eIF3d: A driver of noncanonical cap-dependent translation of specific mRNAs and a trigger of biological/pathological processes.

Eukaryotic initiation factor 3d (eIF3d), a known RNA-binding subunit of the eIF3 complex, is a 66 to 68-kDa protein with an RNA-binding motif and a cap-binding domain. Compared with other eIF3 subunits, eIF3d is relatively understudied. However, recent progress in studying eIF3d has revealed a number of intriguing findings on its role in maintaining eIF3 complex integrity, global protein synthesis, and in biological and pathological processes. It has also been reported that eIF3d has noncanonical functions in regulating translation of a subset of mRNAs by binding to 5'-UTRs or interacting with other proteins independent of the eIF3 complex and additional functions in regulating protein stability. The noncanonical regulation of mRNA translation or protein stability may contribute to the role of eIF3d in biological processes such as metabolic stress adaptation and in disease onset and progression including severe acute respiratory syndrome coronavirus 2 infection, tumorigenesis, and acquired immune deficiency syndrome. In this review, we critically evaluate the recent studies on these aspects of eIF3d and assess prospects in understanding the function of eIF3d in regulating protein synthesis and in biological and pathological processes.

A Functional Switch Between Asperfumene and Fusicoccadiene Synthase and Entrance to Asperfumene Biosynthesis through a Vicinal Deprotonation-Reprotonation Process.

The diterpene synthase AfAS was identified from Aspergillus fumigatiaffinis. Its amino acid sequence and - according to a structural model - active site architecture are highly similar to those of the fusicocca-2,10(14)-diene synthase PaFS, but AfAS produces a structurally much more complex diterpene with a novel 6-5-5-5 tetracyclic skeleton called asperfumene. The cyclisation mechanism of AfAS was elucidated through isotopic labelling experiments and DFT calculations. The reaction cascade proceeds in its initial steps through similar intermediates as for the PaFS cascade, but then diverges through an unusual vicinal deprotonation-reprotonation process that triggers a skeletal rearrangement at the entrance to the steps leading to the unique asperfumene skeleton. The structural model revealed only one major difference between the active sites: The PaFS residue F65 is substituted by I65 in AfAS. Intriguingly, site-directed mutagenesis experiments with both diterpene synthases revealed that position 65 serves as a bidirectional functional switch for the biosynthesis of tetracyclic asperfumene versus structurally less complex diterpenes.

Translation initiation factor eIF3a regulates glucose metabolism and cell proliferation via promoting small GTPase Rheb synthesis and AMPK activation.

Eukaryotic translation initiation factor 3 subunit A (eIF3a), the largest subunit of the eIF3 complex, has been shown to be overexpressed in malignant cancer cells, potentially making it a proto-oncogene. eIF3a overexpression can drive cancer cell proliferation but contributes to better prognosis. While its contribution to prognosis was previously shown to be due to its function in suppressing synthesis of DNA damage repair proteins, it remains unclear how eIF3a regulates cancer cell proliferation. In this study, we show using genetic approaches that eIF3a controls cell proliferation by regulating glucose metabolism via the phosphorylation and activation of AMP-activated protein kinase alpha (AMPKα) at Thr172 in its kinase activation loop. We demonstrate that eIF3a regulates AMPK activation mainly by controlling synthesis of the small GTPase Rheb, largely independent of the well-known AMPK upstream liver kinase B1 and Ca2+/calmodulin-dependent protein kinase kinase 2, and also independent of mammalian target of rapamycin signaling and glucose levels. Our findings suggest that glucose metabolism in and proliferation of cancer cells may be translationally regulated via a novel eIF3a-Rheb-AMPK signaling axis.

A novel survivin dimerization inhibitor without a labile hydrazone linker induces spontaneous apoptosis and synergizes with docetaxel in prostate cancer cells.

