Crystallization of Adenylylsulfate Reductase from Desulfovibrio gigas: A Strategy Based on Controlled Protein Oligomerization

Adenylylsulfate reductase (adenosine 5′-phosphosulfate reductase, APS reductase or APSR, E.C.1.8.99.2) catalyzes the conversion of APS to sulfite in dissimilatory sulfate reduction. APSR was isolated and purified directly from massive anaerobically grown Desulfovibrio gigas, a strict anaerobe, for structure and function investigation. Oligomerization of APSR to form dimers–α_2β_2, tetramers–α_4β_4, hexamers–α_6β_6, and larger oligomers was observed during purification of the protein. Dynamic light scattering and ultracentrifugation revealed that the addition of adenosine monophosphate (AMP) or adenosine 5′-phosphosulfate (APS) disrupts the oligomerization, indicating that AMP or APS binding to the APSR dissociates the inactive hexamers into functional dimers. Treatment of APSR with β-mercaptoethanol decreased the enzyme size from a hexamer to a dimer, probably by disrupting the disulfide Cys156—Cys162 toward the C-terminus of the β-subunit. Alignment of the APSR sequences from D. gigas and A. fulgidus revealed the largest differences in this region of the β-subunit, with the D. gigas APSR containing 16 additional amino acids with the Cys156—Cys162 disulfide. Studies in a pH gradient showed that the diameter of the APSR decreased progressively with acidic pH. To crystallize the APSR for structure determination, we optimized conditions to generate a homogeneous and stable form of APSR by combining dynamic light scattering, ultracentrifugation, and electron paramagnetic resonance methods to analyze the various oligomeric states of the enzyme in varied environments

Spatiotemporal Trends in Oral Cancer Mortality and Potential Risks Associated with Heavy Metal Content in Taiwan Soil

Central and Eastern Taiwan have alarmingly high oral cancer (OC) mortality rates, however, the effect of lifestyle factors such as betel chewing cannot fully explain the observed high-risk. Elevated concentrations of heavy metals in the soil reflect somewhat the levels of exposure to the human body, which may promote cancer development in local residents. This study assesses the space-time distribution of OC mortality in Taiwan, and its association with prime factors leading to soil heavy metal content. The current research obtained OC mortality data from the Atlas of Cancer Mortality in Taiwan, 1972–2001, and derived soil heavy metals content data from a nationwide survey carried out by ROCEPA in 1985. The exploratory data analyses showed that OC mortality rates in both genders had high spatial autocorrelation (Moran’s I = 0.6716 and 0.6318 for males and females). Factor analyses revealed three common factors (CFs) representing the major pattern of soil pollution in Taiwan. The results for Spatial Lag Models (SLM) showed that CF1 (Cr, Cu, Ni, and Zn) was most spatially related to male OC mortality which implicates that some metals in CF1 might play as promoters in OC etiology

Juvenile Dermatomyositis: A 20-year Retrospective Analysis of Treatment and Clinical Outcomes

BackgroundJuvenile dermatomyositis is a rare childhood multisystem autoimmune disease involving primarily the skin and muscles, and it may lead to long-term disability. This study aimed to describe the clinical course of juvenile dermatomyositis and determine if any early clinical or laboratory features could predict outcome.MethodsMedical charts of patients aged ≤18 years and diagnosed with juvenile dermatomyositis (according to the criteria of Bohan and Peter) at the Pediatric Department, National Taiwan University Hospital, between 1989 and 2009 were reviewed. The endpoints for disease assessment were complete clinical response and complete clinical remission. Cox's proportional hazards model was fitted to identify important predictors of complete clinical remission.ResultsA total of 39 patients with juvenile dermatomyositis were reviewed. Two-thirds were females, and the mean age at disease onset was 81.97 ± 46.63 months. The most common initial presentations were Gottron's papule (82.1%) and muscle weakness (82.1%). After excluding one patient with an incomplete record, the remaining 31 patients who had muscle weakness were analyzed; among them, 22 (70.97%) achieved complete clinical response, but only six (19.4%) achieved complete clinical remission. Multivariate analysis showed that female sex, negative Gowers' sign at disease onset, and positive photosensitivity at disease onset were favorable factors to achieve complete clinical remission. Moreover, covariate-adjusted survival curves were drawn for making predictions of complete clinical remission. Only 13 (33.33%) patients were symptom free at the end of follow up, whereas the other 26 suffered from different kinds of complications. None of them developed malignancy, but two (5.13%) patients died during the follow-up period.ConclusionFactors such as male sex and Gowers' sign were unlikely to favor the achievement of complete clinical remission in juvenile dermatomyositis. Certain complications cannot be avoided, and thus more effective treatments and monitoring strategies are needed for better control of juvenile dermatomyositis

