The Tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus.

Emerging evidence suggests that RNA interference (RNAi)-related processes act both in the cytoplasm and in the nucleus. However, the process by which the RNAi machinery is transported into the nucleus remains poorly understood. The Tetrahymena Argonaute protein Twi1p localizes to the nucleus and is crucial for small RNA-directed programmed DNA elimination. In this study, we identify Giw1p, which binds to Twi1p and is required for its nuclear localization. Furthermore, the endoribonuclease (Slicer) activity of Twi1p plays a vital role in the removal of one of the two strands of Twi1p-associated small interfering RNAs (siRNAs), leading to a functionally mature Twi1p-siRNA complex. Slicer activity is also shown to be required for nuclear localization of Twi1p and for its association with Giw1p. These results suggest that Giw1p senses the state of Twi1p-associated siRNAs and selectively transports the mature Twi1p-siRNA complex into the nucleus.

rPTMDetermine: A Fully Automated Methodology for Endogenous Tyrosine Nitration Validation, Site-Localization, and Beyond.

We present herein rPTMDetermine, an adaptive and fully automated methodology for validation of the identification of rarely occurring post-translational modifications (PTMs), using a semisupervised approach with a linear discriminant analysis (LDA) algorithm. With this strategy, verification is enhanced through similarity scoring of tandem mass spectrometry (MS/MS) comparisons between modified peptides and their unmodified analogues. We applied rPTMDetermine to (1) perform fully automated validation steps for modified peptides identified from an in silico database and (2) retrieve potential yet-to-be-identified modified peptides from raw data (that had been missed through conventional database searches). In part (1), 99 of 125 3-nitrotyrosyl-containing (nitrated) peptides obtained from a ProteinPilot search were validated and localized. Twenty nitrated peptides were falsely assigned because of incorrect monoisotopic peak assignments, leading to erroneous identification of deamidation and nitration. Five additional nitrated peptides were, however, validated after performing nonmonoisotopic peak correction. In part (2), an additional 236 unique nitrated peptides were retrieved and localized, containing 113 previously unreported nitration sites; 25 endogenous nitrated peptides with novel sites were selected and verified by comparison with synthetic analogues. In summary, we identified and confidently validated 296 unique nitrated peptides-collectively representing the largest number of endogenously identified 3-nitrotyrosyl-containing peptides from the cerebral cortex proteome of a Macaca fascicularis model of stroke. Furthermore, we harnessed the rPTMDetermine strategy to complement conventional database searching and enhance the confidence of assigning rarely occurring PTMs, while recovering many missed peptides. In a final demonstration, we successfully extended the application of rPTMDetermine to peptides featuring tryptophan oxidation.

Oligomerization of equilibrative nucleoside transporters: a novel regulatory and functional mechanism involving PKC and PP1.

Equilibrative nucleoside transporters (ENTs) translocate nucleosides and nucleobases across plasma membranes, as well as a variety of anti-cancer, -viral, and -parasite nucleoside analogs. They are also key members of the purinome complex and regulate the protective and anti-inflammatory effects of adenosine. Despite their important role, little is known about the mechanisms involved in their regulation. We conducted membrane yeast 2-hybrid and coimmunoprecipitation studies and identified, for the first time to our knowledge, the existence of protein-protein interactions between human ENT1 and ENT2 (hENT1 and hENT2) proteins in human cells and the formation of hetero- and homo-oligomers at the plasma membrane and the submembrane region. The use of NanoLuc Binary Technology allowed us to analyze changes in the oligomeric status of hENT1 and hENT2 and how they rapidly modify the uptake profile for nucleosides and nucleobases and allow cells to respond promptly to external signals or changes in the extracellular environment. These changes in hENTs oligomerization are triggered by PKC activation and subsequent action of protein phosphatase 1.-Grañe-Boladeras, N., Williams, D., Tarmakova, Z., Stevanovic, K., Villani, L. A., Mehrabi, P., Siu, K. W. M., Pastor-Anglada, M., Coe, I. R. Oligomerization of equilibrative nucleoside transporters: a novel regulatory and functional mechanism involving PKC and PP1.

Dissociative electron transfer of copper(ii) complexes of glycyl(glycyl/alanyl)tryptophan in vacuo: IRMPD action spectroscopy provides evidence of transition from zwitterionic to non-zwitterionic peptide structures.