Survivin, a member of the inhibitor of apoptosis protein family, exists as a homodimer and is aberrantly upregulated in a wide spectrum of cancers. It was thought to be an ideal target due to its lack of expression in most adult normal tissues and importance in cancer cell survival. However, it has been challenging to target survivin due to its "undruggable" nature. We previously attempted to target its dimerization domain with a hypothesis that inhibiting survivin dimerization would promote its degradation in proteasome, which led to identification of a lead small-molecule inhibitor, LQZ-7F. LQZ-7F consists of a flat tetracyclic aromatic core with labile hydrazone linking a 1,2,5-oxadiazole moiety. In this study, we tested the hypothesis that LQZ-7F could be developed as a prodrug because the labile hydrazone linker could be hydrolyzed, releasing the tetracyclic aromatic core. To this end, we synthesized the tetracyclic aromatic core (LQZ-7F1) using reported procedure and tested LQZ-7F1 for its biological activities. Here we show that LQZ-7F1 has a significantly improved potency with submicromolar IC50's and induces spontaneous apoptosis in prostate cancer cells. It also more effectively inhibits survivin dimerization and induces survivin degradation in a proteasome-dependent manner than LQZ-7F. We also show that the combination of LQZ-7F1 and docetaxel have strong synergism in inhibiting prostate cancer cell survival. Together, we conclude that the hydrazone linker with the oxadiazole tail is dispensable for survivin inhibition and the survivin dimerization inhibitor, LQZ-7F, may be developed as a prodrug for prostate cancer treatment and to overcome docetaxel resistance.

High Thermoelectric Performance of In4Se3-Based Materials and the Influencing Factors.

Materials that can directly convert electricity into heat, i.e., thermoelectric materials, have attracted renewed attention globally for sustainable energy applications. As one of the state-of-the-art thermoelectric materials, In4Se3 features an interesting crystal structure of quasi-two-dimensional sheets comprising In/Se chains that provide a platform to achieve a Peierls distortion and support a charge density wave instability. Single-crystal In4Se3-δ (δ = 0.65) shows strong anisotropy in its thermoelectric properties with a very high ZT of 1.48 at 705 K in the b-c plane (one of the highest values for an n-type thermoelectric material to date) but a much lower ZT of approximately 0.5 in the a-b plane. Because of the random dispersion of grains and the grain boundary effect, the electrical transport properties of polycrystalline In4Se3 are poor, which is the main impediment to improve their performance. The In4-site in the In4Se3 unit cell is substitutional for dopants such as Pb, which increases the carrier concentration by 2 orders of magnitude and the electrical conductivity to 143 S/cm. Furthermore, the electrical conductivity markedly increases to approximately 160 S/cm when Cu is doped into the interstitial site but remains as low as 30 S/cm with In1/In2/In3-site dopants, e.g., Ni, Zn, Ga, and Sn. In particular, the In4-site dopant ytterbium introduces a pinning level that highly localizes the charge carriers; thus, the electrical conductivity is maintained within an order of magnitude of 30 S/cm. Meanwhile, ytterbium also creates resonance states around the Fermi level that increase the Seebeck coefficient to -350 µV/K, the highest value at the ZT peak. However, the maximum solubility of the dopant may be limited by the Se-vacancy concentration. In addition, a Se vacancy also destroys the regular lattice vibrations and weakens phonon transport. Finally, nanoinclusions can effectively scatter the middle wavelength phonons, resulting in a decrease in the lattice thermal conductivity. Because of the multiple-dopant strategy, polycrystalline materials are competitive with single crystals regarding ZT values; for instance, Pb/Sn-co-doped In4Pb0.01Sn0.04Se3 has ZT = 1.4 at 733 K, whereas In4Se2.95(CuI)0.01 has ZT = 1.34 at 723 K. These properties illustrate the promise of polycrystalline In4Se3-based materials for various applications. Finally, the ZT values of all single crystalline and polycrystalline In4Se3 materials have been summarized as a function of the doping strategy applied at the different lattice sites. Additionally, the correlations between the electrical conductivity and the Seebeck coefficient of all the polycrystalline materials are presented. These insights may provide new ideas in the search for and selection of new thermoelectric compounds in the In/Se and related In/Te, Sn/Se, and Sn/Te systems.

FASN regulates cellular response to genotoxic treatments by increasing PARP-1 expression and DNA repair activity via NF-κB and SP1.

