Determinants of the interaction between the 5'-leader of HIV-1 genome and human lysyl-tRNA synthetase in reverse transcription primer release process.

Reverse transcription of human immunodeficiency virus type 1 (HIV-1) initiates from the 3' end of human tRNALys3. The primer tRNALys3 is selectively packaged into the virus in the form of a complex with human lysyl-tRNA synthetase (LysRS). To facilitate reverse transcription initiation, part of the 5' leader (5'L) of HIV-1 genomic RNA (gRNA) evolves a tRNA anticodon-like element (TLE), which binds LysRS and releases tRNALys3 for primer annealing and reverse transcription initiation. Although TLE has been identified as a key element in 5'L responsible for LysRS binding, how the conformations and various hairpin structures of 5'L regulate 5'L-LysRS interaction is not fully understood. Here, these factors have been individually investigated using direct and competitive fluorescence anisotropy binding experiments. Our data showed that the conformation of 5'L significantly influences its binding affinity with LysRS. The 5'L conformation favoring gRNA dimerization and packaging exhibits much weaker binding affinity with LysRS compared to the alternative 5'L conformation that is not selected for packaging. Additionally, dimerization of 5'L impairs LysRS-5'L interaction. Furthermore, among various regions of 5'L, both the primer binding site/TLE domain and the stem-loop 3 are important for LysRS interaction, whereas the dimerization initiation site and the splicing donor plays a minor role. In contrast, the presence of the transacting responsive and the polyadenylation signal hairpins slightly inhibit LysRS binding. These findings reveal that the conformation and various regions of the 5'L of HIV-1 genome regulate its interaction with human LysRS and the reverse transcription primer release process.

How Placenta Promotes the Successful Reproduction in High-Altitude Populations: A Transcriptome Comparison between Adaptation and Acclimatization.

As the best adapted high altitude population, Tibetans feature a relatively high offspring survival rate. Genome-wide studies have identified hundreds of candidate SNPs related to high altitude adaptation of Tibetans, although most of them have unknown functional relevance. To explore the mechanisms behind successful reproduction at high altitudes, we compared the placental transcriptomes of Tibetans, sea level Hans (SLHan), and Han immigrants (ImHan). Among the three populations, placentas from ImHan showed a hyperactive gene expression pattern. Their increased activation demonstrates a hypoxic stress response similar to sea level individuals experiencing hypoxic conditions. Unlike ImHan, Tibetan placentas were characterized by the significant up-regulation of placenta-specific genes, and the activation of autophagy and the tricarboxylic acid (TCA) cycle. Certain conserved hypoxia response functions, including the antioxidant system and angiogenesis, were activated in both ImHan and Tibetans, but mediated by different genes. The coherence of specific transcriptome features linked to possible genetic contribution was observed in Tibetans. Furthermore, we identified a novel Tibetan-specific EPAS1 isoform with a partial deletion at exon six, which may be involved in the adaption to hypoxia through the EPAS1-centred gene network in the placenta. Overall, our results show that the placenta grants successful pregnancies in Tibetans by strengthening the natural functions of the placenta itself. On the other hand, the placenta of ImHan was in an inhabiting time-dependent acclimatization process representing a common hypoxic stress response pattern.

Hypomethylation in HBV integration regions aids non-invasive surveillance to hepatocellular carcinoma by low-pass genome-wide bisulfite sequencing.

BACKGROUND: Circulating cell-free DNA (cfDNA) methylation has been demonstrated to be a promising approach for non-invasive cancer diagnosis. However, the high cost of whole genome bisulfite sequencing (WGBS) hinders the clinical implementation of a methylation-based cfDNA early detection biomarker. We proposed a novel strategy in low-pass WGBS (~ 5 million reads) to detect methylation changes in circulating cell-free DNA (cfDNA) from patients with liver diseases and hepatocellular carcinoma (HCC). METHODS: The effective small sequencing depth were determined by 5 pilot cfDNA samples with relative high-depth WGBS. CfDNA of 51 patients with hepatitis, cirrhosis, and HCC were conducted using low-pass WGBS. The strategy was validated in an independent WGBS cohort of 32 healthy individuals and 26 early-stage HCC patients. Fifteen paired tumor tissue and buffy coat samples were used to characterize the methylation of hepatitis B virus (HBV) integration regions and genome distribution of cfDNA. RESULTS: A significant enrichment of cfDNA in intergenic and repeat regions, especially in previously reported HBV integration sites were observed, as a feature of cfDNA and the bias of cfDNA release. Methylation profiles nearby HBV integration sites were a better indicator for hypomethylation of tumor genome comparing to Alu and LINE (long interspersed nuclear element) repeats, and were able to facilitate the cfDNA-based HCC prediction. Hypomethylation nearby HBV integration sites (5 kb flanking) was detected in HCC patients, but not in patients with hepatitis and cirrhosis (MethylHBV5k, median:0.61 vs 0.72, P = 0.0003). Methylation levels of integration sites certain candidate regions exhibited an area under the receiver operation curve (AUC) value > 0.85 to discriminate HCC from non-HCC samples. The validation cohort achieved the prediction performance with an AUC of 0.954. CONCLUSIONS: Hypomethylation around viral integration sites aids low-pass cfDNA WGBS to serve as a non-invasive approach for early HCC detection, and inspire future efforts on tumor surveillance for oncovirus with integration activity.