We report herein the first detailed study of the mechanism of redox reactions occurring during the gas-phase dissociative electron transfer of prototypical ternary [CuII(dien)M]˙2+ complexes (M, peptide). The two final products are (i) the oxidized non-zwitterionic π-centered [M]˙+ species with both the charge and spin densities delocalized over the indole ring of the tryptophan residue and with a C-terminal COOH group intact, and (ii) the complementary ion [CuI(dien)]+. Infrared multiple photon dissociation (IRMPD) action spectroscopy and low-energy collision-induced dissociation (CID) experiments, in conjunction with density functional theory (DFT) calculations, revealed the structural details of the mass-isolated precursor and product cations. Our experimental and theoretical results indicate that the doubly positively charged precursor [CuII(dien)M]˙2+ features electrostatic coordination through the anionic carboxylate end of the zwitterionic M moiety. An additional interaction exists between the indole ring of the tryptophan residue and one of the primary amino groups of the dien ligand; the DFT calculations provided the structures of the precursor ion, intermediates, and products, and enabled us to keep track of the locations of the charge and unpaired electron. The dissociative one-electron transfer reaction is initiated by a gradual transition of the M tripeptide from the zwitterionic form in [CuII(dien)M]˙2+ to the non-zwitterionic M intermediate, through a cascade of conformational changes and proton transfers. In the next step, the highest energy intermediate is formed; here, the copper center is 5-coordinate with coordination from both the carboxylic acid group and the indole ring. A subsequent switch back to 4-coordination to an intermediate IM1, where attachment to GGW occurs through the indole ring only, creates the structure that ultimately undergoes dissociation.

The plasma peptides of breast versus ovarian cancer.

BACKGROUND: There is a need to demonstrate a proof of principle that proteomics has the capacity to analyze plasma from breast cancer versus other diseases and controls in a multisite clinical trial design. The peptides or proteins that show a high observation frequency, and/or precursor intensity, specific to breast cancer plasma might be discovered by comparison to other diseases and matched controls. The endogenous tryptic peptides of breast cancer plasma were compared to ovarian cancer, female normal, sepsis, heart attack, Alzheimer's and multiple sclerosis along with the institution-matched normal and control samples collected directly onto ice. METHODS: Endogenous tryptic peptides were extracted from individual breast cancer and control EDTA plasma samples in a step gradient of acetonitrile, and collected over preparative C18 for LC-ESI-MS/MS with a set of LTQ XL linear quadrupole ion traps working together in parallel to randomly and independently sample clinical populations. The MS/MS spectra were fit to fully tryptic peptides or phosphopeptides within proteins using the X!TANDEM algorithm. The protein observation frequency was counted using the SEQUEST algorithm after selecting the single best charge state and peptide sequence for each MS/MS spectra. The observation frequency was subsequently tested by Chi Square analysis. The log10 precursor intensity was compared by ANOVA in the R statistical system. RESULTS: Peptides and/or phosphopeptides of common plasma proteins such as APOE, C4A, C4B, C3, APOA1, APOC2, APOC4, ITIH3 and ITIH4 showed increased observation frequency and/or precursor intensity in breast cancer. Many cellular proteins also showed large changes in frequency by Chi Square (χ2 > 100, p < 0.0001) in the breast cancer samples such as CPEB1, LTBP4, HIF-1A, IGHE, RAB44, NEFM, C19orf82, SLC35B1, 1D12A, C8orf34, HIF1A, OCLN, EYA1, HLA-DRB1, LARS, PTPDC1, WWC1, ZNF562, PTMA, MGAT1, NDUFA1, NOGOC, OR1E1, OR1E2, CFI, HSA12, GCSH, ELTD1, TBX15, NR2C2, FLJ00045, PDLIM1, GALNT9, ASH2L, PPFIBP1, LRRC4B, SLCO3A1, BHMT2, CS, FAM188B2, LGALS7, SAT2, SFRS8, SLC22A12, WNT9B, SLC2A4, ZNF101, WT1, CCDC47, ERLIN1, SPFH1, EID2, THOC1, DDX47, MREG, PTPRE, EMILIN1, DKFZp779G1236 and MAP3K8 among others. The protein gene symbols with large Chi Square values were significantly enriched in proteins that showed a complex set of previously established functional and structural relationships by STRING analysis. An increase in mean precursor intensity of peptides was observed for QSER1 as well as SLC35B1, IQCJ-SCHIP1, MREG, BHMT2, LGALS7, THOC1, ANXA4, DHDDS, SAT2, PTMA and FYCO1 among others. In contrast, the QSER1 peptide QPKVKAEPPPK was apparently specific to ovarian cancer. CONCLUSION: There was striking agreement between the breast cancer plasma peptides and proteins discovered by LC-ESI-MS/MS with previous biomarkers from tumors, cells lines or body fluids by genetic or biochemical methods. The results indicate that variation in plasma peptides from breast cancer versus ovarian cancer may be directly discovered by LC-ESI-MS/MS that will be a powerful tool for clinical research. It may be possible to use a battery of sensitive and robust linear quadrupole ion traps for random and independent sampling of plasma from a multisite clinical trial.

