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1.  Analysis of rapidly synthesized guest-filled porous complexes with synchrotron radiation: practical guidelines for the crystalline sponge method 
This report describes complete practical guidelines and insights for the crystalline sponge method, which have been derived through the first use of synchrotron radiation on these systems, and includes a procedure for faster synthesis of the sponges. These guidelines will be applicable to crystal sponge data collected at synchrotrons or in-house facilities, and will allow researchers to obtain reliable high-quality data and construct chemically and physically sensible models for guest structural determination.
A detailed set of synthetic and crystallographic guidelines for the crystalline sponge method based upon the analysis of expediently synthesized crystal sponges using third-generation synchrotron radiation are reported. The procedure for the synthesis of the zinc-based metal–organic framework used in initial crystal sponge reports has been modified to yield competent crystals in 3 days instead of 2 weeks. These crystal sponges were tested on some small molecules, with two being unexpectedly difficult cases for analysis with in-house diffractometers in regard to data quality and proper space-group determination. These issues were easily resolved by the use of synchrotron radiation using data-collection times of less than an hour. One of these guests induced a single-crystal-to-single-crystal transformation to create a larger unit cell with over 500 non-H atoms in the asymmetric unit. This led to a non-trivial refinement scenario that afforded the best Flack x absolute stereochemical determination parameter to date for these systems. The structures did not require the use of PLATON/SQUEEZE or other solvent-masking programs, and are the highest-quality crystalline sponge systems reported to date where the results are strongly supported by the data. A set of guidelines for the entire crystallographic process were developed through these studies. In particular, the refinement guidelines include strategies to refine the host framework, locate guests and determine occupancies, discussion of the proper use of geometric and anisotropic displacement parameter restraints and constraints, and whether to perform solvent squeezing/masking. The single-crystal-to-single-crystal transformation process for the crystal sponges is also discussed. The presented general guidelines will be invaluable for researchers interested in using the crystalline sponge method at in-house diffraction or synchrotron facilities, will facilitate the collection and analysis of reliable high-quality data, and will allow construction of chemically and physically sensible models for guest structural determination.
doi:10.1107/S2053273314019573
PMCID: PMC4283468  PMID: 25537388
X-ray crystallography; crystalline sponge method; metal–organic framework; single-crystal-to-single-crystal transformation; synchrotron radiation
2.  FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis 
Cell Research  2014;24(12):1403-1419.
The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adipogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5′- and 3′-splice sites, spatially overlapping with mRNA splicing regulatory serine/arginine-rich (SR) protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis.
doi:10.1038/cr.2014.151
PMCID: PMC4260349  PMID: 25412662
N6-methyladenosine (m6A); METTL3; FTO; mRNA splicing; adipogenesis
3.  New localized/delocalized emitting state of Eu2+ in orange-emitting hexagonal EuAl2O4 
Scientific Reports  2014;4:7101.
Eu2+-activated phosphors are being widely used in illuminations and displays. Some of these phosphors feature an extremely broad and red-shifted Eu2+ emission band; however, convincing explanation of this phenomenon is lacking. Here we report a new localized/delocalized emitting state of Eu2+ ions in a new hexagonal EuAl2O4 phosphor whose Eu2+ luminescence exhibits a very large bandwidth and an extremely large Stokes shift. At 77 K, two luminescent sites responsible for 550 nm and 645 nm broadband emissions are recognized, while at room temperature only the 645 nm emission band emits. The 645 nm emission exhibits a typical radiative lifetime of 1.27 μs and an unusually large Stokes shift of 0.92 eV. We identify the 645 nm emission as originating from a new type of emitting state whose composition is predominantly that of localized 4f65d character but which also contains a complementary component with delocalized conduction-band-like character. This investigation provides new insights into a unique type of Eu2+ luminescence in solids whose emission exhibits both a very large bandwidth and an extremely large Stokes shift.
doi:10.1038/srep07101
PMCID: PMC4235305  PMID: 25403911
4.  The synthesis of open-shell, bimetallic Mn/Fe trinuclear clusters 
Journal of the American Chemical Society  2013;135(38):10.1021/ja408003d.
