Diploid and tetraploid genomes of Acorus and the evolution of monocots.

Monocots are a major taxon within flowering plants, have unique morphological traits, and show an extraordinary diversity in lifestyle. To improve our understanding of monocot origin and evolution, we generate chromosome-level reference genomes of the diploid Acorus gramineus and the tetraploid Ac. calamus, the only two accepted species from the family Acoraceae, which form a sister lineage to all other monocots. Comparing the genomes of Ac. gramineus and Ac. calamus, we suggest that Ac. gramineus is not a potential diploid progenitor of Ac. calamus, and Ac. calamus is an allotetraploid with two subgenomes A, and B, presenting asymmetric evolution and B subgenome dominance. Both the diploid genome of Ac. gramineus and the subgenomes A and B of Ac. calamus show clear evidence of whole-genome duplication (WGD), but Acoraceae does not seem to share an older WGD that is shared by most other monocots. We reconstruct an ancestral monocot karyotype and gene toolkit, and discuss scenarios that explain the complex history of the Acorus genome. Our analyses show that the ancestors of monocots exhibit mosaic genomic features, likely important for that appeared in early monocot evolution, providing fundamental insights into the origin, evolution, and diversification of monocots.

Probing differences among Aß oligomers with two triangular trimers derived from Aß.

The assembly of the ß-amyloid peptide (Aß) to form oligomers and fibrils is closely associated with the pathogenesis and progression of Alzheimer's disease. Aß is a shape-shifting peptide capable of adopting many conformations and folds within the multitude of oligomers and fibrils the peptide forms. These properties have precluded detailed structural elucidation and biological characterization of homogeneous, well-defined Aß oligomers. In this paper, we compare the structural, biophysical, and biological characteristics of two different covalently stabilized isomorphic trimers derived from the central and C-terminal regions Aß. X-ray crystallography reveals the structures of the trimers and shows that each trimer forms a ball-shaped dodecamer. Solution-phase and cell-based studies demonstrate that the two trimers exhibit markedly different assembly and biological properties. One trimer forms small soluble oligomers that enter cells through endocytosis and activate capase-3/7-mediated apoptosis, while the other trimer forms large insoluble aggregates that accumulate on the outer plasma membrane and elicit cellular toxicity through an apoptosis-independent mechanism. The two trimers also exhibit different effects on the aggregation, toxicity, and cellular interaction of full-length Aß, with one trimer showing a greater propensity to interact with Aß than the other. The studies described in this paper indicate that the two trimers share structural, biophysical, and biological characteristics with oligomers of full-length Aß. The varying structural, assembly, and biological characteristics of the two trimers provide a working model for how different Aß trimers can assemble and lead to different biological effects, which may help shed light on the differences among Aß oligomers.

Elucidating the Oligomerization and Cellular Interactions of a Trimer Derived from Aß through Fluorescence and Mass Spectrometric Studies.

Aß oligomers play a central role in the neurodegeneration observed with Alzheimer's disease. Our laboratory has developed covalently stabilized trimers derived from residues 17-36 of Aß as model systems for studying Aß oligomers. In the current study, we apply the emerging techniques of fluorescence lifetime imaging microscopy (FLIM) and native mass spectrometry (native MS) to better understand the assembly and interactions of the oligomer model system 2AT-L in aqueous solutions and with cells. 2AT-L and fluorescently labeled 2AT-L analogues assemble in the membrane-like environment of SDS-PAGE, showing diffuse bands of oligomers in equilibrium. Native ion mobility-mass spectrometry (native IM-MS) of 2AT-L allows for the identification of discrete oligomers in solution and shows similar patterns of oligomer formation between 2AT-L and fluorescently labeled analogues. Fluorescence microscopy with SH-SY5Y cells reveals that fluorescently labeled 2AT-L analogues colocalize within lysosomes. FLIM studies with phasor analysis further elucidate the assembly of 2AT-L within cells and establish the occurrence of FRET, indicating the presence of oligomers within cells. Collectively, these multiple complementary techniques help better understand the complex behavior of the 2AT-L model system.

Genomes of leafy and leafless Platanthera orchids illuminate the evolution of mycoheterotrophy.