Orthodontic plate for management of obstructive sleep apnoea in infants with Pierre Robin sequence: experience and protocol in Hong Kong.

OBJECTIVE: To present the application of the pre-epiglottic baton plate (PEBP) in infants with Pierre Robin sequence (PRS) in the Southern Chinese population (Hong Kong) and to present the diagnosis and management protocol of these infants in our centre. DESIGN: Retrospective case series of three patients with PRS. SETTING: Neonatal Intensive Care Unit in Kwong Wah Hospital and Craniofacial Orthodontic Centre in United Christian Hospital, Hong Kong. PARTICIPANTS: Three new-born infants (two girls, one boy) with PRS and upper airway obstruction due to glossoptosis. METHODS: A protocol for the diagnosis and management of these infants in the Southern Chinese population (Hong Kong) was presented. The three patients received nasal high-flow oxygen and/or continuous positive airway pressure (CPAP) as first-line respiratory support, followed by PEBP for 3-5 months. A two-stage approach was undertaken to ensure accurate positioning of the PEBP. RESULTS: All three infants had improvement in clinical signs, symptoms and polysomnography upon discharge. PEBP and other respiratory aids were weaned off at 3-6 months. CONCLUSIONS: The PEBP, combined with other respiratory support, is a useful modality in the treatment of obstructive sleep apnoea in infants with PRS.

Quantitative mass spectrometry of human reticulocytes reveal proteome-wide modifications during maturation.

Erythropoiesis is marked by progressive changes in morphological, biochemical and mechanical properties of erythroid precursors to generate red blood cells (RBC). The earliest enucleated forms derived in this process, known as reticulocytes, are multi-lobular and spherical. As reticulocytes mature, they undergo a series of dynamic cytoskeletal re-arrangements and the expulsion of residual organelles, resulting in highly deformable biconcave RBCs (normocytes). To understand the significant, yet neglected proteome-wide changes associated with reticulocyte maturation, we undertook a quantitative proteomics approach. Immature reticulocytes (marked by the presence of surface transferrin receptor, CD71) and mature RBCs (devoid of CD71) were isolated from human cord blood using a magnetic separation procedure. After sub-fractionation into triton-extracted membrane proteins and luminal samples (isobaric tags for relative and absolute quantitation), quantitative mass spectrometry was conducted to identify more than 1800 proteins with good confidence and coverage. While most structural proteins (such as Spectrins, Ankyrin and Band 3) as well as surface glycoproteins were conserved, proteins associated with microtubule structures, such as Talin-1/2 and ß-Tubulin, were detected only in immature reticulocytes. Atomic force microscopy (AFM)-based imaging revealed an extended network of spectrin filaments in reticulocytes (with an average length of 48 nm), which shortened during reticulocyte maturation (average spectrin length of 41 nm in normocytes). The extended nature of cytoskeletal network may partly account for increased deformability and shape changes, as reticulocytes transform to normocytes.

The plasma peptides of ovarian cancer.

