General Stations: 1 to 2 (Health Risk: Low)

Roadside Stations: 2 (Health Risk: Low)

(Fundação de Amparo à Pesquisa do Estado de São Paulo) Thanks to its magnetic properties, the material -- zinc-doped manganese chromite -- can be used in a range of products, from gas sensors to data storage devices.

(University of Southampton) Scientists have published highly detailed images of lab-grown neurons using Extreme Ultraviolet radiation that could aid the analysis of neurodegenerative diseases.

(Dartmouth College) Annual award supports the research and teaching careers of talented young faculty in the chemical sciences.

(University of Science and Technology of China) Researchers from University of science and technology of China has shed new lights on the topic of solid-phase ion migration. Researchers demonstrated a unique in-situ strategy for visualizing the dynamic solid-phase ion migration between nanostructures with nanogap at the atomic scale. The research article entitled "Real-Time Visualization of Solid-Phase Ion Migration Kinetics on Nanowire Monolayer" was published in Journal of the American Chemical Society on April 29th.

(University of Jyväskylä - Jyväskylän yliopisto) Researchers at the universities of Jyvaskyla and Xiamen discovered a novel way to make functional macroscopic crystalline materials out of nanometer-size 34-atom silver-gold intermetallic clusters. The cluster material has a highly anisotropic electrical conductivity, being a semiconductor in one direction and an electrical insulator in other directions. The research was published in Nature Communications on May 6, 2020.

(Johannes Gutenberg Universitaet Mainz) Scientists at Johannes Gutenberg University Mainz (JGU) and the Helmholtz Institute Mainz (HIM) in Germany have presented a non-contact method for detecting the state of charge and any defects in lithium-ion batteries.

(To watch the full press conference with sign language interpretation, click here.)

The relaxation of anti-epidemic measures is a step in the right direction, Chief Executive Carrie Lam said today.

Mrs Lam made the statement at a press conference this afternoon.

She said the Government will relax the requirement limiting group gatherings in public places to a maximum of four people. Starting from Friday, up to eight people can gather in public places.

"So raising the number from four to eight for the catering business and also for the prohibition against group gatherings under Cap 599G is not an exact science, but this is a step in the right direction of relaxation.

"Maybe in another 14 days’ time we will raise the number of eight to 10, to 12, to 15 and so on."

Regarding bars and pubs, Mrs Lam said these venues will be able to reopen but the Government will put in place requirements to prevent physical interactions.

"We have decided that perhaps to strike a pragmatic balance is to allow them to reopen for business but to put in far more stringent requirements."

Such requirements include no live music, band performances or dancing in bar premises.

"That would be another way to keep the social distance and prevent as much as possible physical interactions."

Click here for the latest measures.

(To watch the full press conference with sign language interpretation, click here.)

Secretary for Education Kevin Yeung today said the Government is confident that classes can resume on May 27.

Mr Yeung made the statement at a press conference this afternoon.

He said: “We are not announcing class resumption right away. We are giving advance notice of about three weeks for all the stakeholders to get prepared for the school resumption.”

The Government will continue to monitor the situation during this period and adjust the school resumption plan if necessary, he added.

"At this stage we are still pretty confident that we should be able to resume classes on May 27."

Chief Executive Carrie Lam inspected one of the CuMask production sites in Tsuen Wan today and expressed gratitude to those manufacturing the reusable masks for Hong Kong residents to fight against the COVID-19 epidemic.

She chatted with the staff and noted that many of them are retired textile industry workers who have re-joined the production workforce to combat the virus.

Mrs Lam praised them for their commitment to serving the community and thanked them for their hard work.

The masks produced in the workshop will be delivered to a clean workspace for sterilising with ozone and packing before distribution. There is no need to wash the mask before it is used for the first time.

In the face of the tight supply of masks amid the epidemic, the Government set aside funds to subsidise projects on technology applications of reusable masks under the first round of the Anti-epidemic Fund.

The CuMask complies with the American Society for Testing & Materials F2100 Level 1 standard and can be washed up to 60 times. It can also be used for a longer period after replacing the filter.

Mrs Lam said she is pleased to note that CuMask is well received by the public and the registration process has been very smooth.

“CuMask is a home-grown scientific research achievement with local application. The whole process has involved co-operation between the Government, industry, academia and the research sector and is an outstanding example of the use of technology to improve people’s lives.

“It will also help solve the problem of the supply of face masks during an epidemic in the long run. I highly commend all the people who have participated in the relevant work and I am fully confident in the development of innovation and technology in Hong Kong.”

Mrs Lam appealed to the public to make use of the registration quota of up to six people to minimise delivery resources and enhance efficiency.

