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Quantitative, Multiplexed Assays for Low Abundance Proteins in Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution
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Toward a Comprehensive Atlas of the Physical Interactome of Saccharomyces cerevisiae
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A Human Protein Atlas for Normal and Cancer Tissues Based on Antibody Proteomics
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Quantitative Mass Spectrometric Multiple Reaction Monitoring Assays for Major Plasma Proteins
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The Human Plasma Proteome: History, Character, and Diagnostic Prospects
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The focal adhesion protein kindlin-2 controls mitotic spindle assembly by inhibiting histone deacetylase 6 and maintaining {alpha}-tubulin acetylation [Signal Transduction]
Kindlins are focal adhesion proteins that regulate integrin activation and outside-in signaling. The kindlin family consists of three members, kindlin-1, -2, and -3. Kindlin-2 is widely expressed in multiple cell types, except those from the hematopoietic lineage. A previous study has reported that the Drosophila Fit1 protein (an ortholog of kindlin-2) prevents abnormal spindle assembly; however, the mechanism remains unknown. Here, we show that kindlin-2 maintains spindle integrity in mitotic human cells. The human neuroblastoma SH-SY5Y cell line expresses only kindlin-2, and we found that when SH-SY5Y cells are depleted of kindlin-2, they exhibit pronounced spindle abnormalities and delayed mitosis. Of note, acetylation of α-tubulin, which maintains microtubule flexibility and stability, was diminished in the kindlin-2–depleted cells. Mechanistically, we found that kindlin-2 maintains α-tubulin acetylation by inhibiting the microtubule-associated deacetylase histone deacetylase 6 (HDAC6) via a signaling pathway involving AKT Ser/Thr kinase (AKT)/glycogen synthase kinase 3β (GSK3β) or paxillin. We also provide evidence that prolonged hypoxia down-regulates kindlin-2 expression, leading to spindle abnormalities not only in the SH-SY5Y cell line, but also cell lines derived from colon and breast tissues. The findings of our study highlight that kindlin-2 regulates mitotic spindle assembly and that this process is perturbed in cancer cells in a hypoxic environment.
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Structural and mutational analyses of the bifunctional arginine dihydrolase and ornithine cyclodeaminase AgrE from the cyanobacterium Anabaena [Enzymology]
In cyanobacteria, metabolic pathways that use the nitrogen-rich amino acid arginine play a pivotal role in nitrogen storage and mobilization. The N-terminal domains of two recently identified bacterial enzymes: ArgZ from Synechocystis and AgrE from Anabaena, have been found to contain an arginine dihydrolase. This enzyme provides catabolic activity that converts arginine to ornithine, resulting in concomitant release of CO2 and ammonia. In Synechocystis, the ArgZ-mediated ornithine–ammonia cycle plays a central role in nitrogen storage and remobilization. The C-terminal domain of AgrE contains an ornithine cyclodeaminase responsible for the formation of proline from ornithine and ammonia production, indicating that AgrE is a bifunctional enzyme catalyzing two sequential reactions in arginine catabolism. Here, the crystal structures of AgrE in three different ligation states revealed that it has a tetrameric conformation, possesses a binding site for the arginine dihydrolase substrate l-arginine and product l-ornithine, and contains a binding site for the coenzyme NAD(H) required for ornithine cyclodeaminase activity. Structure–function analyses indicated that the structure and catalytic mechanism of arginine dihydrolase in AgrE are highly homologous with those of a known bacterial arginine hydrolase. We found that in addition to other active-site residues, Asn-71 is essential for AgrE's dihydrolase activity. Further analysis suggested the presence of a passage for substrate channeling between the two distinct AgrE active sites, which are situated ∼45 Å apart. These results provide structural and functional insights into the bifunctional arginine dihydrolase–ornithine cyclodeaminase enzyme AgrE required for arginine catabolism in Anabaena.