BACKGROUND: It may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma by using liquid chromatography and tandem mass spectrometry to identify, quantify and compare the peptides cleaved ex vivo from different clinical populations. The endogenous tryptic peptides of ovarian cancer plasma were compared to breast cancer and female cancer normal controls, other diseases with their matched or normal controls, plus ice cold plasma to control for pre-analytical variation. METHODS: The endogenous tryptic peptides or tryptic phospho peptides (i.e. without exogenous digestion) were analyzed from 200 µl of EDTA plasma. The plasma peptides were extracted by a step gradient of organic/water with differential centrifugation, dried, and collected over C18 for analytical HPLC nano electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) with a linear quadrupole ion trap. The endogenous peptides of ovarian cancer were compared to multiple disease and normal samples from different institutions alongside ice cold controls. Peptides were randomly and independently sampled by LC-ESI-MS/MS. Precursor ions from peptides > E4 counts were identified by the SEQUEST and X!TANDEM algorithms, filtered in SQL Server, before testing of frequency counts by Chi Square (χ2), for analysis with the STRING algorithm, and comparison of precursor intensity by ANOVA in the R statistical system with the Tukey-Kramer Honestly Significant Difference (HSD) test. RESULTS: Peptides and/or phosphopeptides of common plasma proteins such as HPR, HP, HPX, and SERPINA1 showed increased observation frequency and/or precursor intensity in ovarian cancer. Many cellular proteins showed large changes in frequency by Chi Square (χ2 > 60, p < 0.0001) in the ovarian cancer samples such as ZNF91, ZNF254, F13A1, LOC102723511, ZNF253, QSER1, P4HA1, GPC6, LMNB2, PYGB, NBR1, CCNI2, LOC101930455, TRPM5, IGSF1, ITGB1, CHD6, SIRT1, NEFM, SKOR2, SUPT20HL1, PLCE1, CCDC148, CPSF3, MORN3, NMI, XTP11, LOC101927572, SMC5, SEMA6B, LOXL3, SEZ6L2, and DHCR24. The protein gene symbols with large Chi Square values were significantly enriched in proteins that showed a complex set of previously established functional and structural relationships by STRING analysis. Analysis of the frequently observed proteins by ANOVA confirmed increases in mean precursor intensity in ZFN91, TRPM5, SIRT1, CHD6, RIMS1, LOC101930455 (XP_005275896), CCDC37 and GIMAP4 between ovarian cancer versus normal female and other diseases or controls by the Tukey-Kramer HSD test. CONCLUSION: Here we show that separation of endogenous peptides with a step gradient of organic/water and differential centrifugation followed by random and independent sampling by LC-ESI-MS/MS with analysis of peptide frequency and intensity by SQL Server and R revealed significant difference in the ex vivo cleavage of peptides between ovarian cancer and other clinical treatments. There was striking agreement between the proteins discovered from cancer plasma versus previous biomarkers discovered in tumors by genetic or biochemical methods. The results indicate that variation in plasma proteins from ovarian cancer may be directly discovered by LC-ESI-MS/MS that will be a powerful tool for clinical research.

The plasma peptidome.