To improve our understanding of the origin and evolution of mycoheterotrophic plants, we here present the chromosome-scale genome assemblies of two sibling orchid species: partially mycoheterotrophic Platanthera zijinensis and holomycoheterotrophic Platanthera guangdongensis. Comparative analysis shows that mycoheterotrophy is associated with increased substitution rates and gene loss, and the deletion of most photoreceptor genes and auxin transporter genes might be linked to the unique phenotypes of fully mycoheterotrophic orchids. Conversely, trehalase genes that catalyse the conversion of trehalose into glucose have expanded in most sequenced orchids, in line with the fact that the germination of orchid non-endosperm seeds needs carbohydrates from fungi during the protocorm stage. We further show that the mature plant of P. guangdongensis, different from photosynthetic orchids, keeps expressing trehalase genes to hijack trehalose from fungi. Therefore, we propose that mycoheterotrophy in mature orchids is a continuation of the protocorm stage by sustaining the expression of trehalase genes. Our results shed light on the molecular mechanism underlying initial, partial and full mycoheterotrophy.

Surface-induced dissociation of protein complexes on a cyclic ion mobility spectrometer.

We describe the implementation of a simple three-electrode surface-induced dissociation (SID) cell on a cyclic ion mobility spectrometer (cIMS) and demonstrate the utility of multipass mobility separations for resolving multiple conformations of protein complexes generated during collision-induced and surface-induced unfolding (CIU & SIU) experiments. In addition to CIU and SIU, SID of protein complexes is readily accomplished within the native instrument software and with no additional external power supplies by entering a single SID collision energy, a simplification in user experience compared to prior implementations. A set of cyclic homomeric protein complexes and a heterohexamer with known CID and SID behavior were analyzed to investigate mass and mobility resolution improvements, the latter of which improved by 20-50% (median: 33%) compared to a linear travelling wave device. Multiple passes of intact complexes, or their SID fragments, increased the mobility resolution by an average of 15% per pass, with the racetrack effect being observed after ∼3 or 4 passes, depending on the drift time spread of the analytes. Even with modest improvements to apparent mobility resolving power, multipass experiments were particularly useful for separating conformations produced from CIU and SIU experiments. We illustrate several examples where either (1) multipass experiments revealed multiple overlapping conformations previously unobserved or obscured due to limited mobility resolution, or (2) CIU or SIU conformations that appeared 'native' in a single pass experiment were actually slightly compacted or expanded, with the change only being measurable through multipass experiments. The work conducted here, the first utilization of multipass cyclic ion mobility for CIU, SIU, and SID of protein assemblies and a demonstration of a wholly integrated SIU/SID workflow, paves the way for widespread adoption of SID technology for native mass spectrometry and also improves our understanding of gas-phase protein complex CIU and SIU conformationomes.

Wolfberry genomes and the evolution of Lycium (Solanaceae).

Wolfberry Lycium, an economically important genus of the Solanaceae family, contains approximately 80 species and shows a fragmented distribution pattern among the Northern and Southern Hemispheres. Although several herbaceous species of Solanaceae have been subjected to genome sequencing, thus far, no genome sequences of woody representatives have been available. Here, we sequenced the genomes of 13 perennial woody species of Lycium, with a focus on Lycium barbarum. Integration with other genomes provides clear evidence supporting a whole-genome triplication (WGT) event shared by all hitherto sequenced solanaceous plants, which occurred shortly after the divergence of Solanaceae and Convolvulaceae. We identified new gene families and gene family expansions and contractions that first appeared in Solanaceae. Based on the identification of self-incompatibility related-gene families, we inferred that hybridization hotspots are enriched for genes that might be functioning in gametophytic self-incompatibility pathways in wolfberry. Extremely low expression of LOCULE NUBER (LC) and COLORLESS NON-RIPENING (CNR) orthologous genes during Lycium fruit development and ripening processes suggests functional diversification of these two genes between Lycium and tomato. The existence of additional flowering locus C-like MADS-box genes might correlate with the perennial flowering cycle of Lycium. Differential gene expression involved in the lignin biosynthetic pathway between Lycium and tomato likely illustrates woody and herbaceous differentiation. We also provide evidence that Lycium migrated from Africa into Asia, and subsequently from Asia into North America. Our results provide functional insights into Solanaceae origins, evolution and diversification.

Tandem surface-induced dissociation of protein complexes on an ultrahigh resolution platform.