She added that relevant departments will deliver the masks as soon as possible.

The Government urged District Councillors to focus on livelihood issues and discuss matters rationally, adding that it will continue to co-operate with the District Council under the principles of mutual respect, observation of order and rational discussion.

The Government issued the statement after a number of Central & Western District Council members today entered the office area of the Central & Western District Office without consent.

The statement noted that the members shouted loudly and knocked on the door of the office.

Despite repeated responses and an appeal from the District Office staff, the members still refused to leave.

The statement added that the members stayed in the District Office for a long time, seriously affecting its operation.

The Government expressed regret over their acts.

Secretary for Home Affairs Caspar Tsui visited the Physical Fitness Association of Hong Kong, China today to inspect its work in implementing the Fitness Centre Subsidy Scheme launched under the second phase of the Anti-epidemic Fund.

Mr Tsui said the scheme aims to provide a one-off subsidy of $100,000 to fitness centres to tide businesses over financial difficulties arising from anti-epidemic measures.

He thanked the association for handling the scheme’s applications.

Mr Tsui also expressed gratitude to the fitness industry for complying with the Government’s preventive measures, including suspension of business, in the fight against the virus.

Given the stabilising epidemic situation, the Government has conducted a health risk assessment and will allow premises, including fitness centres, to resume operations, Mr Tsui said, adding that he hopes the fitness industry will soon regain vitality.

The Home Affairs Bureau commissioned the association to assist in implementing the scheme, which opened for applications on May 4.

As of May 7, the association received 397 applications, of which more than half of them have been initially found to be eligible, involving subsidies of about $20 million.

The application period for the scheme will end on June 3.

Call 2302 9089 or send an email for enquiries.

The 2020 Mathematical Art Exhibition Awards were made at the Joint Mathematics Meetings last week "for aesthetically pleasing works that combine mathematics and art." The chosen works were selected from the exhibition of juried works in various media by over 90 mathematicians and artists from around the world.

"Suspended Helical Stair," by Mark Donohue (California College of the Arts, San Francisco, CA), was awarded Best textile, sculpture, or other medium. "A unique cable system to suspend a stair was developed in collaboration with a leading structural engineer. The suspended cables form a double helicoid nested within an ascending spiral hyperboloid to create the necessary points of support for the gravity loads and lateral bracing for the seismic loads. Each concrete stair tread was designed as an independent element that is strung together with the stairs above and below it to form a single spiral stair when the steel cables that run through them are post tensioned. The entire stair tread and suspension cable system can be understood as a play of ruled surfaces with each part related to the other through their shared geometric lineage." The work is string and plywood,45 x 23 x 23 cm, 2018.

2018

"A Unit Domino," by Douglas McKenna (Mathemaesthetics, Inc., Boulder, CO), was awarded Best photograph, painting, or print. "This piece is based upon an artist-discovered "half-domino" space-filling curve. The drawing comprises some half-million connected line segments, arranged in two perfectly recursive levels of double-spiral pairs, slowly changing color, in a single, over-one-mile-long self-avoiding path from lower left to lower right (the lower right square that sticks out is an integral part of its self-negative structure). The limiting curve covers a self-similar gasket tile with an infinitely long, almost-everywhere linear border. With an upside-down copy of itself, two such gaskets of unit area exactly cover a 1x2 domino, without overlap. The artist's app/eBook "Hilbert Curves" for iPad/iPhone explains how he discovered these beautiful constructions." The work is a glicée print,106 x 66 cm, 2015.

"Computational Wings," by David Bachman (Pitzer College, Claremont, CA), received Honorable Mention. "The body of this dragonfly is taken from a photograph, while the wings were computationally generated. A variety of algorithms were used to create them. First, a set of points were randomly populated across each wing and moved by a circle packing algorithm, where the radius of each circle was inversely proportional to the distance from the body. Next, those points were used to create a Voronoi diagram. Main veins were located by a shortest walk algorithm through the edges of this diagram, and those veins were given a variable thickness according to the distance travelled as you traverse them outward from the body." The work is laser etched acrylic, 23 x 35 x 3 cm, 2019.

(Click on the thumbnails to see larger versions of the images.)

The Mathematical Art Exhibition Award "for aesthetically pleasing works that combine mathematics and art" was established in 2008 through an endowment provided to the American Mathematical Society by an anonymous donor who wishes to acknowledge those whose works demonstrate the beauty and elegance of mathematics expressed in a visual art form. The awards are $400 for Best photograph, painting, or print; $400 for Best textile, sculpture, or other medium; and $200 for Honorable Mention. The Mathematical Art Exhibition of juried works in various media is held at the annual Joint Mathematics Meetings of the American Mathematical Society (AMS) and Mathematical Association of America (MAA). a gallery of works in the 2020 exhibition will be on AMS Mathematical Imagery.

