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N6-methyladenosine (m6A) is a fundamentally important RNA modification for gene regulation, whose function is achieved through m6A readers. However, whether and how m6A readers play regulatory roles during fruit ripening and quality formation remains unclear. Here, we characterized SlYTH2 as a tomato m6A reader protein and profiled the binding sites of SlYTH2 at the transcriptome-wide level. SlYTH2 undergoes liquid-liquid phase separation and promotes RNA-protein condensate formation. The target mRNAs of SlYTH2, namely m6A-modified SlHPL and SlCCD1B associated with volatile synthesis, are enriched in SlYTH2-induced condensates. Through polysome profiling assays and proteomic analysis, we demonstrate that knockout of SlYTH2 expedites the translation process of SlHPL and SlCCD1B, resulting in augmented production of aroma-associated volatiles. This aroma enrichment significantly increased consumer preferences for CRISPR-edited fruit over wild type. These findings shed light on the underlying mechanisms of m6A in plant RNA metabolism and provided a promising strategy to generate fruits that are more attractive to consumers. [ABSTRACT FROM AUTHOR]

Seamless integration of microstructures and circuits on three-dimensional (3D) complex surfaces is of significance and is catalyzing the emergence of many innovative 3D curvy electronic devices. However, patterning fine features on arbitrary 3D targets remains challenging. Here, we propose a facile charge-driven electrohydrodynamic 3D microprinting technique that allows micron- and even submicron-scale patterning of functional inks on a couple of 3D-shaped dielectrics via an atmospheric-pressure cold plasma jet. Relying on the transient charging of exposed sites arising from the weakly ionized gas jet, the specified charge is programmably deposited onto the surface as a virtual electrode with spatial and time spans of ~mm in diameter and ~µs in duration to generate a localized electric field accordantly. Therefore, inks with a wide range of viscosities can be directly drawn out from micro-orifices and deposited on both two-dimensional (2D) planar and 3D curved surfaces with a curvature radius down to ~1 mm and even on the inner wall of narrow cavities via localized electrostatic attraction, exhibiting a printing resolution of ~450 nm. In addition, several conformal electronic devices were successfully printed on 3D dielectric objects. Self-aligned 3D microprinting, with stacking layers up to 1400, is also achieved due to the electrified surfaces. This microplasma-induced printing technique exhibits great advantages such as ultrahigh resolution, excellent compatibility of inks and substrates, antigravity droplet dispersion, and omnidirectional printing on 3D freeform surfaces. It could provide a promising solution for intimately fabricating electronic devices on arbitrary 3D surfaces. [ABSTRACT FROM AUTHOR]

In temperate and boreal regions, perennial plants adapt their annual growth cycle to the change of seasons. In natural forests, juvenile seedlings usually display longer growth seasons compared to adult trees to ensure their establishment and survival under canopy shade. However, how trees adjust their annual growth according to their age is not known. In this study, we show that age-dependent seasonal growth cessation is genetically controlled and found that the miR156-SPL3/5 module, a key regulon of vegetative phase change (VPC), also triggers age-dependent growth cessation in Populus trees. We show that miR156 promotes shoot elongation during vegetative growth, and its targets SPL3/5s function in the same pathway but as repressors. We find that the miR156-SPL3/5s regulon controls growth cessation in both leaves and shoot apices and through multiple pathways, but with a different mechanism compared to how the miR156-SPL regulon controls VPC in annual plants. Taken together, our results reveal an age-dependent genetic network in mediating seasonal growth cessation, a key phenological process in the climate adaptation of perennial trees. [ABSTRACT FROM AUTHOR]

Nanosized zero-valent iron (nZVI) is a promising persulfate (PS) activator, however, its structurally dense oxide shell seriously inhibited electrons transfer for O-O bond cleavage of PS. Herein, we introduced sulfidation and phosphorus-doped biochar for breaking the pristine oxide shell with formation of FeS and FePO4-containing mixed shell. In this case, the faster diffusion rate of iron atoms compared to shell components triggered multiple Kirkendall effects, causing inward fluxion of vacancies with further coalescing into radial nanocracks. Exemplified by trichloroethylene (TCE) removal, such a unique "lemon-slice-like" nanocrack structure favored fast outward transfer of electrons and ferrous ions across the mixed shell to PS activation for high-efficient generation and utilization of reactive species, as evidenced by effective dechlorination (90.6%) and mineralization (85.4%) of TCE. O2·- contributed most to TCE decomposition, moreover, the SnZVI@PBC gradually became electron-deficient and thus extracted electrons from TCE with achieving nonradical-based degradation. Compared to nZVI/PS process, the SnZVI@PBC/PS system could significantly reduce catalyst dosage (87.5%) and PS amount (68.8%) to achieve nearly complete TCE degradation, and was anti-interference, stable, and pH-universal. This study advanced mechanistic understandings of multiple Kirkendall effects-triggered nanocrack formation on nZVI with corresponding rational design of Fenton-like catalysts for organics degradation. [ABSTRACT FROM AUTHOR]