An improved protein extraction method applied to cotton leaves is compatible with 2-DE and LC-MS.

BACKGROUND: Two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are widely used in plant proteomics research. However, these two techniques cannot be simultaneously satisfied by traditional protein extraction methods when investigate cotton leaf proteome. RESULTS: Here, we evaluated the efficiency of three different protein extraction methods for 2-DE and LC-MS/MS analyses of total proteins obtained from cotton leaves. The protein yield of the borax/PVPP/phenol (BPP) method (0.14%) was significantly lower than the yields of the trichloroacetic acid/acetone (TCA) precipitation method (1.42%) and optimized TCA combined with BPP (TCA-B) method (0.47%). The BPP method was failed to get a clear 2-DE electrophoretogram. Fifty pairs of protein spots were randomly selected from the 2-DE gels of TCA- and TCA-B-extracted proteins for identification by MALDI TOF/TOF, and the results of 42 pairs were consistent. High-throughput proteomic analysis showed that 6339, 9282 and 9697 unique proteins were identified from the total cotton leaf proteins extracted by the TCA, BPP and TCA-B methods, respectively. Gene Ontology (GO) analysis revealed that the proteins specifically identified by TCA method were primarily distributed in the plasma membrane, while BPP and TCA-B methods specific proteins distributed in the cytosol, indicating the sub-cellular preference of different protein extraction methods. Further, ATP-dependent zinc metalloprotease FTSH 8 could be observed in the 2-DE gels of TCA and TCA-B methods, and could only be detected in the LC-MS/MS results of the BPP and TCA-B methods, showing that TCA-B method might be the optimized choice for both 2-DE and LC-MS/MS. CONCLUSION: Our data provided an improved TCA-B method for protein extraction that is compatible with 2-DE and LC-MS/MS for cotton leaves and similar plant tissues which is rich in polysaccharides and polyphenols.

Increased expression diversity buffers the loss of adaptive potential caused by reduction of genetic diversity in new unfavourable environments.

Mechanisms underlying adaptation to rapid environmental change are issues in evolutionary biology. It is widely accepted that reduction in genetic diversity when suddenly exposed to an unfavourable environment limits the adaptive potential of populations. With growing empirical evidence that expression diversity is likely to increase in the new environment, the role that expression diversity plays in adaptation needs to be theorized. Here, we first established a negative exponential relationship between expression diversity and genetic diversity using a phenomenological differential equation. We then derived a complex trade-off relationship between the changes of expression and genetic diversity, which followed a combination of exponential functions. Furthermore, we found the increase in expression diversity could buffer the loss of adaptive potential as genetic diversity decreased to a certain extent. These theoretical deductions were validated by transcriptomic data of Miscanthus lutarioriparius grown in two experimental fields and supported by good fit and random simulation. These results suggest that increased expression diversity may compensate the loss of genetic diversity and allow the populations to maintain a certain level of phenotypic variation to cope with sudden environmental change. This may buffer the quick diminishing of adaptive potential and consequently increases the change of adaptation to the new environment.

Comparative Proteomic Analysis of Molecular Differences between Leaves of Wild-Type Upland Cotton and Its Fuzzless-Lintless Mutant.

Fuzzless-lintless mutant (fl) ovules of upland cotton have been used to investigate cotton fiber development for decades. However, the molecular differences of green tissues between fl and wild-type (WT) cotton were barely reported. Here, we found that gossypol content, the most important secondary metabolite of cotton leaves, was higher in Gossypium hirsutum L. cv Xuzhou-142 (Xu142) WT than in fl. Then, we performed comparative proteomic analysis of the leaves from Xu142 WT and its fl. A total of 4506 proteins were identified, of which 103 and 164 appeared to be WT- and fl-specific, respectively. In the 4239 common-expressed proteins, 80 and 74 were preferentially accumulated in WT and fl, respectively. Pathway enrichment analysis and protein-protein interaction network analysis of both variety-specific and differential abundant proteins showed that secondary metabolism and chloroplast-related pathways were significantly enriched. Quantitative real-time PCR confirmed that the expression levels of 12 out of 16 selected genes from representative pathways were consistent with their protein accumulation patterns. Further analyses showed that the content of chlorophyll a in WT, but not chlorophyll b, was significantly increased compared to fl. This work provides the leaf proteome profiles of Xu142 and its fl mutant, indicating the necessity of further investigation of molecular differences between WT and fl leaves.