Find out more about the Mathematical Art Exhibition Award and see past recipients.

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The American Mathematical Society is dedicated to advancing research and connecting the diverse global mathematical community through our publications, meetings and conferences, MathSciNet, professional services, advocacy, and awareness programs.

VOLUME 285 (2010) PAGES 13742–13747In Fig. 1E, passage 10, the splicing of a non-adjacent lane from the same immunoblot was not marked. This error has now been corrected and does not affect the results or conclusions of this work.jbc;295/16/5533/F1F1F1Figure 1E.

Extracytoplasmic sugar decoration of glycopolymer components of the bacterial cell wall contributes to their structural diversity. Typically, the molecular mechanism that underpins such a decoration process involves a three-component glycosylation system (TGS) represented by an undecaprenyl-phosphate (Und-P) sugar-activating glycosyltransferase (Und-P GT), a flippase, and a polytopic glycosyltransferase (PolM GT) dedicated to attaching sugar residues to a specific glycopolymer. Here, using bioinformatic analyses, CRISPR-assisted recombineering, structural analysis of cell wall–associated polysaccharides (CWPS) through MALDI-TOF MS and methylation analysis, we report on three such systems in the bacterium Lactococcus lactis. On the basis of sequence similarities, we first identified three gene pairs, csdAB, csdCD, and csdEF, each encoding an Und-P GT and a PolM GT, as potential TGS component candidates. Our experimental results show that csdAB and csdCD are involved in Glc side-chain addition on the CWPS components rhamnan and polysaccharide pellicle (PSP), respectively, whereas csdEF plays a role in galactosylation of lipoteichoic acid (LTA). We also identified a potential flippase encoded in the L. lactis genome (llnz_02975, cflA) and confirmed that it participates in the glycosylation of the three cell wall glycopolymers rhamnan, PSP, and LTA, thus indicating that its function is shared by the three TGSs. Finally, we observed that glucosylation of both rhamnan and PSP can increase resistance to bacteriophage predation and that LTA galactosylation alters L. lactis resistance to bacteriocin.

Neoantimycins are anticancer compounds of 15-membered ring antimycin-type depsipeptides. They are biosynthesized by a hybrid multimodular protein complex of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), typically from the starting precursor 3-formamidosalicylate. Examining fermentation extracts of Streptomyces conglobatus, here we discovered four new neoantimycin analogs, unantimycins B–E, in which 3-formamidosalicylates are replaced by an unusual 3-hydroxybenzoate (3-HBA) moiety. Unantimycins B–E exhibited levels of anticancer activities similar to those of the chemotherapeutic drug cisplatin in human lung cancer, colorectal cancer, and melanoma cells. Notably, they mostly displayed no significant toxicity toward noncancerous cells, unlike the serious toxicities generally reported for antimycin-type natural products. Using site-directed mutagenesis and heterologous expression, we found that unantimycin productions are correlated with the activity of a chorismatase homolog, the nat-hyg5 gene, from a type I PKS gene cluster. Biochemical analysis confirmed that the catalytic activity of Nat-hyg5 generates 3-HBA from chorismate. Finally, we achieved selective production of unantimycins B and C by engineering a chassis host. On the basis of these findings, we propose that unantimycin biosynthesis is directed by the neoantimycin-producing NRPS–PKS complex and initiated with the starter unit of 3-HBA. The elucidation of the biosynthetic unantimycin pathway reported here paves the way to improve the yield of these compounds for evaluation in oncotherapeutic applications.

Previous studies have shown that sphingosine kinase interacting protein (SKIP) inhibits sphingosine kinase (SK) function in fibroblasts. SK phosphorylates sphingosine producing the potent signaling molecule sphingosine-1-phosphate (S1P). SKIP gene (SPHKAP) expression is silenced by hypermethylation of its promoter in acute myeloid leukemia (AML). However, why SKIP activity is silenced in primary AML cells is unclear. Here, we investigated the consequences of SKIP down-regulation in AML primary cells and the effects of SKIP re-expression in leukemic cell lines. Using targeted ultra-HPLC-tandem MS (UPLC-MS/MS), we measured sphingolipids (including S1P and ceramides) in AML and control cells. Primary AML cells had significantly lower SK activity and intracellular S1P concentrations than control cells, and SKIP-transfected leukemia cell lines exhibited increased SK activity. These findings show that SKIP re-expression enhances SK activity in leukemia cells. Furthermore, other bioactive sphingolipids such as ceramide were also down-regulated in primary AML cells. Of note, SKIP re-expression in leukemia cells increased ceramide levels 2-fold, inactivated the key signaling protein extracellular signal-regulated kinase, and increased apoptosis following serum deprivation or chemotherapy. These results indicate that SKIP down-regulation in AML reduces SK activity and ceramide levels, an effect that ultimately inhibits apoptosis in leukemia cells. The findings of our study contrast with previous results indicating that SKIP inhibits SK function in fibroblasts and therefore challenge the notion that SKIP always inhibits SK activity.