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Specificity and affinity of the N-terminal residues in staphylocoagulase in binding to prothrombin [Computational Biology]
In Staphylococcus aureus–caused endocarditis, the pathogen secretes staphylocoagulase (SC), thereby activating human prothrombin (ProT) and evading immune clearance. A previous structural comparison of the SC(1–325) fragment bound to thrombin and its inactive precursor prethrombin 2 has indicated that SC activates ProT by inserting its N-terminal dipeptide Ile1-Val2 into the ProT Ile16 pocket, forming a salt bridge with ProT's Asp194, thereby stabilizing the active conformation. We hypothesized that these N-terminal SC residues modulate ProT binding and activation. Here, we generated labeled SC(1–246) as a probe for competitively defining the affinities of N-terminal SC(1–246) variants preselected by modeling. Using ProT(R155Q,R271Q,R284Q) (ProTQQQ), a variant refractory to prothrombinase- or thrombin-mediated cleavage, we observed variant affinities between ∼1 and 650 nm and activation potencies ranging from 1.8-fold that of WT SC(1–246) to complete loss of function. Substrate binding to ProTQQQ caused allosteric tightening of the affinity of most SC(1–246) variants, consistent with zymogen activation through occupation of the specificity pocket. Conservative changes at positions 1 and 2 were well-tolerated, with Val1-Val2, Ile1-Ala2, and Leu1-Val2 variants exhibiting ProTQQQ affinity and activation potency comparable with WT SC(1–246). Weaker binding variants typically had reduced activation rates, although at near-saturating ProTQQQ levels, several variants exhibited limiting rates similar to or higher than that of WT SC(1–246). The Ile16 pocket in ProTQQQ appears to favor nonpolar, nonaromatic residues at SC positions 1 and 2. Our results suggest that SC variants other than WT Ile1-Val2-Thr3 might emerge with similar ProT-activating efficiency.
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It takes two (Las1 HEPN endoribonuclease domains) to cut RNA correctly [RNA]
The ribosome biogenesis factor Las1 is an essential endoribonuclease that is well-conserved across eukaryotes and a newly established member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain-containing nuclease family. HEPN nucleases participate in diverse RNA cleavage pathways and share a short HEPN nuclease motif (RφXXXH) important for RNA cleavage. Most HEPN nucleases participate in stress-activated RNA cleavage pathways; Las1 plays a fundamental role in processing pre-rRNA. Underscoring the significance of Las1 function in the cell, mutations in the human LAS1L (LAS1-like) gene have been associated with neurological dysfunction. Two juxtaposed HEPN nuclease motifs create Las1's composite nuclease active site, but the roles of the individual HEPN motif residues are poorly defined. Here using a combination of in vivo experiments in Saccharomyces cerevisiae and in vitro assays, we show that both HEPN nuclease motifs are required for Las1 nuclease activity and fidelity. Through in-depth sequence analysis and systematic mutagenesis, we determined the consensus HEPN motif in the Las1 subfamily and uncovered its canonical and specialized elements. Using reconstituted Las1 HEPN-HEPN' chimeras, we defined the molecular requirements for RNA cleavage. Intriguingly, both copies of the Las1 HEPN motif were important for nuclease function, revealing that both HEPN motifs participate in coordinating the RNA within the Las1 active site. We also established that conformational flexibility of the two HEPN domains is important for proper nuclease function. The results of our work reveal critical information about how dual HEPN domains come together to drive Las1-mediated RNA cleavage.
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Webinar: European Democracy in the Last 100 Years: Economic Crises and Political Upheaval
Members Event Webinar
6 May 2020 - 1:00pm to 2:00pm
Event participants
Pepijn Bergsen, Research Fellow, Europe Programme, Chatham House
Dr Sheri Berman, Professor of Political Science, Barnard College
Chair: Hans Kundnani, Senior Research Fellow, Europe Programme, Chatham House
In the last 100 years, global economic crises from the Great Depression of the 1930s to the 2008 financial crash have contributed to significant political changes in Europe, often leading to a rise in popularity for extremist parties and politics. As Europe contends with a perceived crisis of democracy - now compounded by the varied responses to the coronavirus outbreak - how should we understand the relationship between externally-driven economic crises, political upheaval and democracy?
The panellists will consider the parallels between the political responses to some of the greatest economic crises Europe has experienced in the last century. Given that economic crises often transcend borders, why does political disruption vary between democracies? What can history tell us about the potential political impact of the unfolding COVID-19-related economic crisis? And will the unprecedented financial interventions by governments across Europe fundamentally change the expectations citizens have of the role government should play in their lives?
This event is based on a recent article in The World Today by Hans Kundnani and Pepijn Bergsen who are both researchers in Chatham House's Europe Programme. 'Crawling from the Wreckage' is the first in a series of articles that look at key themes in European political discourse from the last century. You can read the article here.
This event is open to Chatham House Members. Not a member? Find out more.
