The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane anion channel that plays a key role in controlling transepithelial fluid movement. Excessive activation results in intestinal fluid loss during secretory diarrheas, whereas CFTR mutations underlie cystic fibrosis (CF). Anion permeability depends both on how well CFTR channels work (permeation/gating) and on how many are present at the membrane. Recently, treatments with two drug classes targeting CFTR—one boosting ion-channel function (potentiators) and the other increasing plasma membrane density (correctors)—have provided significant health benefits to CF patients. Here, we present an image-based fluorescence assay that can rapidly and simultaneously estimate both CFTR ion-channel function and the protein's proximity to the membrane. We monitor F508del-CFTR, the most common CF-causing variant, and confirm rescue by low temperature, CFTR-targeting drugs and second-site revertant mutation R1070W. In addition, we characterize a panel of 62 CF-causing mutations. Our measurements correlate well with published data (electrophysiology and biochemistry), further confirming validity of the assay. Finally, we profile effects of acute treatment with approved potentiator drug VX-770 on the rare-mutation panel. Mapping the potentiation profile on CFTR structures raises mechanistic hypotheses on drug action, suggesting that VX-770 might allow an open-channel conformation with an alternative arrangement of domain interfaces. The assay is a valuable tool for investigation of CFTR molecular mechanisms, allowing accurate inferences on gating/permeation. In addition, by providing a two-dimensional characterization of the CFTR protein, it could better inform development of single-drug and precision therapies addressing the root cause of CF disease.

Shao-En Ong
May 1, 2002; 1:376-386
Research

Jennifer L. Macdonald
May 1, 2005; 46:1061-1067
Methods

Ran Gu and Bing Gao
Math. Comp. 93 (), 2837-2860.
Abstract, references and article information

Carolyn S. Gordon and Michael R. Jablonski
Trans. Amer. Math. Soc. 377 (), 5673-5704.
Abstract, references and article information

Nathan Brownlowe, Alcides Buss, Daniel Gonçalves, Jeremy B. Hume, Aidan Sims and Michael F. Whittaker
Trans. Amer. Math. Soc. 377 (), 5513-5560.
Abstract, references and article information

Roland Donninger
Bull. Amer. Math. Soc. 61 (), 659-685.
Abstract, references and article information

Forty-six mathematical scientists have been named recipients of AMS-Simons Research Enhancement Grants for Primarily Undergraduate Institution (PUI) Faculty. Each awardee will receive $3,000 per year for three years. 

The grants foster and support research collaboration by full-time mid-career mathematicians at US institutions that do not offer a mathematics doctoral degree.

This year’s grant recipients hail from 42 institutions across 21 US states. The grants will support their research in several different areas, from number theory to applied mathematics.

This is the grant program’s second cohort, said Sarah Bryant, associate vice president of programs. “Over the first two years, we’ve worked with faculty from 75 different institutions, including 19 minority-serving institutions, which shows just how much this program is expanding and making an impact,” Bryant said. She noted that “in the first year, the grants supported 87 trips, helped produce 70 publications and preprints, and gave awardees the resources needed to collaborate and advance their work.”

The grant allows for any activities that will further the awardee’s research program. Expenses include but are not limited to conference participation, institute visits, collaboration travel (awardee or collaborator), computer equipment or software, family-care expenses, and teaching assistants.

Administration of the award by the grantee’s institution is required; annual discretionary funds for a grantee’s department and administrative funds for a grantee's institution will be available at the end of each grant year.

The grants are made possible through funding from the Simons Foundation and the American Mathematical Society (AMS), as well as Eve, Kirsten, Lenore, and Ada of the Menger family.

Applications for the next cohort are anticipated to open on MathPrograms.org on January 9, 2025. Visit the AMS website to view an informational PowerPoint or sign up to receive email updates about the program. Faculty who applied for but did not receive the 2023 or 2024 awards are encouraged to reapply if they are still eligible for the grant. 