We describe instrumentation for conducting tandem surface-induced dissociation (tSID) of native protein complexes on an ultrahigh resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. The two stages of SID are accomplished with split lenses replacing the entrance lenses of the quadrupole mass filter (stage 1, referred to herein as SID-Q) and the collision cell (stage 2, Q-SID). After SID-Q, the scattered projectile ions and subcomplexes formed in transit traverse the 20 mm pre-filter prior to the mass-selecting quadrupole, providing preliminary insights into the SID fragmentation kinetics of noncovalent protein complexes. The isolated SID fragments (subcomplexes) are then fragmented by SID in the collision cell entrance lens (Q-SID), generating subcomplexes of subcomplexes. We show that the ultrahigh resolution of the FT-ICR can be used for deconvolving species overlapping in m/z, which are particularly prominent in tandem SID spectra due to the combination of symmetric charge partitioning and narrow product ion charge state distributions. Various protein complex topologies are explored, including homotetramers, homopentamers, a homohexamer, and a heterohexamer.

Temperature prediction of ultrasonic vibration-assisted milling.

Machining temperature is a key factor in ultrasonic vibration-assisted milling as it can significantly influence tool wear rate and residual thermal stresses. In current study, a physics-based analytical predictive model on machining temperature in ultrasonic vibration-assisted milling is proposed, without resorting to iterative numerical simulations. As the tool periodically loses contact with the workpiece under vibration, three types of tool-workpiece separation criteria are first examined based on the tool trajectory under ultrasonic vibration. Type I criterion examines whether the relative velocity between tool and workpiece in cutting direction is opposite to the tool rotation direction. Type II criterion examines whether the instantaneous vibration displacement in radial direction is larger than instantaneous uncut chip thickness. Type III criterion examines whether there is overlap between current and previous tool paths due to vibration. If no contact, the instantaneous temperature rise is zero. Otherwise, the temperature rise is predicted under shearing heat source in shear zone and secondary rubbing heat source along machined surface. A mirror heat source method is applied to predict temperature rise, considering oblique band heat sources moving in a semi-infinite medium. The proposed predictive temperature model in ultrasonic vibration-assisted milling is validated through comparison to experimental measurements on Al 6063 alloy. The proposed predictive model is able to match the measured temperature with high accuracy of 1.85% average error and 5.22% largest error among all cases. Sensitivity analysis is also conducted to study the influences of cutting and vibration parameters on temperature. The proposed model is valuable in terms of providing an accurate and reliable reference for the prediction of temperature in ultrasonic vibration-assisted milling.

Direct Dynamics Simulations of Fragmentation of a Zn(II)-2Cys-2His Oligopeptide. Comparison with Mass Spectrometry Collision-Induced Dissociation.

Abnormalities in zinc metabolism have been linked to many diseases, including different kinds of cancers and neurological diseases. The present study investigates the fragmentation pathways of a zinc chaperon using a model peptide with the sequence acetyl-His1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7 (analog methanobactin peptide-5, amb5). DFT/M05-2X and B3LYP geometry optimizations of [amb5-3H+Zn(II)]- predicted three lowest energy conformers with different chelating motifs. Direct dynamics simulations, using the PM7 semiempirical electronic structure method, were performed for these conformers, labeled a, b, and c, to obtain their fragmentation pathways at different temperatures in the range 1600-2250 K. The simulation results were compared with negative ion mode mass spectrometry experiments. For conformer a, the number of primary dissociation pathways are 11, 14, 24, 70, and 71 at 1600, 1750, 1875, 2000, and 2250 K, respectively. However, there are only 6, 10, 13, 14, and 19 pathways corresponding to these temperatures that have a probability of 2% or more. For conformer b, there are 67 pathways at 2000 K and 71 pathways at 2250 K. For conformer c, 17 pathways were observed at 2000 K. For conformer a, for two of the most common pathways involving C-C bond dissociation, Arrhenius parameters were calculated. The frequency factors and activation energies are smaller than those for C-C homolytic dissociation in alkanes due to increased stability of the product ions as a result of hydrogen bonding. The activation energies agree with the PM7 barriers for the C-C dissociations. Comparison of the simulation and experimental fragmentation ion yields shows the simulations predict double or triple cleavages of the backbone with Zn(II) retaining its binding sites, whereas the experiment exhibits single cleavages of the backbone accompanied by cleavage of two of the Zn(II) binding sites, resulting in b- and y-type ions.

Weak Acid-Base Interactions of Histidine and Cysteine Affect the Charge States, Tertiary Structure, and Zn(II)-Binding of Heptapeptides.