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Disruption of endoplasmic reticulum structure and integrity in lipotoxic cell death
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Marked reduction in bile acid synthesis in cholesterol 7{alpha}-hydroxylase-deficient mice does not lead to diminished tissue cholesterol turnover or to hypercholesterolemia
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Lipidomics reveals a remarkable diversity of lipids in human plasma
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Fish oils and plasma lipid and lipoprotein metabolism in humans: a critical review
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Identification of multiple subclasses of plasma low density lipoproteins in normal humans
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Direct transesterification of all classes of lipids in a one-step reaction
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The plasma lecithin:cholesterol acyltransferase reaction
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Undercurrents: Episode 17 - Alastair Campbell on New Labour and Brexit, Alistair Darling on the Financial Crisis
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Identification of an Unconventional Subpeptidome Bound to the Behcet's Disease-associated HLA-B*51:01 that is Regulated by Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) [Research]
Human leukocyte antigen (HLA) B*51:01 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly genetically associated with Behcet's disease (BD). Previous studies have defined two subgroups of HLA-B*51 peptidome containing proline (Pro) or alanine (Ala) at position 2 (P2). Little is known about the unconventional non-Pro/Ala2 HLA-B*51-bound peptides. We aimed to study the features of this novel subpeptidome, and investigate its regulation by ERAP1. CRISPR-Cas9 was used to generate an HLA-ABC-triple knockout HeLa cell line (HeLa.ABC-KO), which was subsequently transduced to express HLA-B*51:01 (HeLa.ABC-KO.B51). ERAP1 was silenced using lentiviral shRNA. Peptides bound to HLA-B*51:01 were eluted and analyzed by mass spectrometry. The characteristics of non-Pro/Ala2, Pro2, and Ala2 peptides and their alteration by ERAP1 silencing were investigated. Effects of ERAP1 silencing on cell surface expression of HLA-B*51:01 were studied using flow cytometry. More than 20% of peptides eluted from HLA-B*51:01 lacked Pro or Ala at P2. This unconventional group of HLA-B*51:01-bound peptides was relatively enriched for 8-mers (with relatively fewer 9-mers) compared with the Pro2 and Ala2 subpeptidomes and had similar N-terminal and C-terminal residue usages to Ala2 peptides (with the exception of the less abundant leucine at position ). Knockdown of ERAP1 increased the percentage of non-Pro/Ala2 from 20% to ~40%, increased the percentage of longer (10-mer and 11-mer) peptides eluted from HLA-B*51:01 complexes, and abrogated the predominance of leucine at P1. Interestingly knockdown of ERAP1 altered the length and N-terminal residue usage of non-Ala2&Pro2 and Ala2 but not the Pro2 peptides. Finally, ERAP1 silencing regulated the expression levels of cell surface HLA-B*51 in a cell-type-dependent manner. In conclusion, we have used a novel methodology to identify an unconventional but surprisingly abundant non-Pro/Ala2 HLA-B*51:01 subpeptidome. It is increased by knockdown of ERAP1, a gene affecting the risk of developing BD. This has implications for theories of disease pathogenesis.
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Quantitative Profiling of the Human Substantia Nigra Proteome from Laser-capture Microdissected FFPE Tissue [Research]
Laser-capture microdissection (LCM) allows the visualization and isolation of morphologically distinct subpopulations of cells from heterogeneous tissue specimens. In combination with formalin-fixed and paraffin-embedded (FFPE) tissue it provides a powerful tool for retrospective and clinically relevant studies of tissue proteins in a healthy and diseased context. We first optimized the protocol for efficient LCM analysis of FFPE tissue specimens. The use of SDS containing extraction buffer in combination with the single-pot solid-phase-enhanced sample preparation (SP3) digest method gave the best results regarding protein yield and protein/peptide identifications. Microdissected FFPE human substantia nigra tissue samples (~3,000 cells) were then analyzed, using tandem mass tag (TMT) labeling and LC-MS/MS, resulting in the quantification of >5,600 protein groups. Nigral proteins were classified and analyzed by abundance, showing an enrichment of extracellular exosome and neuron-specific gene ontology (GO) terms among the higher abundance proteins. Comparison of microdissected samples with intact tissue sections, using a label-free shotgun approach, revealed an enrichment of neuronal cell type markers, such as tyrosine hydroxylase and alpha-synuclein, as well as proteins annotated with neuron-specific GO terms. Overall, this study provides a detailed protocol for laser-capture proteomics using FFPE tissue and demonstrates the efficiency of LCM analysis of distinct cell subpopulations for proteomic analysis using low sample amounts.