Fatty acid synthase (FASN), the sole cytosolic mammalian enzyme for de novo lipid synthesis, is crucial for cancer cell survival and associates with poor prognosis. FASN overexpression has been found to cause resistance to genotoxic insults. Here we tested the hypothesis that FASN regulates DNA repair to facilitate survival against genotoxic insults and found that FASN suppresses NF-κB but increases specificity protein 1 (SP1) expression. NF-κB and SP1 bind to a composite element in the poly(ADP-ribose) polymerase 1 (PARP-1) promoter in a mutually exclusive manner and regulate PARP-1 expression. Up-regulation of PARP-1 by FASN in turn increases Ku protein recruitment and DNA repair. Furthermore, lipid deprivation suppresses SP1 expression, which is able to be rescued by palmitate supplementation. However, lipid deprivation or palmitate supplementation has no effect on NF-κB expression. Thus, FASN may regulate NF-κB and SP1 expression using different mechanisms. Altogether, we conclude that FASN regulates cellular response against genotoxic insults by up-regulating PARP-1 and DNA repair via NF-κB and SP1.

Diagnostic accuracy of interleukin 27 for tuberculous pleural effusion: two prospective studies and one meta-analysis.

BACKGROUND: Accurate differentiating diagnosis is essential for choosing treatment for exudative pleural effusions. OBJECTIVE: To establish the diagnostic accuracy of interleukin 27 for tuberculous pleural effusion (TPE). METHODS: First, the concentrations of pleural interleukin 27, interferon-gamma and adenosine deaminase were compared between 51 patients with TPE and 103 with non-TPEs (Beijing cohort), and their diagnostic values were evaluated. These were further verified in another independent population (Wuhan cohort, n=120). In the second part of the study, we performed a meta-analysis. RESULTS: With a cut-off value of 591.4 ng/L in the Beijing cohort, the area under the curve, sensitivity, specificity, positive predictive value and negative predictive value of interleukin 27 to diagnose TPE were 0.983 (95% CI 0.947 to 0.997), 96.1% (86.5% to 99.5%), 99.0% (94.7% to 100%), 98.0 (89.4 to 99.9) and 98.1 (93.3 to 99.8), respectively. Excellent diagnostic accuracy of interleukin 27 was also found in the Wuhan cohort and was further confirmed in the meta-analysis. The diagnostic performance of interleukin 27 was comparable to that of interferon-gamma and was more accurate than that of adenosine deaminase. Since the post-test probability of a negative result was always <0.1%, a negative test was considered to exclude TPE in all tuberculosis prevalence settings. CONCLUSIONS: Interleukin 27 can be used to diagnose TPE in a high prevalence setting, and a negative result can also be reliably used to rule out TPE in all prevalence settings.

Determinants of 14-3-3σ protein dimerization and function in drug and radiation resistance.

Many proteins exist and function as homodimers. Understanding the detailed mechanism driving the homodimerization is important and will impact future studies targeting the "undruggable" oncogenic protein dimers. In this study, we used 14-3-3σ as a model homodimeric protein and performed a systematic investigation of the potential roles of amino acid residues in the interface for homodimerization. Unlike other members of the conserved 14-3-3 protein family, 14-3-3σ prefers to form a homodimer with two subareas in the dimeric interface that has 180° symmetry. We found that both subareas of the dimeric interface are required to maintain full dimerization activity. Although the interfacial hydrophobic core residues Leu(12) and Tyr(84) play important roles in 14-3-3σ dimerization, the non-core residue Phe(25) appears to be more important in controlling 14-3-3σ dimerization activity. Interestingly, a similar non-core residue (Val(81)) is less important than Phe(25) in contributing to 14-3-3σ dimerization. Furthermore, dissociating dimeric 14-3-3σ into monomers by mutating the Leu(12), Phe(25), or Tyr(84) dimerization residue individually diminished the function of 14-3-3σ in resisting drug-induced apoptosis and in arresting cells at G2/M phase in response to DNA-damaging treatment. Thus, dimerization appears to be required for the function of 14-3-3σ.

