with Notch1 signaling leads to severe toxic effects. In the course of our studies to identify γ-secretase inhibitors with selectivity for APP over Notch, 1 [3-(benzyl(isopropyl)amino)-1-(naphthalen-2-yl)propan-1-one] was found to inhibit γ-secretase-mediated Aß production without interfering with γ-secretase-mediated Notch processing in purified enzyme assays. As 1 is chemically unstable, efforts to increase the stability of this compound led to the identification of 2 [naphthalene-2-carboxylic acid benzyl-isopropyl-amide] which showed similar biological activity to compound 1. Synthesis and evaluation of a series of amide analogs resulted in benzofuranyl amide analogs that showed promising Notch-sparing γ-secretase inhibitory effects. This class of compounds may serve as a novel lead series for further study in the development of γ-secretase inhibitors.