BACKGROUND: It may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma using LC-ESI-MS/MS to identify, with a linear quadrupole ion trap to identify, quantify and compare the statistical distributions of peptides cleaved ex vivo from plasma samples from different clinical populations. METHODS: A systematic method for the organic fractionation of plasma peptides was applied to identify and quantify the endogenous tryptic peptides from human plasma from multiple institutions by C18 HPLC followed nano electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) with a linear quadrupole ion trap. The endogenous tryptic peptides, or tryptic phospho peptides (i.e. without exogenous digestion), were extracted in a mixture of organic solvent and water, dried and collected by preparative C18. The tryptic peptides from 6 institutions with 12 different disease and normal EDTA plasma populations, alongside ice cold controls for pre-analytical variation, were characterized by mass spectrometry. Each patient plasma was precipitated in 90% acetonitrile and the endogenous tryptic peptides extracted by a stepwise gradient of increasing water and then formic acid resulting in 10 sub-fractions. The fractionated peptides were manually collected over preparative C18 and injected for 1508 LC-ESI-MS/MS experiments analyzed in SQL Server R. RESULTS: Peptides that were cleaved in human plasma by a tryptic activity ex vivo provided convenient and sensitive access to most human proteins in plasma that show differences in the frequency or intensity of proteins observed across populations that may have clinical significance. Combination of step wise organic extraction of 200 µL of plasma with nano electrospray resulted in the confident identification and quantification ~ 14,000 gene symbols by X!TANDEM that is the largest number of blood proteins identified to date and shows that you can monitor the ex vivo proteolysis of most human proteins, including interleukins, from blood. A total of 15,968,550 MS/MS spectra ≥ E4 intensity counts were correlated by the SEQUEST and X!TANDEM algorithms to a federated library of 157,478 protein sequences that were filtered for best charge state (2+ or 3+) and peptide sequence in SQL Server resulting in 1,916,672 distinct best-fit peptide correlations for analysis with the R statistical system. SEQUEST identified some 140,054 protein accessions, or some ~ 26,000 gene symbols, proteins or loci, with at least 5 independent correlations. The X!TANDEM algorithm made at least 5 best fit correlations to more than 14,000 protein gene symbols with p-values and FDR corrected q-values of ~ 0.001 or less. Log10 peptide intensity values showed a Gaussian distribution from E8 to E4 arbitrary counts by quantile plot, and significant variation in average precursor intensity across the disease and controls treatments by ANOVA with means compared by the Tukey-Kramer test. STRING analysis of the top 2000 gene symbols showed a tight association of cellular proteins that were apparently present in the plasma as protein complexes with related cellular components, molecular functions and biological processes. CONCLUSIONS: The random and independent sampling of pre-fractionated blood peptides by LC-ESI-MS/MS with SQL Server-R analysis revealed the largest plasma proteome to date and was a practical method to quantify and compare the frequency or log10 intensity of individual proteins cleaved ex vivo across populations of plasma samples from multiple clinical locations to discover treatment-specific variation using classical statistics suitable for clinical science. It was possible to identify and quantify nearly all human proteins from EDTA plasma and compare the results of thousands of LC-ESI-MS/MS experiments from multiple clinical populations using standard database methods in SQL Server and classical statistical strategies in the R data analysis system.

Loss of water from protonated polyglycines: interconversion and dissociation of the product imidazolone ions.

Collision-induced dissociation of isotopically labelled protonated pentaglycine produced two abundant [b5]+ ions, the products of the loss of water from the first and second amide groups, labelled [b5]+I and [b5]+II. IRMPD spectroscopy and DFT calculations show that these two [b5]+ ions feature N1-protonated 3,5-dihydro-4H-imidazol-4-one structures. 15N-Labelling established that some interconversion occurs between these two ions but dissociations are preferred. For both ions, DFT calculations show that the barrier to interconversion is slightly higher than those to dissociation. Dehydration of protonated hexaglycine produced three imidazolone ions. Ions [b6]+I and [b6]+II exhibit analogous CID spectra to those from [b5]+I and [b5]+II; however, the spectrum of the [b6]+III ion was dramatically different, showing losses predominantly of a further water molecule or cleavage of the second amide bond to give the glycyloxazolone (a deprotonated [b2]+ ion, labelled GlyGlyox (114 Da)) from the N-terminus. Protonated polyglycines [Glyn + H]+, where n = 7-9, all readily lose at least one water molecule. The corresponding [bn]+ ions lose either a further water molecule, an oxazolone from the N-terminus or a truncated peptide from the C-terminus. The number of amino acid residues in the latter two eliminated neutral molecules provides insight into the location of the imidazolone in the peptide chain and which oxygen was lost in the initial dehydration reaction. From this analysis, it appears that water loss from the longer protonated polyglycines is predominantly from the central residues.

Bacteriology and risk factors associated with periprosthetic joint infection after primary total knee arthroplasty: retrospective study of 2543 cases.