Insight into conformational change for 14-3-3σ protein by molecular dynamics simulation.

14-3-3σ is a member of a highly conserved family of 14-3-3 proteins that has a double-edged sword role in human cancers. Former reports have indicated that the 14-3-3 protein may be in an open or closed state. In this work, we found that the apo-14-3-3σ is in an open state compared with the phosphopeptide bound 14-3-3σ complex which is in a more closed state based on our 80 ns molecular dynamics (MD) simulations. The interaction between the two monomers of 14-3-3σ in the open state is the same as that in the closed state. In both open and closed states, helices A to D, which are involved in dimerization, are stable. However, large differences are found in helices E and F. The hydrophobic contacts and hydrogen bonds between helices E and G in apo-14-3-3σ are different from those in the bound 14-3-3σ complex. The restrained and the mutated (Arg56 or Arg129 to alanine) MD simulations indicate that the conformation of four residues (Lys49, Arg56, Arg129 and Tyr130) may play an important role to keep the 14-3-3σ protein in an open or closed state. These results would be useful to evaluate the 14-3-3σ protein structure-function relationship.

Gap-App: A sex-distinct AI-based predictor for pancreatic ductal adenocarcinoma survival as a web application open to patients and physicians.

In this study, using RNA-Seq gene expression data and advanced machine learning techniques, we identified distinct gene expression profiles between male and female pancreatic ductal adenocarcinoma (PDAC) patients. Building upon this insight, we developed sex-specific 3-year survival predictive models along with a single comprehensive model. These sex-specific models outperformed the single general model despite the smaller sample sizes. We further refined our models by using the most important features extracted from these initial models. The refined sex-specific predictive models achieved improved accuracies of 92.62% for males and 91.96% for females, respectively, versus an accuracy of 87.84% from the refined comprehensive model, further highlighting the value of sex-specific analysis. Based on these findings, we created Gap-App, a web application that enables the use of individual gene expression profiles combined with sex information for personalized survival predictions. Gap-App, the first online tool aiming to bridge the gap between complex genomic data and clinical application and facilitating more precise and individualized cancer care, marks a significant advancement in personalized prognosis. The study not only underscores the importance of acknowledging sex differences in personalized prognosis, but also sets the stage for the shift from traditional one-size-fits-all to more personalized and targeted medicine. The GAP-App service is freely available at www.gap-app.org.

Smart exosomes enhance PDAC targeted therapy.

Exosomes continue to attract interest as a promising nanocarrier drug delivery technology. They are naturally derived nanoscale extracellular vesicles with innate properties well suited to shuttle proteins, lipids, and nucleic acids between cells. Nonetheless, their clinical utility is currently limited by several major challenges, such as their inability to target tumor cells and a high proportion of clearance by the mononuclear phagocyte system (MPS) of the liver and spleen. To overcome these limitations, we developed "Smart Exosomes" that co-display RGD and CD47p110-130 through CD9 engineering (ExoSmart). The resultant ExoSmart demonstrates enhanced binding capacity to αvß3 on pancreatic ductal adenocarcinoma (PDAC) cells, resulting in amplified cellular uptake in in vitro and in vivo models and increased chemotherapeutic efficacies. Simultaneously, ExoSmart significantly reduced liver and spleen clearance of exosomes by inhibiting macrophage phagocytosis via CD47p110-130 interaction with signal regulatory proteins (SIRPα) on macrophages. These studies demonstrate that an engineered exosome drug delivery system increases PDAC therapeutic efficacy by enhancing active PDAC targeting and prolonging circulation times, and their findings hold tremendous translational potential for cancer therapy while providing a concrete foundation for future work utilizing novel peptide-engineered exosome strategies.

Small Molecule Inhibitors Targeting the "Undruggable" Survivin: The Past, Present, and Future from a Medicinal Chemist's Perspective.