The transcription factor forkhead box P3 (FOXP3) is a biomarker for regulatory T cells and can also be expressed in cancer cells, but its function in cancer appears to be divergent. The role of hepatocyte-expressed FOXP3 in hepatocellular carcinoma (HCC) is unknown. Here, we collected tumor samples and clinical information from 115 HCC patients and used five human cancer cell lines. We examined FOXP3 mRNA sequences for mutations, used a luciferase assay to assess promoter activities of FOXP3's target genes, and employed mouse tumor models to confirm in vitro results. We detected mutations in the FKH domain of FOXP3 mRNAs in 33% of the HCC tumor tissues, but in none of the adjacent nontumor tissues. None of the mutations occurred at high frequency, indicating that they occurred randomly. Notably, the mutations were not detected in the corresponding regions of FOXP3 genomic DNA, and many of them resulted in amino acid substitutions in the FKH region, altering FOXP3's subcellular localization. FOXP3 delocalization from the nucleus to the cytoplasm caused loss of transcriptional regulation of its target genes, inactivated its tumor-inhibitory capability, and changed cellular responses to histone deacetylase (HDAC) inhibitors. More complex FKH mutations appeared to be associated with worse prognosis in HCC patients. We conclude that mutations in the FKH domain of FOXP3 mRNA frequently occur in HCC and that these mutations are caused by errors in transcription and are not derived from genomic DNA mutations. Our results suggest that transcriptional mutagenesis of FOXP3 plays a role in HCC.

Prostate cancer (PCa) cells heavily rely on an active androgen receptor (AR) pathway for their survival. Enzalutamide (MDV3100) is a second-generation antiandrogenic drug that was approved by the Food and Drug Administration in 2012 to treat patients with castration-resistant prostate cancer (CRPC). However, emergence of resistance against this drug is inevitable, and it has been a major challenge to develop interventions that help manage enzalutamide-resistant CRPC. Erythropoietin-producing human hepatocellular (Eph) receptors are targeted by ephrin protein ligands and have a broad range of functions. Increasing evidence indicates that this signaling pathway plays an important role in tumorigenesis. Overexpression of EPH receptor B4 (EPHB4) has been observed in multiple types of cancer, being closely associated with proliferation, invasion, and metastasis of tumors. Here, using RNA-Seq analyses of clinical and preclinical samples, along with several biochemical and molecular methods, we report that enzalutamide-resistant PCa requires an active EPHB4 pathway that supports drug resistance of this tumor type. Using a small kinase inhibitor and RNAi-based gene silencing to disrupt EPHB4 activity, we found that these disruptions re-sensitize enzalutamide-resistant PCa to the drug both in vitro and in vivo. Mechanistically, we found that EPHB4 stimulates the AR by inducing proto-oncogene c-Myc (c-Myc) expression. Taken together, these results provide critical insight into the mechanism of enzalutamide resistance in PCa, potentially offering a therapeutic avenue for enhancing the efficacy of enzalutamide to better manage this common malignancy.

Telomeres are specific nucleoprotein structures that are located at the ends of linear eukaryotic chromosomes and play crucial roles in genomic stability. Telomere DNA consists of simple repeats of a short G-rich sequence: TTAGGG in mammals and TTTAGGG in most plants. In recent years, the mammalian telomeric G-rich repeats have been shown to form G-quadruplex (G4) structures, which are crucial for modulating telomere functions. Surprisingly, even though plant telomeres are essential for plant growth, development, and environmental adaptions, only few reports exist on plant telomeric G4 DNA (pTG4). Here, using bulk and single-molecule assays, including CD spectroscopy, and single-molecule FRET approaches, we comprehensively characterized the structure and dynamics of a typical plant telomeric sequence, d[GGG(TTTAGGG)3]. We found that this sequence can fold into mixed G4s in potassium, including parallel and antiparallel structures. We also directly detected intermediate dynamic transitions, including G-hairpin, parallel G-triplex, and antiparallel G-triplex structures. Moreover, we observed that pTG4 is unfolded by the AtRecQ2 helicase but not by AtRecQ3. The results of our work shed light on our understanding about the existence, topological structures, stability, intermediates, unwinding, and functions of pTG4.