Concomitant deprotonation and metallation of hexadentate ligand platform tbsLH6 (tbsLH6 = 1,3,5-C6H9(NHC6H4-o-NHSiMe2 tBu)3) with divalent transition metal starting materials Fe2(Mes)4 (Mes = mesityl) or Mn3(Mes)6 in the presence of tetrahydrofuran (THF) resulted in isolation of homotrinuclear complexes (tbsL)Fe3(THF) and (tbsL)Mn3(THF) respectively. In the absence of coordinating solvent (THF) the deprotonation and metallation exclusively afforded dinuclear complexes of the type (tbsLH2)M2 (M = Fe or Mn). The resulting dinuclear species were utilized as synthons to prepare bimetallic trinuclear clusters. Treatment of (tbsLH2)Fe2 complex with divalent Mn source (Mn2(N(SiMe3)2)4) afforded the bimetallic complex (tbsL)Fe2Mn(THF) which established the ability of hexamine ligand tbsLH6 to support mixed metal clusters. The substitutional homogeneity of (tbsL)Fe2Mn(THF) was determined by 1H NMR, 57Fe Mössbauer, and X-ray fluorescence. Anomalous scattering measurements were critical for the unambiguous assignment of the trinuclear core composition. Heating a solution of (tbsLH2)Mn2 with a stoichiometric amount of Fe2(Mes)4 (0.5 mol equiv) affords a mixture of both (tbsL)Mn2Fe(THF) and (tbsL)Fe2Mn(THF) as a result of the thermodynamic preference for heavier metal substitution within the hexa-anilido ligand framework. These results demonstrate for the first time the assembly of mixed metal cluster synthesis in an unbiased ligand platform.
doi:10.1021/ja408003d
PMCID: PMC3822335  PMID: 23984911
5.  Hepatitis B virus vaccination booster does not provide additional protection in adolescents: a cross-sectional school-based study 
BMC Public Health  2014;14(1):991.
Background
Current consensus does not support the use of a universal booster of hepatitis B virus (HBV) vaccine because there is an anamnestic response in almost all children 15 years after universal infant HBV vaccination. We aimed to provide a booster strategy among adolescents as a result of their changes in lifestyle and sexual activity.
Methods
This study comprised a series of cross-sectional serological surveys of HBV markers in four age groups between 2004 and 2012. The seropositivity rates of hepatitis B surface antigen (HBsAg) and its reciprocal antibody (anti-HBs) for each age group were collected. There were two parts to this study; age-specific HBV seroepidemiology and subgroup analysis, including effects of different vaccine types, booster response for immunogenicity at 15 years of age, and longitudinal follow-up to identify possible additional protection by HBV booster.
Results
Within the study period, data on serum anti-HBs and HBsAg in a total of 6950 students from four age groups were collected. The overall anti-HBs and HBsAg seropositivity rates were 44.3% and 1.2%, respectively. The anti-HBs seropositivity rate in the plasma-derived subgroup was significantly higher in both 15- and 18-year age groups. Overall response rate in the double-seronegative recipients at 15 years of age was 92.5% at 6 weeks following one recombinant HBV booster dose. Among the 24 recipients showing anti-HBs seroconversion at 6 weeks after booster, seven subjects (29.2%) had lost their anti-HBs seropositivity again within 3 years. Increased seropositivity rates and titers of anti-HBs did not provide additional protective effects among subjects comprehensively vaccinated against HBV in infancy.
Conclusions
HBV booster strategy at 15 years of age was the main contributor to the unique age-related phenomenon of anti-HBs seropositivity rate and titer. No increase in HBsAg seropositivity rates within different age groups was observed. Vaccination with plasma-derived HBV vaccines in infancy provided higher anti-HBs seropositivity at 15–18 years of age. Overall booster response rate was 92.5% and indicated that intact immunogenicity persisted at least 15 years after primary HBV vaccination in infancy. Booster vaccination of HBV did not confer additional protection against HBsAg carriage in our study.
doi:10.1186/1471-2458-14-991
PMCID: PMC4246462  PMID: 25248369
HBV booster; Adolescents; Anamnestic response; Infant HBV vaccination
6.  Contemporary X-ray electron-density studies using synchrotron radiation 
IUCrJ  2014;1(Pt 5):267-280.