Zinc fingers are proteins that are characterized by the coordination of zinc ions by an amino acid sequence that commonly contains two histidines and two cysteines (2His-2Cys motif). Investigations of oligopeptides that contain the 2His-2Cys motif, e.g., acetyl-His1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7, have discovered they exhibit pH-dependent Zn(II) chelation and have redox activities with Cu(I/II), forming a variety of metal complexes. To further understand how these 2His-2Cys oligopeptides bind these metal ions, we have undertaken a series of ion mobility-mass spectrometry and B3LYP/LanL2DZ computational studies of structurally related heptapeptides. Starting with the sequence above, we have modified the potential His, Cys, or C-terminus binding sites and report how these changes in primary structure affect the oligopeptides positive and negative charge states, conformational structure, collision-induced breakdown energies, and how effectively Zn(II) binds to these sequences. The results show evidence that the weak acid-base properties of Cys-His are intrinsically linked and can result in an intramolecular salt-bridged network that affects the oligopeptide properties.

Investigation on Drug Resistance Threshold of Newly Found HIV Infected Persons in Shaanxi / 现代检验医学杂志

Objective To analyze the resistance gene sequences of newly untreated HIV infected people,and to understand the prevalence and epidemic characteristics of HIV drug-resistant strains in Shaanxi Province.Methods According to the rec-ommended by the World Health Organization HIV drug resisitance threshold(HIVDR-TS),sera from 47 newly diagnosed HIV infected patients aged 16~25 years were analyzed.In-house determination of pol gene partial sequence,using online CPR software to determine whether there was transmission of resistant mutants,and submitted to Stanford University in the United States online HIV drug resistance database for the extent of the drug resistance analysis,the phylogenetic tree was constructed using mega 5.0 software,analysis of subtypes.Results No spread drug mutations were found in 47 samples,but there were 14 samples that were found to have associated with resistance gene mutation sites,including the RT region T69N and E138A,V179D/E mutations,PR A71T/V,L10I/V mutation sites and in subtype CRF01_B a like this in the site K103R,with V179E and there was a synergistic effect,resulting in accordance with efavirenz(EFV)and nevirapine(NVP) drugs such as moderately resistant.The mutations found in the remaining 13 samples would lead to a low degree of resist-ance to some of the drugs,and there was no clinical significance.Conclusion The prevalence rate of HIV resistant strains in Shaanxi province was <5%,which be longed to the low epidemic level.It is not necessary to detect drug resistance before individual treatment,and it is necessary to carry out the investigation of drug resistance.

Genome-wide identification and characterization of TCP genes involved in ovule development of Phalaenopsis equestris.

TEOSINTE-BRANCHED/CYCLOIDEA/PCF (TCP) proteins are plant-specific transcription factors known to have a role in multiple aspects of plant growth and development at the cellular, organ and tissue levels. However, there has been no related study of TCPs in orchids. Here we identified 23 TCP genes from the genome sequence of Phalaenopsis equestris Phylogenetic analysis distinguished two homology classes of PeTCP transcription factor families: classes I and II. Class II was further divided into two subclasses, CIN and CYC/TB1. Spatial and temporal expression analysis showed that PePCF10 was predominantly expressed in ovules at early developmental stages and PeCIN8 had high expression at late developmental stages in ovules, with overlapping expression at day 16 after pollination. Subcellular localization and protein-protein interaction analyses revealed that PePCF10 and PeCIN8 could form homodimers and localize in the nucleus. However, PePCF10 and PeCIN8 could not form heterodimers. In transgenic Arabidopsis thaliana plants (overexpression and SRDX, a super repression motif derived from the EAR-motif of the repression domain of tobacco ETHYLENE-RESPONSIVE ELEMENT-BINDING FACTOR 3 and SUPERMAN, dominantly repressed), the two genes helped regulate cell proliferation. Together, these results suggest that PePCF10 and PeCIN8 play important roles in orchid ovule development by modulating cell division.

2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (HKL-1) induces G2/M arrest and mitotic catastrophe in human leukemia HL-60 cells.

2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (HKL-1), a 2-phenyl-1,8-naphthyridin-4-one (2-PN) derivative, was synthesized and evaluated as an effective antimitotic agent in our laboratory. However, the molecular mechanisms are uncertain. In this study, HKL-1 was demonstrated to induce multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human leukemia HL-60 cells. Western blotting showed that HKL-1 induces mitotic catastrophe in HL-60 cells through regulating mitotic phase-specific kinases (down-regulating CDK1, cyclin B1, CENP-E, and aurora B) and regulating the expression of Bcl-2 family proteins (down-regulating Bcl-2 and up-regulating Bax and Bak), followed by caspase-9/-3 cleavage. These findings suggest that HKL-1 appears to exert its cytotoxicity toward HL-60 cells in culture by inducing mitotic catastrophe.