Most mammalian cells have an intrinsic circadian clock that coordinates metabolic activity with the daily rest and wake cycle. The circadian clock is known to regulate cell differentiation, but how continuous daily oscillations of the internal clock can control a much longer, multiday differentiation process is not known. Here, we simultaneously monitor circadian clock and adipocyte-differentiation progression live in single cells. Strikingly, we find a bursting behavior in the cell population whereby individual preadipocytes commit to differentiate primarily during a 12-h window each day, corresponding to the time of rest. Daily gating occurs because cells irreversibly commit to differentiate within only a few hours, which is much faster than the rest phase and the overall multiday differentiation process. The daily bursts in differentiation commitment result from a differentiation-stimulus driven variable and slow increase in expression of PPARG, the master regulator of adipogenesis, overlaid with circadian boosts in PPARG expression driven by fast, clock-driven PPARG regulators such as CEBPA. Our finding of daily bursts in cell differentiation only during the circadian cycle phase corresponding to evening in humans is broadly relevant, given that most differentiating somatic cells are regulated by the circadian clock. Having a restricted time each day when differentiation occurs may open therapeutic strategies to use timed treatment relative to the clock to promote tissue regeneration. [ABSTRACT FROM AUTHOR]

Chronic infection with liver flukes (such as Clonorchis sinensis) can induce severe biliary injuries, which can cause cholangitis, biliary fibrosis, and even cholangiocarcinoma. The release of extracellular vesicles by C. sinensis (CsEVs) is of importance in the long-distance communication between the hosts and worms. However, the biological effects of EVs from liver fluke on biliary injuries and the underlying molecular mechanisms remain poorly characterized. In the present study, we found that CsEVs induced M1-like activation. In addition, the mice that were administrated with CsEVs showed severe biliary injuries associated with remarkable activation of M1-like macrophages. We further characterized the signatures of miRNAs packaged in CsEVs and identified a miRNA Csi-let-7a-5p, which was highly enriched. Further study showed that Csi-let-7a-5p facilitated the activation of M1-like macrophages by targeting Socs1 and Clec7a; however, CsEVs with silencing Csilet-7a-5p showed a decrease in proinflammatory responses and biliary injuries, which involved in the Socs1- and Clec7a-regulated NF-κB signaling pathway. Our study demonstrates that Csi-let-7a-5p delivered by CsEVs plays a critical role in the activation of M1-like macrophages and contributes to the biliary injuries by targeting the Socs1- and Clec7a-mediated NF-κB signaling pathway, which indicates a mechanism contributing to biliary injuries caused by fluke infection. However, molecules other than Csi-let-7a-5p from CsEVs that may also promote M1-like polarization and exacerbate biliary injuries are not excluded. [ABSTRACT FROM AUTHOR]

Numerous animal lineages have maternally inherited symbionts that are required for host reproduction and growth. Endosymbionts also pose a risk to their hosts because of the mutational decay of their genomes through genetic drift or to selfish mutations that favor symbiont fitness over host fitness. One model for heritable endosymbiosis is the association of aphids with their obligate bacterial symbiont, Buchnera. We experimentally established heteroplasmic pea aphid matrilines containing pairs of closely related Buchnera haplotypes and used deep sequencing of diagnostic markers to measure haplotype frequencies in successive host generations. These frequencies were used to estimate the effective population size of Buchnera within hosts (i.e., the transmission bottleneck size) and the extent of within-host selection. The within-host effective population size was in the range of 10 to 20, indicating a strong potential for genetic drift and fixation of deleterious mutations. Remarkably, closely related haplotypes were subject to strong within-host selection, with selection coefficients as high as 0.5 per aphid generation. In one case, the direction of selection depended on the thermal environment and went in the same direction as between-host selection. In another, a new mutant haplotype had a strong within-host advantage under both environments but had no discernible effect on host-level fitness under laboratory conditions. Thus, within-host selection can be strong, resulting in a rapid fixation of mutations with little impact on host-level fitness. Together, these results show that within-host selection can drive evolution of an obligate symbiont, accelerating sequence evolution. [ABSTRACT FROM AUTHOR]