Haploinsufficiency of Meis homeobox 2 (MEIS2), encoding a transcriptional regulator, is associated with human cleft palate, and Meis2 inactivation leads to abnormal palate development in mice, implicating MEIS2 functions in palate development. However, its functional mechanisms remain unknown. Here we observed widespread MEIS2 expression in the developing palate in mice. Wnt1Cre-mediated Meis2 inactivation in cranial neural crest cells led to a secondary palate cleft. Importantly, about half of the Wnt1Cre;Meis2f/f mice exhibited a submucous cleft, providing a model for studying palatal bone formation and patterning. Consistent with complete absence of palatal bones, the results from integrative analyses of MEIS2 by ChIP sequencing, RNA-Seq, and an assay for transposase-accessible chromatin sequencing identified key osteogenic genes regulated directly by MEIS2, indicating that it plays a fundamental role in palatal osteogenesis. De novo motif analysis uncovered that the MEIS2-bound regions are highly enriched in binding motifs for several key osteogenic transcription factors, particularly short stature homeobox 2 (SHOX2). Comparative ChIP sequencing analyses revealed genome-wide co-occupancy of MEIS2 and SHOX2 in addition to their colocalization in the developing palate and physical interaction, suggesting that SHOX2 and MEIS2 functionally interact. However, although SHOX2 was required for proper palatal bone formation and was a direct downstream target of MEIS2, Shox2 overexpression failed to rescue the palatal bone defects in a Meis2-mutant background. These results, together with the fact that Meis2 expression is associated with high osteogenic potential and required for chromatin accessibility of osteogenic genes, support a vital function of MEIS2 in setting up a ground state for palatal osteogenesis.

Prevention of aberrant cutaneous wound repair and appropriate regeneration of an intact and functional integument require the coordinated timing of fibroblast and keratinocyte migration. Here, we identified a mechanism whereby opposing cell-specific motogenic functions of a multifunctional intracellular and extracellular protein, the receptor for hyaluronan-mediated motility (RHAMM), coordinates fibroblast and keratinocyte migration speed and ensures appropriate timing of excisional wound closure. We found that, unlike in WT mice, in Rhamm-null mice, keratinocyte migration initiates prematurely in the excisional wounds, resulting in wounds that have re-surfaced before the formation of normal granulation tissue, leading to a defective epidermal architecture. We also noted aberrant keratinocyte and fibroblast migration in the Rhamm-null mice, indicating that RHAMM suppresses keratinocyte motility but increases fibroblast motility. This cell context–dependent effect resulted from cell-specific regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and expression of a RHAMM target gene encoding matrix metalloprotease 9 (MMP-9). In fibroblasts, RHAMM promoted ERK1/2 activation and MMP-9 expression, whereas in keratinocytes, RHAMM suppressed these activities. In keratinocytes, loss of RHAMM function or expression promoted epidermal growth factor receptor–regulated MMP-9 expression via ERK1/2, which resulted in cleavage of the ectodomain of the RHAMM partner protein CD44 and thereby increased keratinocyte motility. These results identify RHAMM as a key factor that integrates the timing of wound repair by controlling cell migration.

Hypersecretion of glucagon from pancreatic α-cells strongly contributes to diabetic hyperglycemia. Moreover, failure of α-cells to increase glucagon secretion in response to falling blood glucose concentrations compromises the defense against hypoglycemia, a common complication in diabetes therapy. However, the mechanisms underlying glucose regulation of glucagon secretion are poorly understood and likely involve both α-cell–intrinsic and intraislet paracrine signaling. Among paracrine factors, glucose-stimulated release of the GABA metabolite γ-hydroxybutyric acid (GHB) from pancreatic β-cells might mediate glucose suppression of glucagon release via GHB receptors on α-cells. However, the direct effects of GHB on α-cell signaling and glucagon release have not been investigated. Here, we found that GHB (4–10 μm) lacked effects on the cytoplasmic concentrations of the secretion-regulating messengers Ca2+ and cAMP in mouse α-cells. Glucagon secretion from perifused mouse islets was also unaffected by GHB at both 1 and 7 mm glucose. The GHB receptor agonist 3-chloropropanoic acid and the antagonist NCS-382 had no effects on glucagon secretion and did not affect stimulation of secretion induced by a drop in glucose from 7 to 1 mm. Inhibition of endogenous GHB formation with the GABA transaminase inhibitor vigabatrin also failed to influence glucagon secretion at 1 mm glucose and did not prevent the suppressive effect of 7 mm glucose. In human islets, GHB tended to stimulate glucagon secretion at 1 mm glucose, an effect mimicked by 3-chloropropanoic acid. We conclude that GHB does not mediate the inhibitory effect of glucose on glucagon secretion.