The use of synchrotron radiation for experimental electron-density determination during the last decade is reviewed. Possible future directions of this field are examined.
Synchrotron radiation has many compelling advantages over conventional radiation sources in the measurement of accurate Bragg diffraction data. The variable photon energy and much higher flux may help to minimize critical systematic effects such as absorption, extinction and anomalous scattering. Based on a survey of selected published results from the last decade, the benefits of using synchrotron radiation in the determination of X-ray electron densities are discussed, and possible future directions of this field are examined.
doi:10.1107/S2052252514018570
PMCID: PMC4174870  PMID: 25295169
electron-density studies; synchrotron radiation; X-ray diffraction
7.  Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase 
Cell Research  2014;24(2):177-189.
The methyltransferase like 3 (METTL3)-containing methyltransferase complex catalyzes the N6-methyladenosine (m6A) formation, a novel epitranscriptomic marker; however, the nature of this complex remains largely unknown. Here we report two new components of the human m6A methyltransferase complex, Wilms' tumor 1-associating protein (WTAP) and methyltransferase like 14 (METTL14). WTAP interacts with METTL3 and METTL14, and is required for their localization into nuclear speckles enriched with pre-mRNA processing factors and for catalytic activity of the m6A methyltransferase in vivo. The majority of RNAs bound by WTAP and METTL3 in vivo represent mRNAs containing the consensus m6A motif. In the absence of WTAP, the RNA-binding capability of METTL3 is strongly reduced, suggesting that WTAP may function to regulate recruitment of the m6A methyltransferase complex to mRNA targets. Furthermore, transcriptomic analyses in combination with photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) illustrate that WTAP and METTL3 regulate expression and alternative splicing of genes involved in transcription and RNA processing. Morpholino-mediated knockdown targeting WTAP and/or METTL3 in zebrafish embryos caused tissue differentiation defects and increased apoptosis. These findings provide strong evidence that WTAP may function as a regulatory subunit in the m6A methyltransferase complex and play a critical role in epitranscriptomic regulation of RNA metabolism.
doi:10.1038/cr.2014.3
PMCID: PMC3915904  PMID: 24407421
WTAP; m6A methyltransferase; METTL3; METTL14; mRNA
8.  Synthesis of Binucleating Macrocycles and their Nickel(II) Hydroxo- and Cyano-Bridged Complexes with Divalent Ions: Anatomical Variation of Ligand Features 
Inorganic chemistry  2012;51(20):11017-11029.
The planar NNN-pincer complexes [MII(pyN Me2)(OH)]1− (MII = Ni, Cu) fix CO2 in η1-OCO2H complexes; results for the copper system are described. MnII, FeII, CoII, and ZnII behave differently, forming [MII(pyN2Me)2]2− with N4O2 coordination. Incorporation of the NiII pincer into binucleating macrocycle 2 containing a triamino MII locus connected by two 1,3-biphenylene groups affords proximal NiII and MII sites for investigation of the synthesis, structure, and reactivity of Ni-X-M bridge units. This ligand structure is taken as a reference for variations in MII atoms and binding sites and bridges X = OH− and CN− to produce additional members of the macrocyclic family with improved properties. Macrocycle 2 with a 22-membered ring is shown to bind MII = Mn, Fe, and Cu with hydroxo bridges. Introduction of the 4-BuiO group (macrocycle 3) improves the solubility of neutral complexes such as those with NiII-OH-CuII and NiII-CN-FeII bridges. The syntheses of macrocycle 5 with a 7-Me-[12]aneSN3 and macrocycle 6 with a 1,8-Me2-[14]aneN4 MII binding site are described to together with hydoxo-bridged Ni-Cu and cyano-bridged Ni-Fe complexes. This work was motivated by the presence of a Ni⋯(HO)-Fe bridge grouping in a reactive state of carbon monoxide dehydrogenase. Attempted decrease in Ni-(OH)-M distances (3.70-3.87 Å) to smaller values observed in the enzyme by use of macrocycle 4 having 1,2-biphenylene connectors led to a mononuclear octahedral NiII complex. Bridge structural units are summarized and the structures of fourteen macrocyclic complexes including eight with bridges are described.