DNA replication is dramatically slowed down under replication stress. The regulation of replication speed is a conserved response in eukaryotes and, in fission yeast, requires the checkpoint kinases Rad3ATR and Cds1Chk2. However, the underlying mechanism of this checkpoint regulation remains unresolved. Here, we report that the Rad3ATR-Cds1Chk2 checkpoint directly targets the Cdc45-MCM-GINS (CMG) replicative helicase under replication stress. When replication forks stall, the Cds1Chk2 kinase directly phosphorylates Cdc45 on the S275, S322, and S397 residues, which significantly reduces CMG helicase activity. Furthermore, in cds1Chk2-mutated cells, the CMG helicase and DNA polymerases are physically separated, potentially disrupting replisomes and collapsing replication forks. This study demonstrates that the intra-S phase checkpoint directly regulates replication elongation, reduces CMG helicase processivity, prevents CMG helicase delinking from DNA polymerases, and therefore helps preserve the integrity of stalled replisomes and replication forks. [ABSTRACT FROM AUTHOR]

The thermal tolerance of an organism limits its ecological and geographic ranges and is potentially affected by dependence on temperature-sensitive symbiotic partners. Aphid species vary widely in heat sensitivity, but almost all aphids are dependent on the nutrient-provisioning intracellular bacterium Buchnera, which has evolved with aphids for 100 million years and which has a reduced genome potentially limiting heat tolerance. We addressed whether heat sensitivity of Buchnera underlies variation in thermal tolerance among 5 aphid species. We measured how heat exposure of juvenile aphids affects later survival, maturation time, and fecundity. At one extreme, heat exposure of Aphis gossypii enhanced fecundity and had no effect on the Buchnera titer. In contrast, heat suppressed Buchnera populations in Aphis fabae, which suffered elevated mortality, delayed development and reduced fecundity. Likewise, in Acyrthosiphon kondoi and Acyrthosiphon pisum, heat caused rapid declines in Buchnera numbers, as well as reduced survivorship, development rate, and fecundity. Fecundity following heat exposure is severely decreased by a Buchnera mutation that suppresses the transcriptional response of a gene encoding a small heat shock protein. Similarly, absence of this Buchnera heat shock gene may explain the heat sensitivity of Ap. fabae. Fluorescent in situ hybridization revealed heat-induced deformation and shrinkage of bacteriocytes in heat-sensitive species but not in heat-tolerant species. Sensitive and tolerant species also differed in numbers and transcriptional responses of heat shock genes. These results show that shifts in Buchnera heat sensitivity contribute to host variation in heat tolerance. [ABSTRACT FROM AUTHOR]

DNA replication forks in eukaryotic cells stall at a variety of replication barriers. Stalling forks require strict cellular regulations to prevent fork collapse. However, the mechanism underlying these cellular regulations is poorly understood. In this study, a cellular mechanism was uncovered that regulates chromatin structures to stabilize stalling forks. When replication forks stall, H2BK33, a newly identified acetylation site, is deacetylated and H3K9 trimethylated in the nucleosomes surrounding stalling forks, which results in chromatin compaction around forks. Acetylation-mimic H2BK33Q and its deacetylase clr6-1 mutations compromise this fork stalling-induced chromatin compaction, cause physical separation of replicative helicase and DNA polymerases, and significantly increase the frequency of stalling fork collapse. Furthermore, this fork stalling-induced H2BK33 deacetylation is independent of checkpoint. In summary, these results suggest that eukaryotic cells have developed a cellular mechanism that stabilizes stalling forks by targeting nucleosomes and inducing chromatin compaction around stalling forks. This mechanism is named the "Chromsfork" control: Chromatin Compaction Stabilizes Stalling Replication Forks. [ABSTRACT FROM AUTHOR]

The synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) is an active ingredient of thousands of commercial herbicides. Multiple species of bacteria degrade 2,4-D via a pathway initiated by the Fe(II) and α-ketoglutarate (Fe/αKG)-dependent aryloxyalkanoate dioxygenases (AADs). Recently, genes encoding 2 AADs have been deployed commercially in herbicide-tolerant crops. Some AADs can also inactivate chiral phenoxypropionate and aryloxyphenoxypropionate (AOPP) herbicides, albeit with varying substrate enantio-selectivities. Certain AAD enzymes, such as AAD-1, have expanded utility in weed control systems by enabling the use of diverse modes of action with a single trait. Here, we report 1) the use of a genomic context-based approach to identify 59 additional members of the AAD class, 2) the biochemical characterization of AAD-2 from Bradyrhizobium diazoefficiens USDA 110 as a catalyst to degrade (S)-stereoisomers of chiral synthetic auxins and AOPP herbicides, 3) spectroscopic data that demonstrate the canonical ferryl complex in the AAD-1 reaction, and 4) crystal structures of representatives of the AAD class. Structures of AAD-1, an (R)-enantiomer substrate-specific enzyme, in complexes with a phenoxypropionate synthetic auxin or with AOPP herbicides and of AAD-2, which has the opposite (S)-enantiomeric substrate specificity, reveal the structural basis for stereoselectivity and provide insights into a common catalytic mechanism. [ABSTRACT FROM AUTHOR]

DNA methylation is an important epigenetic mark involved in many biological processes. The genome of the climacteric tomato fruit undergoes a global loss of DNA methylation due to active DNA demethylation during the ripening process. It is unclear whether the ripening of other fruits is also associated with global DNA demethylation. We characterized the single-base resolution DNA methylomes of sweet orange fruits. Compared with immature orange fruits, ripe orange fruits gained DNA methylation at over 30,000 genomic regions and lost DNA methylation at about 1,000 genomic regions, suggesting a global increase in DNA methylation during orange fruit ripening. This increase in DNA methylation was correlated with decreased expression of DNA demethylase genes. The application of a DNA methylation inhibitor interfered with ripening, indicating that the DNA hypermethylation is critical for the proper ripening of orange fruits. We found that ripening-associated DNA hypermethylation was associated with the repression of several hundred genes, such as photosynthesis genes, and with the activation of hundreds of genes, including genes involved in abscisic acid responses. Our results suggest important roles of DNA methylation in orange fruit ripening. [ABSTRACT FROM AUTHOR]

Nucleation and growth of hydrogen nanobubbles are key initial steps in electrochemical water splitting. These processes remain largely unexplored due to a lack of proper tools to probe the nanobubble's interfacial structure with sufficient spatial and temporal resolution. We report the use of superresolution microscopy to image transient formation and growth of single hydrogen nanobubbles at the electrode/solution interface during electrocatalytic water splitting.We found hydrogen nanobubbles can be generated even at very early stages in water electrolysis, i.e., ~500 mV before reaching its thermodynamic reduction potential. The ability to image single nanobubbles on an electrode enabled us to observe in real time the process of hydrogen spillover from ultrathin gold nanocatalysts supported on indium-tin oxide. [ABSTRACT FROM AUTHOR]

Cortical interneurons (cINs) modulate excitatory neuronal activity by providing local inhibition. During fetal development, several cIN subtypes derive from the medial ganglionic eminence (MGE), a transient ventral telencephalic structure. While altered cIN development contributes to neurodevelopmental disorders, the inaccessibility of human fetal brain tissue during development has hampered efforts to define molecular networks controlling this process. Here, we modified protocols for directed differentiation of human embryonic stem cells, obtaining efficient, accelerated production of MGE-like progenitors and MGE-derived cIN subtypes with the expected electrophysiological properties. We defined transcriptome changes accompanying this process and integrated these data with direct transcriptional targets of NKX2-1, a transcription factor controlling MGE specification. This analysis defined NKX2-1-associated genes with enriched expression during MGE specification and cIN differentiation, including known and previously unreported transcription factor targets with likely roles in MGE specification, and other target classes regulating cIN migration and function. NKX2-1-associated peaks were enriched for consensus binding motifs for NKX2-1, LHX, and SOX transcription factors, suggesting roles in coregulating MGE gene expression. Among the NKX2-1 direct target genes with cIN-enriched expression was CHD2, which encodes a chromatin remodeling protein mutated to cause human epilepsies. Accordingly, CHD2 deficiency impaired cIN specification and altered later electrophysiological function, while CHD2 coassociated with NKX2-1 at cis-regulatory elements and was required for their transactivation by NKX2-1 in MGE-like progenitors. This analysis identified several aspects of gene-regulatory networks underlying human MGE specification and suggested mechanisms by which NKX2-1 acts with chromatin remodeling activities to regulate gene expression programs underlying cIN development. [ABSTRACT FROM AUTHOR]