INTRODUCTION: Periprosthetic joint infection after total knee arthroplasty is a serious complication. This study aimed to identify risk factors and bacteriological features associated with periprosthetic joint infection after primary total knee arthroplasty performed at a teaching hospital. METHODS: We reviewed 2543 elective primary total knee arthroplasties performed at our institution from 1993 to 2013. Data were collected from the Hong Kong Hospital Authority's Clinical Data Analysis and Reporting System, the Infection Control Team, and the joint replacement division registry. The association between potential risk factors and periprosthetic joint infection was examined by univariable analysis and multivariable logistic regression. Univariable analyses were also performed to examine the association between potential risk factors and bacteriology and between potential risk factors, including bacteriology, and early-onset infection. RESULTS: The incidence of periprosthetic joint infection in our series was 1.34% (n=34). The incidence of early-onset infection was 0.39% (n=24). Of the periprosthetic joint infections, 29.4% were early-onset infections. In both univariable and multivariable analyses, only rheumatoid arthritis was a significant predictor of periprosthetic joint infection. Methicillin-sensitive Staphylococcus aureus was the most common causative organism. We did not identify any significant association between potential risk factors and bacteriology. Periprosthetic joint infection caused by skin flora was positively associated with early-onset infection but the association was not statistically significant. CONCLUSION: The incidence of periprosthetic joint infection after elective primary total knee arthroplasty performed at our institution from 1993 to 2013 was 1.34%. Rheumatoid arthritis was a significant risk factor for periprosthetic joint infection.

Characterization of the Molecular Mechanisms Underlying the Chronic Phase of Stroke in a Cynomolgus Monkey Model of Induced Cerebral Ischemia.

Stroke is one of the main causes of mortality and long-term disability worldwide. The pathophysiological mechanisms underlying this disease are not well understood, particularly in the chronic phase after the initial ischemic episode. In this study, a Macaca fascicularis stroke model consisting of two sample groups, as determined by MRI-quantified infarct volumes as a measure of the stroke severity 28 days after the ischemic episode, was evaluated using qualitative and quantitative proteomics analyses. By using multiple online multidimensional liquid chromatography platforms, 8790 nonredundant proteins were identified that condensed to 5223 protein groups at 1% global false discovery rate (FDR). After the application of a conservative criterion (5% local FDR), 4906 protein groups were identified from the analysis of cerebral cortex. Of the 2068 quantified proteins, differential proteomic analyses revealed that 31 and 23 were dysregulated in the elevated- and low-infarct-volume groups, respectively. Neurogenesis, synaptogenesis, and inflammation featured prominently as the cellular processes associated with these dysregulated proteins. Protein interaction network analysis revealed that the dysregulated proteins for inflammation and neurogenesis were highly connected, suggesting potential cross-talk between these processes in modulating the cytoskeletal structure and dynamics in the chronic phase poststroke. Elucidating the long-term consequences of brain tissue injuries from a cellular prospective, as well as the molecular mechanisms that are involved, would provide a basis for the development of new potentially neurorestorative therapies.

Isomerization versus dissociation of phenylalanylglycyltryptophan radical cations.

Four isomers of the radical cation of tripeptide phenylalanylglycyltryptophan, in which the initial location of the radical center is well defined, have been isolated and their collision-induced dissociation (CID) spectra examined. These ions, the π-centered [FGWπ˙]+, α-carbon- [FGα˙W]+, N-centered [FGWN˙]+ and ζ-carbon- [Fζ˙GW]+ radical cations, were generated via collision-induced dissociation (CID) of transition metal-ligand-peptide complexes, side chain fragmentation of a π-centered radical cation, homolytic cleavage of a labile nitrogen-nitrogen single bond, and laser induced dissociation of an iodinated peptide, respectively. The π-centered and tryptophan N-centered peptide radical cations produced almost identical CID spectra, despite the different locations of their initial radical sites, which indicated that interconversion between the π-centered and tryptophan N-centered radical cations is facile. By contrast, the α-carbon-glycyl radical [FGα˙W]+, and ζ-phenyl radical [Fζ˙GW]+, featured different dissociation product ions, suggesting that the interconversions among α-carbon, π-centered (or tryptophan N-centered) and ζ-carbon-radical cations have higher barriers than those to dissociation. Density functional theory calculations have been used to perform systematic mechanistic investigations on the interconversions between these isomers and to study selected fragmentation pathways for these isomeric peptide radical cations. The results showed that the energy barrier for interconversion between [FGWπ˙]+ and [FGWN˙]+ is only 31.1 kcal mol-1, much lower than the barriers to their dissociation (40.3 kcal mol-1). For the [FGWπ˙]+, [FGα˙W]+, and [Fζ˙GW]+, the barriers to interconversion are higher than those to dissociation, suggesting that interconversions among these isomers are not competitive with dissociations. The [z3 - H]˙+ ions isolated from [FGα˙W]+ and [Fζ˙GW]+ show distinctly different fragmentation patterns, indicating that the structures of these ions are different and this result is supported by the DFT calculations.