doi:10.1021/ic301506x
PMCID: PMC3483644  PMID: 23030366
9.  Risk Factors for Small Airway Obstruction among Chinese Island Residents: A Case-Control Study 
PLoS ONE  2013;8(7):e68556.
Background
We investigated the prevalence of and risk factors for small airway obstruction (SAO) among Chinese island residents to establish means to prevent and treat SAO.
Methods
From October 17, 2011 to November 1, 2011, a total of 2,873 residents aged >20 years who lived on the Huangqi Peninsula of Fujian were recruited by random cluster sampling. They were asked to complete a Burden of Obstructive Lung Disease (BOLD) questionnaire and underwent physical examinations and lung function evaluations. SAO was defined as a forced expiratory flow at 50% of vital capacity, Vmax50%, of less than 70% of predicted. Risk factors for SAO were assessed from among demographic and anthropometric variables, blood chemistry results, and questionnaire response items.
Results
A total of 216 (7.52%) Chinese island residents were identified as having SAO (95 males; 121 females). Their survey and test results were compared with 432 age and sex-matched healthy controls (192 males; 240 females) for SAO risk factors. Among numerous factors investigated, only diabetes mellitus (p = 0.039), smoking index (SI, p<0.001 for SI>600), second hand smoke (p = 0.002), and lack of regular exercise (p<0.001) were significant risk factors for SAO.
Conclusions
The risk factors for SAO among Chinese island residents appeared to be similar to those among people who live in high-density urban environments and impoverished rural areas. Public health policies and medical practices directed toward improving respiratory health for island residents should be comparable to those used for urban and rural dwellers.
doi:10.1371/journal.pone.0068556
PMCID: PMC3715483  PMID: 23874670
10.  Receptor-Mediated Activation of a Proinsulin-Transferrin Fusion Protein in Hepatoma Cells 
A proinsulin-transferrin (ProINS-Tf) recombinant fusion protein was designed and characterized for the sustained release of an active form of insulin (INS) by hepatoma cells. During incubation with H4IIE hepatoma cells, a gradual decline of ProINS-Tf concentration, with a concomitant generation of the immuno-reactive insulin-transferrin (irINS-Tf), was detected in the culture medium by using INS- or proinsulin (ProINS)-specific radioimmunoassay (RIA) system. Further studies indicated that the conversion of ProINS-Tf to irINS-Tf was a transferrin receptor (TfR) mediated process that was pH-sensitive, and temperature- and microtubule-dependent. These results suggest that the conversion occurred during the slow recycling route of transferrin (Tf)-TfR pathway, possibly processed by proteases in the slow recycling compartments juxtaposed to the trans-Golgi network (TGN). ProINS-Tf exhibited little activity in the short-term promotion of glucose uptake in adipocytes, indicating that it was in an inactive form similar to ProINS. Stimulation of Akt phosphorylation by ProINS-Tf was detected only after prolonged incubation with H4IIE cells. On the other hand, ProINS-Tf pre-incubated with H4IIE cells for 24 h acquired an immediate activity of stimulating Akt phosphorylation. Furthermore, ProINS-Tf elicited a strong activity in inhibition of glucose production following 24 h incubation with H4IIE cells. Based on these findings, we conclude that the Tf-TfR endocytosis and recycling pathway enables the conversion and release of ProINS-Tf in an active form of irINS-Tf. Results from this study suggest that the Tf-TfR pathway can be exploited for the design of prohormone-Tf fusion proteins as protein prodrugs for their sustained and targeted activation.