Myostatin (or growth/differentiation factor 8 (GDF8)) is a member of the transforming growth factor β superfamily of growth factors and negatively regulates skeletal muscle growth. Its dysregulation is implicated in muscle wasting diseases. SRK-015 is a clinical-stage mAb that prevents extracellular proteolytic activation of pro- and latent myostatin. Here we used integrated structural and biochemical approaches to elucidate the molecular mechanism of antibody-mediated neutralization of pro-myostatin activation. The crystal structure of pro-myostatin in complex with 29H4-16 Fab, a high-affinity variant of SRK-015, at 2.79 Å resolution revealed that the antibody binds to a conformational epitope in the arm region of the prodomain distant from the proteolytic cleavage sites. This epitope is highly sequence-divergent, having only limited similarity to other closely related members of the transforming growth factor β superfamily. Hydrogen/deuterium exchange MS experiments indicated that antibody binding induces conformational changes in pro- and latent myostatin that span the arm region, the loops contiguous to the protease cleavage sites, and the latency-associated structural elements. Moreover, negative-stain EM with full-length antibodies disclosed a stable, ring-like antigen–antibody structure in which the two Fab arms of a single antibody occupy the two arm regions of the prodomain in the pro- and latent myostatin homodimers, suggesting a 1:1 (antibody:myostatin homodimer) binding stoichiometry. These results suggest that SRK-015 binding stabilizes the latent conformation and limits the accessibility of protease cleavage sites within the prodomain. These findings shed light on approaches that specifically block the extracellular activation of growth factors by targeting their precursor forms.

Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme involved in ATP generation and critical for cancer metabolism. PKM2 is expressed in many human cancers and is regulated by complex mechanisms that promote tumor growth and proliferation. Therefore, it is considered an attractive therapeutic target for modulating tumor metabolism. Various stimuli allosterically regulate PKM2 by cycling it between highly active and less active states. Several small molecules activate PKM2 by binding to its intersubunit interface. Serine and cysteine serve as an activator and inhibitor of PKM2, respectively, by binding to its amino acid (AA)-binding pocket, which therefore represents a potential druggable site. Despite binding similarly to PKM2, how cysteine and serine differentially regulate this enzyme remains elusive. Using kinetic analyses, fluorescence binding, X-ray crystallography, and gel filtration experiments with asparagine, aspartate, and valine as PKM2 ligands, we examined whether the differences in the side-chain polarity of these AAs trigger distinct allosteric responses in PKM2. We found that Asn (polar) and Asp (charged) activate PKM2 and that Val (hydrophobic) inhibits it. The results also indicate that both Asn and Asp can restore the activity of Val-inhibited PKM2. AA-bound crystal structures of PKM2 displayed distinctive interactions within the binding pocket, causing unique allosteric effects in the enzyme. These structure-function analyses of AA-mediated PKM2 regulation shed light on the chemical requirements in the development of mechanism-based small-molecule modulators targeting the AA-binding pocket of PKM2 and provide broader insights into the regulatory mechanisms of complex allosteric enzymes.

Aldehyde oxidases (AOXs) are a small group of enzymes belonging to the larger family of molybdo-flavoenzymes, along with the well-characterized xanthine oxidoreductase. The two major types of reactions that are catalyzed by AOXs are the hydroxylation of heterocycles and the oxidation of aldehydes to their corresponding carboxylic acids. Different animal species have different complements of AOX genes. The two extremes are represented in humans and rodents; whereas the human genome contains a single active gene (AOX1), those of rodents, such as mice, are endowed with four genes (Aox1-4), clustering on the same chromosome, each encoding a functionally distinct AOX enzyme. It still remains enigmatic why some species have numerous AOX enzymes, whereas others harbor only one functional enzyme. At present, little is known about the physiological relevance of AOX enzymes in humans and their additional forms in other mammals. These enzymes are expressed in the liver and play an important role in the metabolisms of drugs and other xenobiotics. In this review, we discuss the expression, tissue-specific roles, and substrate specificities of the different mammalian AOX enzymes and highlight insights into their physiological roles.