Survivin, a homodimeric protein and a member of the IAP family, plays a vital function in cell survival and cycle progression by interacting with various proteins and complexes. Its expression is upregulated in cancers but not detectable in normal tissues. Thus, it has been regarded and validated as an ideal cancer target. However, survivin is "undruggable" due to its lack of enzymatic activities or active sites for small molecules to bind/inhibit. Academic and industrial laboratories have explored different strategies to overcome this hurdle over the past two decades, with some compounds advanced into clinical testing. These strategies include inhibiting survivin expression, its interaction with binding partners and homodimerization. Here, we provide comprehensive analyses of these strategies and perspective on different small molecule survivin inhibitors to help drug discovery targeting "undruggable" proteins in general and survivin specifically with a true survivin inhibitor that will prevail in the foreseeable future.

Proline Isomerization: From the Chemistry and Biology to Therapeutic Opportunities.

Proline isomerization, the process of interconversion between the cis- and trans-forms of proline, is an important and unique post-translational modification that can affect protein folding and conformations, and ultimately regulate protein functions and biological pathways. Although impactful, the importance and prevalence of proline isomerization as a regulation mechanism in biological systems have not been fully understood or recognized. Aiming to fill gaps and bring new awareness, we attempt to provide a wholistic review on proline isomerization that firstly covers what proline isomerization is and the basic chemistry behind it. In this section, we vividly show that the cause of the unique ability of proline to adopt both cis- and trans-conformations in significant abundance is rooted from the steric hindrance of these two forms being similar, which is different from that in linear residues. We then discuss how proline isomerization was discovered historically followed by an introduction to all three types of proline isomerases and how proline isomerization plays a role in various cellular responses, such as cell cycle regulation, DNA damage repair, T-cell activation, and ion channel gating. We then explore various human diseases that have been linked to the dysregulation of proline isomerization. Finally, we wrap up with the current stage of various inhibitors developed to target proline isomerases as a strategy for therapeutic development.

Dearomatizing [2+2+1] Spiroannulation of Indoles with Alkynes.

A palladium-catalyzed dearomatizing [2+2+1] spiroannulation of indoles with two molecular internal alkynes is developed in the presence of Cu(OAc)2/O2 as the oxidant, in which a domino sequence including C-H activation of indole followed by consecutive Heck reactions is involved. A range of 3,3'-spiroindolines bearing tetrasubstituted cyclopentadiene moieties and exocyclic C═C bonds at C2 are obtained in moderate to excellent yields.

Reversible promoter demethylation of PDGFD confers gemcitabine resistance through STAT3 activation and RRM1 upregulation.

Drug resistance is a major problem in cancer treatment with traditional or targeted therapeutics. Gemcitabine is approved for several human cancers and the first line treatment for locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC). However, gemcitabine resistance frequently occurs and is a major problem in successful treatments of these cancers and the mechanism of gemcitabine resistance remains largely unknown. In this study, we identified 65 genes that had reversible methylation changes in their promoters in gemcitabine resistant PDAC cells using whole genome Reduced Representation Bisulfite Sequencing analyses. One of these genes, PDGFD, was further studied in detail for its reversible epigenetic regulation in expression and shown to contribute to gemcitabine resistance in vitro and in vivo via stimulating STAT3 signaling in both autocrine and paracrine manners to upregulate RRM1 expression. Analyses of TCGA datasets showed that PDGFD positively associates with poor outcome of PDAC patients. Together, we conclude that the reversible epigenetic upregulation plays an important role in gemcitabine resistance development and targeting PDGFD signaling alleviates gemcitabine resistance for PDAC treatment.

Do products from healthier vending machines on a university campus sell?

ObjectiveThis is a cross-sectional study that compares the sales of "healthy" and "unhealthy" vending machines following the introduction of healthier vending machines on a university campus. Method: Healthy ("green" and "amber" category), competitively priced snacks and beverages in vending machines (n = 4) called Grab Goodness were placed alongside standard vending machines (n = 11). The monthly sales data from all vending machines were captured electronically for 20 months. Results: Assortment of snacks and beverages offered by standard vending machines were of low nutritional quality, with only 16% of all products categorized as "green." The new Grab Goodness machines accounted for 28% of all vending machine purchases over 20 months, and 50% of all products purchased through these machines were "green" category items. Conclusions: The purchases of healthier snack options demonstrate encouraging patterns that support more nutritious and healthy alternatives in vending machines.