MCRS2 represses the transactivation activities of Nrf1

<p>Abstract</p> <p>Background</p> <p>Nrf1 [p45 nuclear factor-erythroid 2 (p45 NF-E2)-related factor 1], a member of the CNC-bZIP (CNC basic region leucine zipper) family, is known to be a transcriptional activator by dimerization with distinct partners, such as Maf, FosB, c-Jun, JunD, etc. The transcriptional roles of CNC-bZIP family are demonstrated to be involved in globin gene expression as well as the antioxidant response. For example, CNC-bZIP factors can regulate the expression of detoxification proteins through AREs, such as expression of human gamma-glutamylcysteine synthetases (GCS), glutathione S-transferases (GST), UDP-glucuronosyl transferase (UDP-GT), NADP (H) quinone oxidoreductase (NQOs), etc. To further explore other factor(s) in cells related to the function of Nrf1, we performed a yeast two-hybrid screening assay to identify any Nrf1-interacting proteins. In this study, we isolated a cDNA encoding residues 126–475 of MCRS2 from the HeLa cell cDNA library. Some functions of MCRS1 and its splice variant-MSP58 and MCRS2 have been previously identified, such as transforming, nucleolar sequestration, ribosomal gene regulation, telomerase inhibition activities, etc. Here, we demonstrated MCRS2 can function as a repressor on the Nrf1-mediated transactivation using both in vitro and in vivo systems.</p> <p>Results</p> <p>To find other proteins interacting with the CNC bZIP domain of Nrf1, the CNC-bZIP region of Nrf1 was used as a bait in a yeast two-hybrid screening assay. MCRS2, a splicing variant of p78/MCRS1, was isolated as the Nrf1-interacting partner from the screenings. The interaction between Nrf1 and MCRS2 was confirmed <it>in vitro </it>by GST pull-down assays and <it>in vivo </it>by co-immunoprecipitation. Further, the Nrf1-MCRS2 interaction domains were mapped to the residues 354–447 of Nrf1 as well as the residues 314–475 of MCRS2 respectively, by yeast two-hybrid and GST pull-down assays. By immunofluorescence, MCRS2-FLAG was shown to colocalize with HA-Nrf1 in the nucleus and didn't result in the redistribution of Nrf1. This suggested the existence of Nrf1-MCRS2 complex in vivo. To further confirm the biological function, a reporter driven by CNC-bZIP protein binding sites was also shown to be repressed by MCRS2 in a transient transfection assay. An artificial reporter gene activated by LexA-Nrf1 was also specifically repressed by MCRS2.</p> <p>Conclusion</p> <p>From the results, we showed MCRS2, a new Nrf1-interacting protein, has a repression effect on Nrf1-mediated transcriptional activation. This was the first ever identified repressor protein related to Nrf1 transactivation.</p

New primers for methylation-specific polymerase chain reaction enhance specificity of detecting STAT1 methylation

AbstractObjectiveSignal transducer and activator of transcription (STAT)1 is a key tumor suppressor, which is always methylated in a variety of human cancers. However, nonspecific primers for the detection of specific promoter hypermethylation of STAT1 gene can lead to false-positive or false-negative results for gene methylation.Materials and MethodsWe designed new primers for the detection of STAT1 methylation and compared the sensitivities and specificities of these new primers with prior published primers by methylation-specific polymerase chain reaction (PCR) from ovarian clear cell carcinomas. The mRNA expression levels of STAT1 in these cancerous tissues were also evaluated by reverse-transcriptase PCR and correlated with the results of promoter methylation of STAT1 gene.ResultsNine (39%) of the 23 samples detected by the new primers and 13 samples (56%) detected by prior published primers showed STAT1 methylation. A direct DNA sequencing test revealed that four of the 13 samples (30.8%) showed false positivity for STAT1 methylation using the prior published primers. In contrast, none of the nine samples was false-positive for the detection of STAT1 methylation using the new primers. The new primers for the detection of STAT1 methylation showed 100% specificity and 100% sensitivity without false positivity.ConclusionSpecific primers for methylation-specific PCR are mandatory for the accurate detection of STAT1 gene methylation. Besides, specific primers can generate correct interpretation of STAT1 gene methylation, and its correlation with the clinicopathological characteristics and outcome of cancer patients

Role of the Diphosphine Chelate in Emissive, Charge-Neutral Iridium(III) Complexes

A class of neutral tris-bidentate Ir(III) metal complexes incorporating a diphosphine as a chelate is prepared and characterized here for the first time. Treatment of [Ir(dppb)(tht)Cl3] (1) with fppzH afforded the dichloride complexes, trans-(Cl,Cl)[Ir(dppb)(fppz)Cl2] (2) and cis-(Cl,Cl)[Ir(dppb)(fppz)Cl2] (3). The reaction of 3 with the dianionic chelate precursor bipzH2 or mepzH2, in DMF gave the complex [Ir(dppb)(fppz)(bipz)] (4) or [Ir(dppb)(fppz)(mepz)] (5), respectively. In contrast, a hydride complex [Ir(dppb)(fppz)(bipzH)H] (6) was isolated instead of 4 in protic solvent, namely: DGME. All complexes 2 - 6 are luminescent in powder forms and thin films where the dichlorides (2, 3) emit with maxima at 590-627 nm (orange) and quantum yields (Q.Y.s) up to 90% whereas the tris-bidentate (4, 5) and hydride (6) complexes emit at 455-458 nm (blue) with Q.Y.s up to 70%. Hybrid TD-DFT calculations showed considerable MLCT contribution to the orange-emitting 2 and 3 but substantial ligand-centered 3ππ* transition character in the blue-emitting 4 - 6. The dppb does not participate to these radiative transitions in 4 - 6, but it provides the rigidity and steric bulk needed to promote the luminescence by suppressing the self-quenching in the solid state. Fabrication of an OLED with dopant 5 gave a deep blue CIE chromaticity of (0.16, 0.15). Superior blue emitters, which are vital in OLED applications, may be found in other neutral Ir(III) complexes containing phosphine chelates

Can machine learning algorithms associated with text mining from internet data improve housing price prediction performance?

Housing frenzies in China have attracted widespread global attention over the past few years, but the key is how to more accurately forecast housing prices in order to establish an effective real estate policy. Based on the ubiquitousness and immediacy of Internet data, this research adopts a broader version of text mining to search for keywords in relation to housing prices and then evaluates the predictive abilities using machine learning algorithms. Our findings indicate that this new method, especially random forest, not only detects turning points, but also offers prediction ability that clearly outperforms traditional regression analysis. Overall, the prediction based on online search data through a machine learning mechanism helps us better understand the trends of house prices in China. First published online 10 June 202