β-1,3-d-Glucan is a ubiquitous glucose polymer produced by plants, bacteria, and most fungi. It has been used as a diagnostic tool in patients with invasive mycoses via a highly-sensitive reagent consisting of the blood coagulation system of horseshoe crab. However, no method is currently available for measuring β-1,6-glucan, another primary β-glucan structure of fungal polysaccharides. Herein, we describe the development of an economical and highly-sensitive and specific assay for β-1,6-glucan using a modified recombinant endo-β-1,6-glucanase having diminished glucan hydrolase activity. The purified β-1,6-glucanase derivative bound to the β-1,6-glucan pustulan with a KD of 16.4 nm. We validated the specificity of this β-1,6-glucan probe by demonstrating its ability to detect cell wall β-1,6-glucan from both yeast and hyphal forms of the opportunistic fungal pathogen Candida albicans, without any detectable binding to glucan lacking the long β-1,6-glucan branch. We developed a sandwich ELISA-like assay with a low limit of quantification for pustulan (1.5 pg/ml), and we successfully employed this assay in the quantification of extracellular β-1,6-glucan released by >250 patient-derived strains of different Candida species (including Candida auris) in culture supernatant in vitro. We also used this assay to measure β-1,6-glucan in vivo in the serum and in several organs in a mouse model of systemic candidiasis. Our work describes a reliable method for β-1,6-glucan detection, which may prove useful for the diagnosis of invasive fungal infections.

For successful infection of their hosts, pathogenic bacteria recognize host-derived signals that induce the expression of virulence factors in a spatiotemporal manner. The fulminating food-borne pathogen Vibrio vulnificus produces a cytolysin/hemolysin protein encoded by the vvhBA operon, which is a virulence factor preferentially expressed upon exposure to murine blood and macrophages. The Fe-S cluster containing transcriptional regulator IscR activates the vvhBA operon in response to nitrosative stress and iron starvation, during which the cellular IscR protein level increases. Here, electrophoretic mobility shift and DNase I protection assays revealed that IscR directly binds downstream of the vvhBA promoter PvvhBA, which is unusual for a positive regulator. We found that in addition to IscR, the transcriptional regulator HlyU activates vvhBA transcription by directly binding upstream of PvvhBA, whereas the histone-like nucleoid-structuring protein (H-NS) represses vvhBA by extensively binding to both downstream and upstream regions of its promoter. Of note, the binding sites of IscR and HlyU overlapped with those of H-NS. We further substantiated that IscR and HlyU outcompete H-NS for binding to the PvvhBA regulatory region, resulting in the release of H-NS repression and vvhBA induction. We conclude that concurrent antirepression by IscR and HlyU at regions both downstream and upstream of PvvhBA provides V. vulnificus with the means of integrating host-derived signal(s) such as nitrosative stress and iron starvation for precise regulation of vvhBA transcription, thereby enabling successful host infection.

The canonical pathway of eicosanoid production in most mammalian cells is initiated by phospholipase A2-mediated release of arachidonic acid, followed by its enzymatic oxidation resulting in a vast array of eicosanoid products. However, recent work has demonstrated that the major phospholipase in mitochondria, iPLA2γ (patatin-like phospholipase domain containing 8 (PNPLA8)), possesses sn-1 specificity, with polyunsaturated fatty acids at the sn-2 position generating polyunsaturated sn-2-acyl lysophospholipids. Through strategic chemical derivatization, chiral chromatographic separation, and multistage tandem MS, here we first demonstrate that human platelet-type 12-lipoxygenase (12-LOX) can directly catalyze the regioselective and stereospecific oxidation of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) and 2-arachidonoyl-lysophosphatidylethanolamine (2-AA-LPE). Next, we identified these two eicosanoid-lysophospholipids in murine myocardium and in isolated platelets. Moreover, we observed robust increases in 2-AA-LPC, 2-AA-LPE, and their downstream 12-LOX oxidation products, 12(S)-HETE-LPC and 12(S)-HETE-LPE, in calcium ionophore (A23187)-stimulated murine platelets. Mechanistically, genetic ablation of iPLA2γ markedly decreased the calcium-stimulated production of 2-AA-LPC, 2-AA-LPE, and 12-HETE-lysophospholipids in mouse platelets. Importantly, a potent and selective 12-LOX inhibitor, ML355, significantly inhibited the production of 12-HETE-LPC and 12-HETE-LPE in activated platelets. Furthermore, we found that aging is accompanied by significant changes in 12-HETE-LPC in murine serum that were also markedly attenuated by iPLA2γ genetic ablation. Collectively, these results identify previously unknown iPLA2γ-initiated signaling pathways mediated by direct 12-LOX oxidation of 2-AA-LPC and 2-AA-LPE. This oxidation generates previously unrecognized eicosanoid-lysophospholipids that may serve as biomarkers for age-related diseases and could potentially be used as targets in therapeutic interventions.