doi:10.1016/j.jconrel.2011.06.029
PMCID: PMC3196267  PMID: 21756950
Proinsulin; transferrin; insulin; prohormone conversion; receptor-mediated endocytosis and recycling; bifunctional fusion protein
11.  Local injection of lentivirus-delivered livinshRNA suppresses lung adenocarcinoma growth by inducing a G0/G1 phase cell cycle arrest 
The inhibitor of apoptosis protein (IAP) plays an important role in tumorigenesis and may be a potential target for cancer therapy. Livin, which belongs to this family, is highly expressed in various tumors. The previous study demonstrated that silencing Livin gene promoted lung cancer cell apoptosis; however, the effects on tumor growth suppression by targeting this gene in vivo, to thereby determine the efficacy of targeting Livin for patient therapy, have not been determined. This study injected lentivirus-delivered livinshRNA into established xenograft tumors derived from the lung adenocarcinoma cell line SPC-A-1 in BALB/C nude mice, the result showed that LivinshRNA down-regulated Livin expression effectively, induced tumor cell apoptosis, reduced tumor cell proliferation, and suppressed tumor growth dramatically, with a tumor volume inhibitory rate of (58.65±4.82)% and a tumor weight inhibitory rate of (47.44±1.64)%, but with less severe adverse reaction to the mouse. This study further demonstrated that Livin gene silencing induced a G0/G1-phase cell cycle arrest and cyclin D1 downregulation, which is a key regulator of the G0/G1- to S-phase transition. These findings suggest that LivinshRNA local injection may serve as a therapeutic method for patient treatment, and that LivinshRNA may suppress tumor growth by arresting the cell cycle in the G0/G1-phase.
PMCID: PMC3466986  PMID: 23071862
Livin gene; RNA interference; lung adenocarcinoma; xenograft tumor model; cell cycle
12.  Optimizing the accuracy and precision of the single-pulse Laue technique for synchrotron photo-crystallography 
Journal of Synchrotron Radiation  2010;17(Pt 4):479-485.
The accuracy achieved in single-pulse pump-probe Laue experiments at beamline 14-ID at APS is estimated to be 3–4%.
The accuracy that can be achieved in single-pulse pump-probe Laue experiments is discussed. It is shown that with careful tuning of the experimental conditions a reproducibility of the intensity ratios of equivalent intensities obtained in different measurements of 3–4% can be achieved. The single-pulse experiments maximize the time resolution that can be achieved and, unlike stroboscopic techniques in which the pump-probe cycle is rapidly repeated, minimize the temperature increase due to the laser exposure of the sample.
doi:10.1107/S0909049510019710
PMCID: PMC2891491  PMID: 20567080
single-pulse diffraction; accuracy; Laue method; RATIO method; photo-crystallography
13.  Time-resolved synchrotron diffraction and theoretical studies of very short-lived photo-induced molecular species 
Excited-state geometries determined by time-resolved synchrotron diffraction are summarized with emphasis on their comparison with a series of theoretical results. The relative merits of monochromatic and polychromatic (Laue) techniques are discussed.
Definitive experimental results on the geometry of fleeting species are at the time of writing still limited to monochromatic data collection, but methods for modifications of the polychromatic Laue data to increase their accuracy and their suitability for pump–probe experiments have been implemented and are reviewed. In the monochromatic experiments summarized, excited-state conversion percentages are small when neat crystals are used, but are higher when photoactive species are embedded in an inert framework in supramolecular crystals. With polychromatic techniques and increasing source brightness, smaller samples down to tenths of a micrometre or less can be used, increasing homogeneity of exposure and the fractional population of the excited species. Experiments described include a series of transition metal complexes and a fully organic example involving excimer formation. In the final section, experimental findings are compared with those from theoretical calculations on the isolated species. Qualitative agreement is generally obtained, but the theoretical results are strongly dependent on the details of the calculation, indicating the need for further systematic analysis.
doi:10.1107/S0108767309055342
PMCID: PMC2824528  PMID: 20164641
pump–probe experiments; time-resolved diffraction; excited-state molecular geometries; excimers

Results 1-13 (13)