Long-term evolution and transmission dynamics of swine influenza A virus.

Swine influenza A viruses (SwIV) cause significant economic losses in animal husbandry as well as instances of human disease and occasionally give rise to human pandemics, including that caused by the H1N1/2009 virus. The lack of systematic and longitudinal influenza surveillance in pigs has hampered attempts to reconstruct the origins of this pandemic. Most existing swine data were derived from opportunistic samples collected from diseased pigs in disparate geographical regions, not from prospective studies in defined locations, hence the evolutionary and transmission dynamics of SwIV are poorly understood. Here we quantify the epidemiological, genetic and antigenic dynamics of SwIV in Hong Kong using a data set of more than 650 SwIV isolates and more than 800 swine sera from 12 years of systematic surveillance in this region, supplemented with data stretching back 34 years. Intercontinental virus movement has led to reassortment and lineage replacement, creating an antigenically and genetically diverse virus population whose dynamics are quantitatively different from those previously observed for human influenza viruses. Our findings indicate that increased antigenic drift is associated with reassortment events and offer insights into the emergence of influenza viruses with epidemic potential in swine and humans.

How well are we managing fragility hip fractures? A narrative report on the review with the attempt to setup a Fragility Fracture Registry in Hong Kong.

INTRODUCTION: In setting up a disease registry for fragility fractures in Hong Kong, we conducted a retrospective systematic study on the management of fragility hip fractures. Patient outcomes were compared with the standards from our orthopaedic working group and those from the British Orthopaedic Association that runs a mature fracture registry in the United Kingdom. METHODS: Clinical data on fragility hip fracture patients admitted to six acute major hospitals in Hong Kong in 2012 were captured. These included demographics, pre- and post-operative assessments, discharge details, complications, and 1-year follow-up information. Analysis was performed according to the local standards with reference to those from the British Orthopaedic Association. RESULTS: Overall, 91.0% of patients received orthopaedic care within 4 hours of admission and 60.5% received surgery within 48 hours. Preoperative geri-orthopaedic co-management was received by 3.5% of patients and was one of the reasons for the delayed surgery in 22% of patients. Only 22.9% were discharged with medication that would promote bone health. Institutionalisation on discharge significantly increased by 16.2% (P<0.001). Only 35.1% of patients attended out-patient follow-up 1 year following fracture, and mobility had deteriorated in 69.9% compared with the premorbid state. Death occurred in 17.3% of patients within a year of surgery compared with 1.6% mortality rate in a Hong Kong age-matched population. CONCLUSIONS: The efficiency and quality of acute care for fragility hip fracture patients was documented. Regular geri-orthopaedic co-management can enhance acute care. Much effort is needed to improve functional recovery, prescription of bone health medications, attendance for follow-up, and to decrease institutionalisation. A Fracture Liaison Service is vital to improve long-term care and prevent secondary fractures.

Identification of 14-3-3zeta associated protein networks in oral cancer.

Advancements in genomics, proteomics, and bioinformatics have improved our understanding of gene/protein networks involved in intra- and intercellular communication and tumor-host interactions. Using proteomics integrated with bioinformatics, previously we reported overexpression of 14-3-3ζ in premalignant oral lesions and oral squamous cell carcinoma tissues in comparison with normal oral epithelium. 14-3-3ζ emerged as a novel molecular target for therapeutics and a potential prognostic marker in oral squamous cell carcinoma patients. However, the role of 14-3-3ζ in development and progression of oral cancer is not known yet. This study aimed to identify the 14-3-3ζ associated protein networks in oral cancer cell lines using IP-MS/MS and bioinformatics. A total of 287 binding partners of 14-3-3ζ were identified in metastatic (MDA1986) and nonmetastatic (SCC4) oral cancer cell lines including other 14-3-3 isoforms (2%), proteins involved in apoptosis (2%), cytoskeleton (9%), metabolism (16%), and maintenance of redox potential (2%). Our bioinformatics analysis revealed involvement of 14-3-3ζ in protein networks regulating cell cycle, proliferation, apoptosis, cellular trafficking, and endocytosis in oral cancer. In conclusion, our data revealed several novel protein interaction networks involving 14-3-3ζ in oral cancer progression and metastasis.