Fluorescence imaging multiplicity of biological systems is an area of intense focus, currently limited to fluorescence channels in the visible and first near-infrared (NIR-I; ~700-900 nm) spectral regions. The development of conjugatable fluorophores with longer wavelength emission is highly desired to afford more targeting channels, reduce background autofluorescence, and achieve deeper tissue imaging depths. We have developed NIR-II (1,000-1,700 nm) molecular imaging agents with a bright NIR-II fluorophore through high-efficiency click chemistry to specific molecular antibodies. Relying on buoyant density differences during density gradient ultracentrifugation separations, highly pure NIR-II fluorophore-antibody conjugates emitting ~1,100 nm were obtained for use as molecular-specific NIR-II probes. This facilitated 3D staining of ~170-µm histological brain tissues sections on a home-built confocal microscope, demonstrating multicolor molecular imaging across both the NIR-I and NIR-II windows (800-1,700 nm). [ABSTRACT FROM AUTHOR]

Hepatitis A virus (HAV) infects ~1.4 million people annually and, although there is a vaccine, there are no licensed therapeutic drugs. HAV is unusually stable (making disinfection problematic) and little is known of how it enters cells and releases its RNA. Here we report a potent HAV-specific monoclonal antibody, R10, which neutralizes HAV infection by blocking attachment to the host cell. High-resolution cryo-EM structures of HAV full and empty particles and of the complex of HAV with R10 Fab reveal the atomic details of antibody binding and point to a receptor recognition site at the pentamer interface. These results, together with our observation that the R10 Fab destabilizes the capsid, suggest the use of a receptor mimic mechanism to neutralize virus infection, providing new opportunities for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Rho GTPases, including the Rho, Cdc42, Rac, and ROP subfamilies, act as pivotal signaling switches in various growth and developmental processes. Compared with the well-defined role of cytoskeletal organization in Rho signaling, much less is known regarding transcriptional regulation. In a mutant screen for phenotypic enhancers of transgenic Arabidopsis plants expressing a constitutively active form of ROP2 (designated CA1-1), we identified RNA polymerase II (Pol II) C-terminal domain (CTD) phosphatase-like 1 (CPL1) as a transcriptional regulator of ROP2 signaling. We show that ROP2 activation inhibits CPL1 activity by promoting its degradation, leading to an increase in CTD Ser5 and Ser2 phosphorylation. We also observed similar modulation of CTD phosphorylation by yeast Cdc42 GTPase and enhanced degradation of the yeast CTD phosphatase Fcp1 by activated ROP2 signaling. Taken together, our results suggest that modulation of the Pol II CTD code by Rho GTPase signaling represents an evolutionarily conserved mechanism in both unicellular and multicellular eukaryotes. [ABSTRACT FROM AUTHOR]

Commercial tomatoes are widely perceived by consumers as lacking flavor. A major part of that problem is a postharvest handling system that chills fruit. Low-temperature storage is widely used to slow ripening and reduce decay. However, chilling results in loss of flavor. Flavor-associated volatiles are sensitive to temperatures below 12 °C, and their loss greatly reduces flavor quality. Here, we provide a comprehensive view of the effects of chilling on flavor and volatiles associated with consumer liking. Reduced levels of specific volatiles are associated with significant reductions in transcripts encoding key volatile synthesis enzymes. Although expression of some genes critical to volatile synthesis recovers after a return to 20 °C, some genes do not. RNAs encoding transcription factors essential for ripening, including RIPENING INHIBITOR (RIN), NONRIPENING, and COLORLESS NONRIPENING are reduced in response to chilling and may be responsible for reduced transcript levels in many downstream genes during chilling. Those reductions are accompanied by major changes in the methylation status of promoters, including RIN. Methylation changes are transient and may contribute to the fidelity of gene expression required to provide maximal beneficial environmental response with minimal tangential influence on broader fruit developmental biology. [ABSTRACT FROM AUTHOR]