Mammalian Asn-linked glycans are extensively processed as they transit the secretory pathway to generate diverse glycans on cell surface and secreted glycoproteins. Additional modification of the glycan core by α-1,6-fucose addition to the innermost GlcNAc residue (core fucosylation) is catalyzed by an α-1,6-fucosyltransferase (FUT8). The importance of core fucosylation can be seen in the complex pathological phenotypes of FUT8 null mice, which display defects in cellular signaling, development, and subsequent neonatal lethality. Elevated core fucosylation has also been identified in several human cancers. However, the structural basis for FUT8 substrate specificity remains unknown.Here, using various crystal structures of FUT8 in complex with a donor substrate analog, and with four distinct glycan acceptors, we identify the molecular basis for FUT8 specificity and activity. The ordering of three active site loops corresponds to an increased occupancy for bound GDP, suggesting an induced-fit folding of the donor-binding subsite. Structures of the various acceptor complexes were compared with kinetic data on FUT8 active site mutants and with specificity data from a library of glycan acceptors to reveal how binding site complementarity and steric hindrance can tune substrate affinity. The FUT8 structure was also compared with other known fucosyltransferases to identify conserved and divergent structural features for donor and acceptor recognition and catalysis. These data provide insights into the evolution of modular templates for donor and acceptor recognition among GT-B fold glycosyltransferases in the synthesis of diverse glycan structures in biological systems.

Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we studied the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that are derived from the closest germline (GL) sequence. Both analyzed fibril structures were seeded with ex-vivo amyloid fibrils purified from the explanted heart of this patient. We find that residues 11-42 and 69-102 adopt β-sheet conformation in patient protein fibrils. We identify arginine-49 as a key residue that forms a salt bridge to aspartate-25 in the patient protein fibril structure. In the germline sequence, this residue is replaced by a glycine. Fibrils from the GL protein and from the patient protein harboring the single point mutation R49G can be both heterologously seeded using patient ex-vivo fibrils. Seeded R49G fibrils show an increased heterogeneity in the C-terminal residues 80-102, which is reflected by the disappearance of all resonances of these residues. By contrast, residues 11-42 and 69-77, which are visible in the MAS solid-state NMR spectra, show 13Cα chemical shifts that are highly like patient fibrils. The mutation R49G thus induces a conformational heterogeneity at the C terminus in the fibril state, whereas the overall fibril topology is retained. These findings imply that patient mutations in FOR005 can stabilize the fibril structure.

Natalie Bruiners
Dec 1, 2020; 61:1617-1628
Research Articles

Functions of the gut microbiome have a growing number of implications for host metabolic health, with diet being one of the most significant influences on microbiome composition. Compelling links between diet and the gut microbiome suggest key roles for various macronutrients, including lipids, yet how individual classes of dietary lipids interact with the microbiome remains largely unknown. Sphingolipids are bioactive components of most foods and are also produced by prominent gut microbes. This makes sphingolipids intriguing candidates for shaping diet–microbiome interactions. Here, we used a click chemistry–based approach to track the incorporation of bioorthogonal dietary omega-alkynyl sphinganine (sphinganine alkyne [SAA]) into the murine gut microbial community (Bioorthogonal labeling). We identified microbial and SAA-specific metabolic products through fluorescence-based sorting of SAA-containing microbes (Sort), 16S rRNA gene sequencing to identify the sphingolipid-interacting microbes (Seq), and comparative metabolomics to identify products of SAA assimilation by the microbiome (Spec). Together, this approach, termed Bioorthogonal labeling-Sort-Seq-Spec (BOSSS), revealed that SAA assimilation is nearly exclusively performed by gut Bacteroides, indicating that sphingolipid-producing bacteria play a major role in processing dietary sphinganine. Comparative metabolomics of cecal microbiota from SAA-treated mice revealed conversion of SAA to a suite of dihydroceramides, consistent with metabolic activities of Bacteroides and Bifidobacterium. Additionally, other sphingolipid-interacting microbes were identified with a focus on an uncharacterized ability of Bacteroides and Bifidobacterium to metabolize dietary sphingolipids. We conclude that BOSSS provides a platform to study the flux of virtually any alkyne-labeled metabolite in diet–microbiome interactions.

The rise of drug-resistant tuberculosis poses a major risk to public health. Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. However, the underlying molecular mechanisms are unknown. In this study, we used an in vitro macrophage infection model to investigate simvastatin’s anti-tubercular activity by systematically inhibiting each branch of the mevalonate pathway and evaluating the effects of the branch-specific inhibitors on mycobacterial growth. The anti-tubercular activity of simvastatin used at clinically relevant doses specifically targeted the cholesterol biosynthetic branch rather than the prenylation branches of the mevalonate pathway. Using Western blot analysis and AMP/ATP measurements, we found that simvastatin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, the AMPK-mTORC1-TFEB axis, and Mycobacterium tuberculosis infection control, and uncover new anti-tubercular therapy targets.