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Nanodomains can persist at physiologic temperature in plasma membrane vesicles and be modulated by altering cell lipids [Research Articles]
The formation and properties of liquid-ordered (Lo) lipid domains (rafts) in the plasma membrane are still poorly understood. This limits our ability to manipulate ordered lipid domain-dependent biological functions. Giant plasma membrane vesicles (GPMVs) undergo large-scale phase separations into coexisting Lo and liquid-disordered lipid domains. However, large-scale phase separation in GPMVs detected by light microscopy is observed only at low temperatures. Comparing Förster resonance energy transfer-detected versus light microscopy-detected domain formation, we found that nanodomains, domains of nanometer size, persist at temperatures up to 20°C higher than large-scale phases, up to physiologic temperature. The persistence of nanodomains at higher temperatures is consistent with previously reported theoretical calculations. To investigate the sensitivity of nanodomains to lipid composition, GPMVs were prepared from mammalian cells in which sterol, phospholipid, or sphingolipid composition in the plasma membrane outer leaflet had been altered by cyclodextrin-catalyzed lipid exchange. Lipid substitutions that stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on the thermal stability of nanodomains and large-scale phase separation in GPMVs, with nanodomains persisting at higher temperatures than large-scale phases for a wide range of lipid compositions. This indicates that it is likely that plasma membrane nanodomains can form under physiologic conditions more readily than large-scale phase separation. We also conclude that membrane lipid substitutions carried out in intact cells are able to modulate the propensity of plasma membranes to form ordered domains. This implies lipid substitutions can be used to alter biological processes dependent upon ordered domains.
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It takes two (Las1 HEPN endoribonuclease domains) to cut RNA correctly [RNA]
The ribosome biogenesis factor Las1 is an essential endoribonuclease that is well-conserved across eukaryotes and a newly established member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain-containing nuclease family. HEPN nucleases participate in diverse RNA cleavage pathways and share a short HEPN nuclease motif (RφXXXH) important for RNA cleavage. Most HEPN nucleases participate in stress-activated RNA cleavage pathways; Las1 plays a fundamental role in processing pre-rRNA. Underscoring the significance of Las1 function in the cell, mutations in the human LAS1L (LAS1-like) gene have been associated with neurological dysfunction. Two juxtaposed HEPN nuclease motifs create Las1's composite nuclease active site, but the roles of the individual HEPN motif residues are poorly defined. Here using a combination of in vivo experiments in Saccharomyces cerevisiae and in vitro assays, we show that both HEPN nuclease motifs are required for Las1 nuclease activity and fidelity. Through in-depth sequence analysis and systematic mutagenesis, we determined the consensus HEPN motif in the Las1 subfamily and uncovered its canonical and specialized elements. Using reconstituted Las1 HEPN-HEPN' chimeras, we defined the molecular requirements for RNA cleavage. Intriguingly, both copies of the Las1 HEPN motif were important for nuclease function, revealing that both HEPN motifs participate in coordinating the RNA within the Las1 active site. We also established that conformational flexibility of the two HEPN domains is important for proper nuclease function. The results of our work reveal critical information about how dual HEPN domains come together to drive Las1-mediated RNA cleavage.
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On a class of singular Sturm–Liouville problems
A. A. Vladimirov
Trans. Moscow Math. Soc. 80 (2020), 211-219.
Abstract, references and article information
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On the solvability of a class of nonlinear integral equations in the problem of a spread of an epidemic
A. G. Sergeev and Kh. A. Khachatryan
Trans. Moscow Math. Soc. 80 (2020), 95-111.
Abstract, references and article information
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Products of conjugacy classes in ????????₂(ℝ)
S. Yu. Orevkov
Trans. Moscow Math. Soc. 80 (2020), 73-81.
Abstract, references and article information
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Barnes no longer Jamaica's coach - Last-minute goal brings sweet victory over Panama
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CBD News: Statement by Ahmed Djoghlaf, Executive Secretary, at the Expert Workshop on Scientific and Technical Guidance on the Use of Biogeographic Classification Systems and Identification of Marine Areas in Need of Protection beyond National Jurisdictio
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CBD News: With four new ratifications in the last week, the Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization has received 66% of the necessary ratifications, with only 17 more rat
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CBD News: It is a great pleasure to welcome you to the city of Montreal, where the Great Lakes Waterway and the Saint Lawrence Seaway meet, on the occasion of the 6th GEF-UNDP-IMO Research and Development Forum and Exhibition on Ballast Water Management.