Lethal infections by strains of the highly-pathogenic avian influenza virus (HPAIV) H5N1 pose serious threats to both the poultry industry and public health worldwide. A lack of confirmed HPAIV epitopes recognized by cytotoxic T lymphocytes (CTLs) has hindered the utilization of CD8+ T-cell–mediated immunity and has precluded the development of effectively diversified epitope-based vaccination approaches. In particular, an HPAIV H5N1 CTL-recognized epitope based on the peptide MHC-I–β2m (pMHC-I) complex has not yet been designed. Here, screening a collection of selected peptides of several HPAIV strains against a specific pathogen-free pMHC-I (pBF2*1501), we identified a highly-conserved HPAIV H5N1 CTL epitope, named HPAIV–PA123–130. We determined the structure of the BF2*1501–PA123–130 complex at 2.1 Å resolution to elucidate the molecular mechanisms of a preferential presentation of the highly-conserved PA123–130 epitope in the chicken B15 lineage. Conformational characteristics of the PA123–130 epitope with a protruding Tyr-7 residue indicated that this epitope has great potential to be recognized by specific TCRs. Moreover, significantly increased numbers of CD8+ T cells specific for the HPAIV–PA123–130 epitope in peptide-immunized chickens indicated that a repertoire of CD8+ T cells can specifically respond to this epitope. We anticipate that the identification and structural characterization of the PA123–130 epitope reported here could enable further studies of CTL immunity against HPAIV H5N1. Such studies may aid in the development of vaccine development strategies using well-conserved internal viral antigens in chickens.

Inter-α-inhibitor is a proteoglycan essential for mammalian reproduction and also plays a less well-characterized role in inflammation. It comprises two homologous “heavy chains” (HC1 and HC2) covalently attached to chondroitin sulfate on the bikunin core protein. Before ovulation, HCs are transferred onto the polysaccharide hyaluronan (HA) to form covalent HC·HA complexes, thereby stabilizing an extracellular matrix around the oocyte required for fertilization. Additionally, such complexes form during inflammatory processes and mediate leukocyte adhesion in the synovial fluids of arthritis patients and protect against sepsis. Here using X-ray crystallography, we show that human HC1 has a structure similar to integrin β-chains, with a von Willebrand factor A domain containing a functional metal ion-dependent adhesion site (MIDAS) and an associated hybrid domain. A comparison of the WT protein and a variant with an impaired MIDAS (but otherwise structurally identical) by small-angle X-ray scattering and analytical ultracentrifugation revealed that HC1 self-associates in a cation-dependent manner, providing a mechanism for HC·HA cross-linking and matrix stabilization. Surprisingly, unlike integrins, HC1 interacted with RGD-containing ligands, such as fibronectin, vitronectin, and the latency-associated peptides of transforming growth factor β, in a MIDAS/cation-independent manner. However, HC1 utilizes its MIDAS motif to bind to and inhibit the cleavage of complement C3, and small-angle X-ray scattering–based modeling indicates that this occurs through the inhibition of the alternative pathway C3 convertase. These findings provide detailed structural and functional insights into HC1 as a regulator of innate immunity and further elucidate the role of HC·HA complexes in inflammation and ovulation.

β-Glucocerebrosidase (GBA) hydrolyzes glucosylceramide (GlcCer) to generate ceramide. Previously, we demonstrated that lysosomal GBA1 and nonlysosomal GBA2 possess not only GlcCer hydrolase activity, but also transglucosylation activity to transfer the glucose residue from GlcCer to cholesterol to form β-cholesterylglucoside (β-GlcChol) in vitro. β-GlcChol is a member of sterylglycosides present in diverse species. How GBA1 and GBA2 mediate β-GlcChol metabolism in the brain is unknown. Here, we purified and characterized sterylglycosides from rodent and fish brains. Although glucose is thought to be the sole carbohydrate component of sterylglycosides in vertebrates, structural analysis of rat brain sterylglycosides revealed the presence of galactosylated cholesterol (β-GalChol), in addition to β-GlcChol. Analyses of brain tissues from GBA2-deficient mice and GBA1- and/or GBA2-deficient Japanese rice fish (Oryzias latipes) revealed that GBA1 and GBA2 are responsible for β-GlcChol degradation and formation, respectively, and that both GBA1 and GBA2 are responsible for β-GalChol formation. Liquid chromatography–tandem MS revealed that β-GlcChol and β-GalChol are present throughout development from embryo to adult in the mouse brain. We found that β-GalChol expression depends on galactosylceramide (GalCer), and developmental onset of β-GalChol biosynthesis appeared to be during myelination. We also found that β-GlcChol and β-GalChol are secreted from neurons and glial cells in association with exosomes. In vitro enzyme assays confirmed that GBA1 and GBA2 have transgalactosylation activity to transfer the galactose residue from GalCer to cholesterol to form β-GalChol. This is the first report of the existence of β-GalChol in vertebrates and how β-GlcChol and β-GalChol are formed in the brain.