Influenza A virus (IAV) mutates rapidly, resulting in antigenic drift and poor year-to-year vaccine effectiveness. One challenge in designing effective vaccines is that genetic mutations frequently cause amino acid variations in IAV envelope protein hemagglutinin (HA) that create new N-glycosylation sequons; resulting N-glycans cause antigenic shielding, allowing viral escape from adaptive immune responses. Vaccine candidate strain selection currently involves correlating antigenicity with HA protein sequence among circulating strains, but quantitative comparison of site-specific glycosylation information may likely improve the ability to design vaccines with broader effectiveness against evolving strains. However, there is poor understanding of the influence of glycosylation on immunodominance, antigenicity, and immunogenicity of HA, and there are no well-tested methods for comparing glycosylation similarity among virus samples. Here, we present a method for statistically rigorous quantification of similarity between two related virus strains that considers the presence and abundance of glycopeptide glycoforms. We demonstrate the strength of our approach by determining that there was a quantifiable difference in glycosylation at the protein level between WT IAV HA from A/Switzerland/9715293/2013 (SWZ13) and a mutant strain of SWZ13, even though no N-glycosylation sequons were changed. We determined site-specifically that WT and mutant HA have varying similarity at the glycosylation sites of the head domain, reflecting competing pressures to evade host immune response while retaining viral fitness. To our knowledge, our results are the first to quantify changes in glycosylation state that occur in related proteins of considerable glycan heterogeneity. Our results provide a method for understanding how changes in glycosylation state are correlated with variations in protein sequence, which is necessary for improving IAV vaccine strain selection. Understanding glycosylation will be especially important as we find new expression vectors for vaccine production, as glycosylation state depends greatly on the host species.

In-depth coverage of proteomic analysis could enhance our understanding to the mechanism of the protein functions. Unfortunately, many highly hydrophobic proteins and low-abundance proteins, which play critical roles in signaling networks, are easily lost during sample preparation, mainly attributed to the fact that very few extractants can simultaneously satisfy the requirements on strong solubilizing ability to membrane proteins and good enzyme compatibility. Thus, it is urgent to screen out ideal extractant from the huge compound libraries in a fast and effective way. Herein, by investigating the interior mechanism of extractants on the membrane proteins solubilization and trypsin compatibility, a molecular dynamics simulation system was established as complement to the experimental procedure to narrow down the scope of candidates for proteomics analysis. The simulation data shows that the van der Waals interaction between cation group of ionic liquid and membrane protein is the dominant factor in determining protein solubilization. In combination with the experimental data, 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) is on the shortlist for the suitable candidates from comprehensive aspects. Inspired by the advantages of C12Im-Cl, an ionic liquid-based filter-aided sample preparation (i-FASP) method was developed. Using this strategy, over 3,300 proteins were confidently identified from 10³ HeLa cells (~100 ng proteins) in a single run, an improvement of 53% over the conventional FASP method. Then the i-FASP method was further successfully applied to the label-free relative quantitation of human liver cancer and para-carcinoma tissues with obviously improved accuracy, reproducibility and coverage than the commonly used urea-based FASP method. The above results demonstrated that the i-FASP method could be performed as a versatile tool for the in-depth coverage proteomic analysis of biological samples.

Complex protein glycosylation occurs through biosynthetic steps in the secretory pathway that create macro- and microheterogeneity of structure and function.  Required for all life forms, glycosylation diversifies and adapts protein interactions with binding partners that underpin interactions at cell surfaces and pericellular and extracellular environments. Because these biological effects arise from heterogeneity of structure and function, it is necessary to measure their changes as part of the quest to understand nature.  Quite often, however, the assumption behind proteomics that post-translational modifications are discrete additions that can be modeled using the genome as a template does not apply to protein glycosylation.  Rather, it is necessary to quantify the glycosylation distribution at each glycosite and to aggregate this information into a population of mature glycoproteins that exist in a given biological system.  To date, mass spectrometric methods for assigning singly glycosylated peptides are well-established.  But it is necessary to quantify glycosylation heterogeneity accurately in order to gauge the alterations that occur during biological processes.  The task is to quantify the glycosylated peptide forms as accurately as possible and then apply appropriate bioinformatics algorithms to the calculation of micro- and macro-similarities.  In this review, we summarize current approaches for protein quantification as they apply to this glycoprotein similarity problem.