The motor function of vertebrate myosin-5a is inhibited by its tail in a Ca2+-dependent manner. We previously demonstrated that the calmodulin (CaM) bound to the first isoleucine-glutamine (IQ) motif (IQ1) of myosin-5a is responsible for the Ca2+-dependent regulation of myosin-5a. We have solved the crystal structure of a truncated myosin-5a containing the motor domain and IQ1 (MDIQ1) complexedwith Ca2+-bound CaM (Ca2+-CaM) at 2.5-Å resolution. Comparedwith the structure of the MD-IQ1 complexed with essential light chain (an equivalent of apo-CaM), MD-IQ1/Ca2+-CaM displays large conformational differences in IQ1/CaM and little difference in the motor domain. In the MD-IQ1/Ca2+-CaM structure, the N-lobe and the C-lobe of Ca2+-CaM adopt an open conformation and grip the C-terminal and the N-terminal portions of the IQ1, respectively. Remarkably, the interlobe linker of CaMin IQ1/Ca2+-CaM is in a position opposite that in IQ1/apo-CaM, suggesting that CaM flip-flops relative to the IQ1 during the Ca2+ transition. We demonstrated that CaM continuously associates with the IQ1 during the Ca2+ transition and that the binding of CaM to IQ1 increases Ca2+ affinity and substantially changes the kinetics of the Ca2+ transition, suggesting that the IQ1/CaM complex functions as an intact Ca2+ sensor responding to distinct calcium signals. [ABSTRACT FROM AUTHOR]

Metallic glass (MG) is an important new category of materials, but very few rigorous laws are currently known for defining its "disordered" structure. Recently we found that under compression, the volume (V) of an MG changes precisely to the 2.5 power of its principal diffraction peak position (1/q1). In the present study, we find that this 2.5 power law holds even through the first-order polyamorphic transition of a Ce68Al10Cu20Co2 MG. This transition is, in effect, the equivalent of a continuous "composition" change of 4f-localized "big Ce" to 4f-itinerant "small Ce," indicating the 2.5 power law is general for tuning with composition. The exactness and universality imply that the 2.5 power law may be a general rule defining the structure of MGs. [ABSTRACT FROM AUTHOR]

Fumarate an d nitrate reduction (FNR) regulatory proteins are O2-sensing bacterial transcription factors that control the switch between aerobic and anaerobic metabolism. Under anaerobic conditions [4Fe-4S]2+-FNR exists as a DNA-binding homodimer. In response to elevated oxygen levels, the [4Fe-4S]2+ cluster undergoes a rapid conversion to a [2Fe-2S]2+ cluster, resulting in a dimer-to-monomer transition and loss of site-specific DNA binding. In this work, resonance Raman and UV-visible absorption/CD spectroscopies and MS were used to characterize the interconversion between [4Fe-4S]2+ and [2Fe-2S]2+ clusters In Escheríchía coli FNR. Selective 34S labeling of the bridging sulfides in the [4Fe-4S]2+ cluster-bound form of FNR facilitated identification of resonantly enhanced Cys32S-34S stretching modes in the resonance Raman spectrum of the O2-exposed [2Fe-2S]2+ duster-bound form of FNR. This result indicates O2-induced oxidation and retention of bridging sulfides in the form of [2Fe-2S]2+ duster-bound cysteine persulfides. MS also demonstrates that multiple cysteine persulfides are formed on O2 exposure of [4Fe-4S]2+-FNR. The [4Fe-4S]2+ cluster in FNR can also be regenerated from the cysteine persulfide-coordinated [2Fe-2S]2+ cluster by anaerobic incubation with DTT and Fe2+ ion in the absence of exogenous sulfide. Resonance Raman data indicate that this type of cluster conversion involving sulfide oxidation is not unique to FNR, because it also occurs in O2-exposed forms of O2-sensitive [4Fe-4S] clusters in radical S-adeno-sylmethionine enzymes. The results provide fresh insight into the molecular mechanism of O2 sensing by FNR and iron-sulfur cluster conversion reactions in general, and suggest unique mechanisms for the assembly or repair of biological [4Fe-4S] clusters. [ABSTRACT FROM AUTHOR]

Aberrant glucose metabolism is one of the hallmarks of cancer that facilitates cancer cell survival and proliferation. Here, we demonstrate that MUC1, a large, type I transmembrane protein that is overexpressed in several carcinomas including pancreatic adenocarcinoma, modulates cancer cell metabolism to facilitate growth properties of cancer cells. MUC1 occupies the promoter elements of multiple genes directly involved in glucose metabolism and regulates their expression. Furthermore, MUC1 expression enhances glycolytic activity in pancreatic cancer cells. We also demonstrate that MUC1 expression enhances in vivo glucose uptake and expression of genes involved in glucose uptake and metabolism in orthotopic implantation models of pancreatic cancer. The MUC1 cytoplasmic tail is known to activate multiple signaling pathways through its interactions with several transcription factors/coregulators at the promoter elements of various genes. Our results indicate that MUC1 acts as a modulator of the hypoxic response in pancreatic cancer cells by regulating the expression/stability and activity of hypoxia-inducible factor-lα (HIF-1α). MUC1 physically interacts with HIF-1α and p300 and stabilizes the former at the protein level. By using a ChIP assay, we demonstrate that MUC1 facilitates recruitment of HIF-1rx and p300 on glycolytic gene promoters in a hypoxia-dependent manner. Also, by metabolomic studies, we demonstrate that MUC1 regulates multiple metabolite intermediates in the glucose and amino acid metabolic pathways. Thus, our studies indicate that MUC1 acts as a master regulator of the metabolic program and facilitates metabolic alterations in the hypoxic environments that help tumor cells survive and proliferate under such conditions. [ABSTRACT FROM AUTHOR]