Lipiodol versus diaphragm in 4D-CBCT-guided stereotactic radiotherapy of hepatocellular carcinomas.

PURPOSE: The purpose of this work was to investigate the potential of lipiodol as a direct tumor surrogate alternative to the diaphragm surrogate on four-dimensional cone-beam computed tomography (4D-CBCT) image guidance for stereotactic radiotherapy of hepatocellular carcinomas. METHODS: A total of 29 hepatocellular carcinomas (HCC) patients treated by stereotactic radiotherapy following transarterial chemoembolization (TACE) with homogeneous or partial defective lipiodol retention were included. In all, 4-7 pretreatment 4D-CBCT scans were selected for each patient. For each scan, either lipiodol or the diaphragm was used for 4D registration. Resulting lipiodol/diaphragm motion ranges and position errors relative to the reconstructed midventilation images were analyzed to obtain the motion variations, and group mean (ΔM), systematic (Σ), and random (σ) errors of the treatment setup. RESULTS: Of the lipiodolized tumors, 55 % qualified for direct localization on the 4D-CBCT. Significant correlations of lipiodol and diaphragm positions were found in the left-right (LR), craniocaudal (CC), and anteroposterior (AP) directions. ΔM and σ obtained with lipiodol and diaphragm were similar, agreed to within 0.5 mm in the LR and AP, and 0.3 mm in the CC directions, and Σ differed by 1.4 (LR), 1.1 (CC), and 0.6 (AP) mm. Variations of diaphragm motion range > 5 mm were not observed with lipiodol and in one patient with diaphragm. The margin required for the tumor prediction error using the diaphragm surrogate was 6.7 (LR), 11.7 (CC), and 4.1 (AP) mm. CONCLUSION: Image-guidance combining lipiodol with 4D-CBCT enabled accurate localization of HCC and thus margin reduction. A major limitation was the degraded lipiodol contrast on 4D-CBCT.

Alkali-Metal-Ion-Assisted Hydrogen Atom Transfer in the Homocysteine Radical.

Intramolecular hydrogen atom transfer (HAT) was examined in homocysteine (Hcy) thiyl radical/alkali metal ion complexes in the gas phase by combination of experimental techniques (ion-molecule reactions and infrared multiple photon dissociation spectroscopy) and theoretical calculations. The experimental results unequivocally show that metal ion complexation (as opposed to protonation) of the regiospecifically generated Hcy thiyl radical promotes its rapid isomerisation into an α-carbon radical via HAT. Theoretical calculations were employed to calculate the most probable HAT pathway and found that in alkali metal ion complexes the activation barrier is significantly lower, in full agreement with the experimental data. This is, to our knowledge, the first example of a gas-phase thiyl radical thermal rearrangement into an α-carbon species within the same amino acid residue and is consistent with the solution phase behaviour of Hcy radical.

Transnitrosylation products of the dipeptide cysteinyl-cysteine: an examination by tandem mass spectrometry and density functional theory.

The fragmentation pathways of protonated mono- and di-nitrosylated derivatives from the dipeptide Cys-Cys obtained by electrospray were examined. Protonated mononitrosylated dipeptide upon loss of ˙NO formed a radical cation, which in turn shows two fragment ions, one from the loss of HS˙ and the other from a neutral loss giving a radical cation of formula C2H5NS˙(+). Protonated dinitrosylated dipeptide dissociated by losing both ˙NO molecules, forming a cyclic structure with a vicinal disulfide bridge whose major dissociation channel was the loss of CO. After CO loss, two pathways were observed (loss of NH3 and C2H3NS) which were preceded by proton exchange occurring between one ß-carbon and the nitrogen atom. DFT calculations did not show significant differences in the energies involved for the loss of the NO radical from either of the cysteine residues of the protonated di-nitrosylated dipeptide. Upon loss of the first NO radical, the thiyl radical afforded the vicinal disulfide product with a small barrier through radical substitution of the remaining NO moiety. The calculated relative energy barriers for the different channels are in good agreement with experimental observations. Structures of the ions obtained after dissociation are suggested on the basis of the proposed mechanisms.