Exploring Transatlantic Responses to Far-right Populism in Europe: Simulation Exercise Research paper sysadmin 1 May 2018

A new paper summarizes the findings of a recent simulation exercise exploring how governments on both sides of the Atlantic might respond to a descent towards populist authoritarianism in an EU member state.

Summary

• To better understand how governments on both sides of the Atlantic might respond to a descent towards populist authoritarianism in an EU member state, Chatham House organized a simulation event involving a group of experts drawn from the public sector, academia and NGOs.
• Simulation exercises enable the testing and modelling of the responses of different actors when presented with specific situations; participants’ interactions in a given set of circumstances are explored, and patterns of negotiation are captured and analysed.
• In this simulation, European, US and multilateral representatives were given the task of managing relations with Baltia, a fictional Eastern European state on the verge of electing a far-right nationalist, Eurosceptic government. They were then challenged to manage their relationship with Baltia after it had elected such a government, which was pushing for a ‘leave’ vote in a planned referendum on the country’s continued EU membership.
• The simulation highlighted a number of issues:
  • Limited instruments are available to liberal democratic governments where there is cause for concern regarding the outcome of an election in an allied country. There are relatively few tools at the disposal of governments to support political allies, or to prevent outcomes that are perceived as threatening democratic norms. The simulation reinforced the view that interventionist moves, either from the European Commission or from individual national governments, would be more likely to come in response to an unfavourable development rather than pre-emptively.
  • The EU, and caucuses of European states, are the main international interlocutors in this type of political crisis involving an EU member state. The US opted to play a limited role in the negotiations; the same was largely true for NATO, aside from its action in sharing intelligence about a potential coup in Baltia. France and Germany formed a natural working partnership, taking meetings together and coordinating policies first before discussing them with a wider European circle, although their positions did not always align.
  • The UK’s capacity to shape the outcome of collective EU discussions appeared more restricted, while Brexit also seemed to shape the response of other EU states to the developing situation in Baltia. Although member states were undoubtedly reluctant to see another country go down this route, they were also resolute in demonstrating a unity of approach and limited flexibility in the face of the new populist government’s attempt to divide them.

MLB Network's countdown of baseball's best players at each position continued with the third installment of the "Top 10 Right Now!" series, featuring the game's top left and center fielders.

PITTSBURGH, Sept. 24, 2024 — Ansys and TSMC today announced a successful pilot with Microsoft that significantly speeds-up the simulation and analysis of silicon photonic components. Together, the companies achieved […]

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Oct. 25, 2024 — Aggregation of proteins underlies many human disorders, including Alzheimer’s. Teams from the New Jersey Institute of Technology and Princeton University joined forces to study how the amyloid […]

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Microsoft and Quantinuum reported the ability to create 12 logical qubits on Quantinuum’s H2 trapped ion system this week and also reported using two logical qubits on an H1 system […]

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Distinctive meanings for a word like “risk” can have a big impact on public messaging, especially when it comes to issues like climate change

One of the most difficult problems with quantum computing relates to increasing the size of the quantum computer. Researchers globally are seeking to solve this “challenge of scale.” To bring […]

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Anthony Atala | Smithsonian Magazine’s 2016 American Ingenuity Award Winner for Life Sciences The director of the Wake Forest Institute for Regenerative Medicine, Atala is a surgeon and leading expert in creating living human tissues and organs to replace those that are defective or damaged. He has spent the past decade attempting to construct living organs using 3-D printing technology. Atala implanted the world’s first laboratory-grown organ into a human in 1999 and, this year, he and his colleagues “printed” cartilage, bone and muscle tissue before successfully implanting them into a lab animal. That’s a crucial first step toward Atala’s long-term goal of overcoming the dire shortage of donated organs with custom-made body parts. Read more about Atala's work: http://smithmag.co/SiiV2J | #IngenuityAwards And more about the American Ingenuity Awards: http://smithmag.co/77xPqy

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