The boronic acid dipeptide bortezomib inhibits the chymotrypsin-like activity of the 26S proteasome and shows significant therapeutic efficacy in multiple myeloma. However, recent studies suggest that bortezomib may have more complex mechanisms of action in treating cancer. We report here that the endocytosis and lysosomal degradation of the receptor tyrosine kinase C-KIT are required for bortezomib- but not tyrosine kinase inhibitor imatinib-caused apoptosis of t(8;21) leukemia and gastrointestinal stromal tumor cells, suggesting that C-KIT may recruit an apoptosis initiator. We show that C-KIT binds and phosphorylates heat shock protein 90β (Hsp90β), which sequestrates apoptotic protease activating factor 1 (Apaf-1). Bortezomib dephosphorylates pHsp90β and releases Apaf-1. Although the activated caspase-3 is not sufficient to cause marked apoptosis, it cleaves the t(8;21) generated acute myeloid leukemia 1-eight twenty one (AML1-ETO) and AML1-ETO9a fusion proteins, with production of cleavage fragments that perturb the functions of the parental oncoproteins and further contribute to apoptosis. Notably, bortezomib exerts potent therapeutic efficacy in mice bearing AML1-ETO9a-driven leukemia. These data show that C-KIT-pHsp90β-Apaf-1 cascade is critical for some malignant cells to evade apoptosis, and the clinical therapeutic potentials of bortezomib in C-KIT-driven neoplasms should be further explored. [ABSTRACT FROM AUTHOR]

The nucleotide NAADP was recently discovered as a second messenger involved in the initiation and propagation of Ca2+ signaling in lymphoma T cells, but its impact on primary T cell function is still unknown. An optimized, synthetic, small molecule inhibitor of NAADP action, termed BZ194, was designed and synthesized. BZ194 neither interfered with Ca2+ mobilization by D-myo-inositol 1,4,5-trisphosphate or cyclic ADP-ribose nor with capacitative Ca2+ entry. BZ194 specifically and effectively blocked NAADP-stimulated [³H]ryanodine binding to the purified type 1 ryanodine receptor. Further, in intact T cells, Ca2+ mobilization evoked by NAADP or by formation of the immunological synapse between primary effector T cells and astrocytes was inhibited by BZ194. Downstream events of Ca2+ mobilization, such as nuclear translocation of "nuclear factor of activated T cells" (NFAT), T cell receptor-driven interleukin-2 production, and proliferation in antigen-experienced CD4+ effector T cells, were attenuated by the NAADP antagonist. Taken together, specific inhibition of the NAADP signaling pathway constitutes a way to specifically and effectively modulate T-cell activation and has potential in the therapy of autoimmune diseases. [ABSTRACT FROM AUTHOR]

The anadromous Chinese sturgeon (Acipenser sinensis) is endangered and listed among the first class of protected animals in China. The possible causes for the decline of this species are the effects of synthetic chemicals, and loss of critical habitat. Chinese sturgeon in the Yangtze River have accumulated triphenyltin (TPT) to 31-128 ng/g wet weigh (ww) in liver, which is greater than the concentrations of tributyltin (<1.0 ng/g ww). Maternal transfer of TPT has resulted in concentrations of 25.5 ± 13.0 ng/g ww in eggs of wild Chinese sturgeon, which poses a significant risk to the larvae naturally fertilized or hatched in the Yangtze River. The incidence of deformities in fry was 7.5%, with 1.2% of individuals exhibiting ocular abnormal development, and 6.3% exhibited skeletal/morphological deformations. The incidences of both ocular and skeletal/morphological deformations were directly proportional to the TPT concentration in the eggs of both the Chinese sturgeon and the Siberian sturgeon (Acipenser baerii) in controlled laboratory studies. The rates of deformities in the controlled studies were consistent with the rates caused at the similar concentrations in eggs collected from the field. Thus, TPT is the causal agent to induce the malformation of larvae of Chinese sturgeon. The incidence of deformed larvae of Chinese sturgeon is an indicator of overall population-level effects of TPT on Chinese sturgeon, because TPT at environmentally relevant concentrations can result in significantly decrease both quality and quantity of eggs and spawning frequency of fish. [ABSTRACT FROM AUTHOR]