Ag9GaSe6: high-pressure-induced Ag migration causes thermoelectric performance irreproducibility and elimination of such instability.

The argyrodite Ag9GaSe6 is a newly recognized high-efficiency thermoelectric material with an ultralow thermal conductivity; however, liquid-like Ag atoms are believed to cause poor stability and performance irreproducibility, which was evidenced even after the 1st measurement run. Herein, we demonstrate the abovementioned instability and irreproducibility are caused by standard thermoelectric sample hot-pressing procedure, during which high pressure promotes the 3-fold-coordinated Ag atoms migrate to 4-fold-coordinated sites with higher-chemical potentials. Such instability can be eliminated by a simple annealing treatment, driving the metastable Ag atoms back to the original sites with lower-chemical potentials as revealed by the valence band X-ray photoelectron chemical potential spectra and single crystal X-ray diffraction data. Furthermore, the hot-pressed-annealed samples exhibit great stability and TE property repeatability. Such a stability and repeatability has never been reported before. This discovery will give liquid-like materials great application potential.

eIF3a regulation of mTOR signaling and translational control via HuR in cellular response to DNA damage.

eIF3a (eukaryotic translation initiation factor 3a), a subunit of the eIF3 complex, has been suggested to play a regulatory role in protein synthesis and in cellular response to DNA-damaging treatments. S6K1 is an effector and a mediator of mTOR complex 1 (mTORC1) in regulating protein synthesis and integrating diverse signals into control of cell growth and response to stress. Here, we show that eIF3a regulates S6K1 activity by inhibiting mTORC1 kinase via regulating Raptor synthesis. The regulation of Raptor synthesis is via eIF3a interaction with HuR (human antigen R) and binding of the eIF3a-HuR complex to the 5'-UTR of Raptor mRNA. Furthermore, mTORC1 may mediate eIF3a function in cellular response to cisplatin by regulating synthesis of NER proteins and NER activity. Taken together, we conclude that the mTOR signaling pathway may also be regulated by translational control and mediate eIF3a regulation of cancer cell response to cisplatin by regulating NER protein synthesis.

Prolyl Isomerization-Mediated Conformational Changes Define ATR Subcellular Compartment-Specific Functions.

ATR is a PI3K-like kinase protein, regulating checkpoint responses to DNA damage and replication stress. Apart from its checkpoint function in the nucleus, ATR actively engages in an antiapoptotic role at mitochondria following DNA damage. The different functions of ATR in the nucleus and cytoplasm are carried out by two prolyl isomeric forms of ATR: trans- and cis-ATR, respectively. The isomerization occurs at the Pin1 Ser428-Pro429 motif of ATR. Here, we investigated the structural basis of the subcellular location-specific functions of human ATR. Using a mass spectrometry-based footprinting approach, the surface accessibility of ATR lysine residues to sulfo-NHS-LC-biotin modification was monitored and compared between the cis- and the trans-isomers. We have identified two biotin-modified lysine residues, K459 and K469, within the BH3-like domain of cis-ATR that were not accessible in trans-ATR, indicating a conformational change around the BH3 domain between cis- and trans-ATR. The conformational alteration also involved the N-terminal domain and the middle HEAT domain. Moreover, experimental results from an array of complementary assays show that cis-ATR with the accessible BH3 domain was able to bind to tBid while trans-ATR could not. In addition, both cis- and trans-ATR can directly form homodimers via their C-terminal domains without ATRIP, while nuclear (trans-ATR) in the presence of ATRIP forms dimer-dimer complexes involving both N- and C-termini of ATR and ATRIP after UV. Structural characteristics around the Ser428-Pro429 motif and the BH3 domain region are also analyzed by molecular modeling and dynamics simulation. In support, cis conformation was found to be significantly more energetically favorable than trans at the Ser428-Pro429 bond in a 20-aa wild-type ATR peptide. Taken together, our results suggest that the isomerization-induced structural changes of ATR define both its subcellular location and compartment-specific functions and play an essential role in promoting cell survival and DNA damage responses.