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CBD News: Plastic is everywhere, a part of our daily lives. However, the convenience of plastics now threatens the very survival of our planet.
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CBD News: Every year, we use some 500 billion plastic bags. Every year, as much as 13 million tons of plastic finds its way into the ocean. Every year, 17 million barrels of oil are used to produce plastic.
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New analytical tools for HDG in elasticity, with applications to elastodynamics
Shukai Du and Francisco-Javier Sayas
Math. Comp. 89 (2019), 1745-1782.
Abstract, references and article information
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Uniqueness for the inverse boundary value problem of piecewise homogeneous anisotropic elasticity in the time domain
Cătălin I. Cârstea, Gen Nakamura and Lauri Oksanen
Trans. Amer. Math. Soc. 373 (2020), 3423-3443.
Abstract, references and article information
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RNLI Severn Class
peterphotographic posted a photo:
Airfix 1:72 RNLI Severn Class - Plymouth Lifeboat
Walthamstow, East London, UK
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Classifying spaces and Bredon (co)homology for transitive groupoids
Carla Farsi, Laura Scull and Jordan Watts
Proc. Amer. Math. Soc. 148 (2020), 2717-2737.
Abstract, references and article information
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Classical solution of a PDE system stemming from auxin transport model for leaf venation
Bin Li and Jieqiong Shen
Proc. Amer. Math. Soc. 148 (2020), 2565-2578.
Abstract, references and article information
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A topology on the set of isomorphism classes of maximal Cohen–Macaulay modules
Naoya Hiramatsu and Ryo Takahashi
Proc. Amer. Math. Soc. 148 (2020), 2359-2369.
Abstract, references and article information
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????-analogues of several ????-formulas
Chuanan Wei
Proc. Amer. Math. Soc. 148 (2020), 2287-2296.
Abstract, references and article information
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Ship's last HK passenger back home
The Security Bureau today said that the last Hong Kong resident who had contracted COVID-19 while aboard the Diamond Princess cruise ship and was hospitalised in Japan has returned to Hong Kong.
Immigration Department staff that assisted Hong Kong residents in Japan have completed their mission and returned to Hong Kong as well.
In early February, a cluster of COVID-19 infection cases occurred on the Diamond Princess cruise docked in Yokohama.
Of some 3,700 passengers and crew, about 370 were from Hong Kong. The 712 confirmed COVID-19 cases associated with the cruise included 76 Hong Kong residents who were hospitalised in Japan for isolation and treatment.
While three Hong Kong residents passed away, the remaining 73 patients returned to Hong Kong or their places of residence after being discharged from the hospital.
As for other Hong Kong residents on the cruise, the bureau noted that the Hong Kong Special Administrative Region Government arranged three flights, between February 19 and 23, to escort 193 of them back to the city.
Upon arrival, they were transferred to the quarantine centre at Chun Yeung Estate to undergo 14 days of quarantine.
Another 144 Hong Kong residents returned on their own via other flights, including 25 close contacts of the patients who had completed quarantine in Japan.
For those who returned to Hong Kong on their own and did not complete 14 days of quarantine in Japan, they were required to complete the remaining quarantine period at a quarantine centre.
Of the 231 cruise passengers admitted to the quarantine centre at Chun Yeung Estate, nine tested positive for COVID-19 and were sent to hospitals for isolation and treatment.
The Hong Kong SAR Government expressed profound condolences on the passing of the Hong Kong patients and its deepest sympathies to their families.
The SAR Government emphasised that the incident could not have been resolved smoothly without the staunch support of the Office of the Commissioner of the Ministry of Foreign Affairs of the People's Republic of China in the Hong Kong Special Administrative Region, the Embassy of the People’s Republic of China in Japan and Japanese authorities.
The SAR Government also thanked Cathay Pacific Airways and the Airport Authority for their assistance, the bureau added.
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John Lewis Partnership selects four startups to reduce plastic waste
The partnership will join hands with four UK startups as it hopes to reduce plastic waste as part of its retail tech initiative, JLAB
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Classification and Identification of Lie Algebras
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