The migration of lymphocytes into the CNS during viral encephalitis is hindered by the blood-brain barrier (BBB) such that most infiltrating cells remain localized to perivascular spaces. This sequestration of leukocytes away from the parenchyma is believed to protect the CNS from immunopathologic injury. Infections of the CNS with highly cytopathic neurotropic viruses, such as West Nile virus (WNV), however, require the parenchymal penetration of T lymphocytes for virus clearance and survival, suggesting that perivascular localization might hinder antiviral immune responses during WNV encephalitis. Using human and murine brain specimens from individuals with WNV encephalitis, we evaluated the expression of CXCL12 and its receptor, CXCR4, at the BBB and tested the hypothesis that inhibition of CXCR4 would promote T lymphocyte entry into the CNS parenchyma and increase viral clearance. Antagonism of CXCR4 significantly improved survival from lethal infection through enhanced intraparenchymal migration of WNV-specific CD8+ T cells within the brain, leading to reduced viral loads and, surprisingly, decreased immunopathology at this site. The benefits of enhanced CD8+ T cell infiltration suggest that pharmacologic targeting of CXCR4 may have therapeutic utility for the treatment of acute viral infections of the CNS. [ABSTRACT FROM AUTHOR]

Using a combination of techniques we developed, we infected zebrafish embryos using pseudotyped retroviruses and mapped the genomic locations of the proviral integrations in the F1 off- spring of the infected fish. From F1 fish, we obtained 2,045 sequences representing 933 unique retroviral integrations. A total of 599 were mappable to the current genomic assembly (Zv6), and 233 of the integrations landed within genes. By inbreeding fish carrying proviral integrations in 25 different genes, we were able to demonstrate that in ∼50% of the gene ‘hits,’ the mRNA transcript levels were reduced by ⩾70%, with the highest probability for mutation occurring if the integration was in an exon or first intron. Based on these data, the mutagenic frequency for the retrovirus is nearly one in five integrations. In addition, a strong mutagenic effect is seen when murine leukemia virus integrates specifically in the first intron of genes but not in other introns. Three of 19 gene inactivation events had embryonic defects. Using the strategy we outlined, it is possible to identify 1 mutagenic event for every 30 sequencing reactions done on the F1 fish. This is a 20- to 30-fold increase in efficiency when compared with the current resequencing approach [targeting induced local lesions in genomes (TILLING)] used in zebrafish for identifying mutations in genes. Combining this increase in efficiency with cryopreservation of sperm samples from the F1 fish, it is now possible to create a stable resource that contains mutations in every known zebrafish gene. [ABSTRACT FROM AUTHOR]

Azotobacter vinelandii flavodoxin hydroquinone (FldHQ) is a physiological reductant to nitrogenase supporting catalysis that is twice as energy efficient (ATP/2e- = 2) as dithionite (ATP/2e- = 4). This catalytic efficiency results from reduction of Fe protein from A. vinelandii (Av2) to the all-ferrous oxidation state ([Fe4S4]0), in contrast to dithionite, which only reduces Av2 to the [Fe4S4]1+ state. Like FldHQ, Ti(III) citrate yields ATP/2e- = 2, and Ti(III)-reduced [Fe4S4]0 Av2 has a S = 4 spin state and characteristic Mossbauer spectrum, a parallel mode g = 16.4 EPR signal, and a shoulder at 520 nm in its UV-vis spectrum, each of which distinguish the S = 4 [Fe4S4]0 Av2 from other states. In this study, we demonstrate that FldHQ makes [Fe4S4]0 Av2, which is sufficiently characterized to demonstrate unique physical properties that distinguish it from the previously characterized Ti(III)-reduced [Fe4S4]0 Av2. In particular, Evans NMR magnetic susceptibility and EPR measurements indicate that FldHQ-reduced [Fe4S4]0 Av2 has an S = 0 spin state (like [Fe4S4]2+ Av2). There is no g = 16.4 EPR signal and no shoulder at 520 nm in its absorbance spectrum, which resembles that of [Fe4S4]1+ Av2. That the physiological reductant to Av2 is capable of forming [Fe4S4]0 Av2 has important implications for in vivo nitrogenase activity. [ABSTRACT FROM AUTHOR]