Coexpression network revealing the plasticity and robustness of population transcriptome during the initial stage of domesticating energy crop Miscanthus lutarioriparius.

KEY MESSAGE: Coexpression network revealing genes with Co-variation Expression pattern (CE) and those with Top rank of Expression fold change (TE) played different roles in responding to new environment of Miscanthus lutarioriparius. Variation in gene expression level, the product of genetic and/or environmental perturbation, determines the robustness-to-plasticity spectrum of a phenotype in plants. Understanding how expression variation of plant population response to a new field is crucial to domesticate energy crops. Weighted Gene Coexpression Network Analysis (WGCNA) was used to explore the patterns of expression variation based on 72 Miscanthus lutarioriparius transcriptomes from two contrasting environments, one near the native habitat and the other in one harsh domesticating region. The 932 genes with Co-variation Expression pattern (CE) and other 932 genes with Top rank of Expression fold change (TE) were identified and the former were strongly associated with the water use efficiency (r ≥ 0.55, P ≤ 10-7). Functional enrichment of CE genes were related to three organelles, which well matched the annotation of twelve motifs identified from their conserved noncoding sequence; while TE genes were mostly related to biotic and/or abiotic stress. The expression robustness of CE genes with high genetic diversity kept relatively stable between environments while the harsh environment reduced the expression robustness of TE genes with low genetic diversity. The expression plasticity of CE genes was increased less than that of TE genes. These results suggested that expression variation of CE genes and TE genes could account for the robustness and plasticity of acclimation ability of Miscanthus, respectively. The patterns of expression variation revealed by transcriptomic network would shed new light on breeding and domestication of energy crops.

Genome-wide investigation and expression profiling of APX gene family in Gossypium hirsutum provide new insights in redox homeostasis maintenance during different fiber development stages.

Ascorbate peroxidase (APX) is a member of heme-containing peroxidases which catalyze the H2O2-dependent oxidation of a wide range of substrates in plants and animals. As is known, H2O2 acts as a signaling molecule in the regulation of fiber development. Our previous work reported that ascorbate peroxidase 1 (GhAPX1) was important for cotton fiber elongation. However, knowledge about APX gene family members and their evolutionary and functional characteristics in cotton is limited. Here, we report 26 GhAPX genes by genome-wide investigation of tetraploid cotton Gossypium hirsutum. Phylogenetic and gene structure analyses classified these APX members into five clades and syntenic analysis suggested two duplication events. Expression profiling of the 26 APXs revealed that ten members are expressed in cotton fibers. Notably, GhAPX10A, GhAPX10D, GhAPX12A, and GhAPX12D showed high expression levels in 30-day fiber, while GhAPX1A/D, GhAPX3A/D, and GhAPX6A/D showed very low expression levels. The enzyme activity and H2O2 content assays revealed that cotton fiber kept high enzyme activity and the lowest H2O2 level in 30-day fibers, indicating that other than GhAPX1, the newly reported APX members are responsible for the reactive oxygen species homeostasis in the cotton fiber maturation stages. Expression profiling of ten fiber-expressed APXs after phytohormone treatments revealed their regulation patterns by different stimuli, suggesting that GhAPX1, GhAPX12A, and GhAPX12D are responsible to most phytohormone treatments. Our data provided evolutionary and functional information of GhAPX gene family members and revealed that different members are responsible to redox homeostasis during different cotton fiber development stages.

Systematic comparison of lncRNAs with protein coding mRNAs in population expression and their response to environmental change.

BACKGROUND: Long non-coding RNA (lncRNA) is a class of non-coding RNA with important regulatory roles in biological process of organisms. The systematic comparison of lncRNAs with protein coding mRNAs in population expression and their response to environmental change are still poorly understood. Here we identified 17,610 lncRNAs and calculated their expression levels based on RNA-seq of 80 individuals of Miscanthus lutarioriparius from two environments, the nearly native habitats and transplanted field, respectively. RESULTS: LncRNAs had significantly higher expression diversity and lower expression frequency in population than protein coding mRNAs in both environments, which suggested that lncRNAs may experience more relaxed selection or divergent evolution in population compared with protein coding RNAs. In addition, the increase of expression diversity for lncRNAs was always significantly higher and the magnitude of fold change of expression in new stress environment was significantly larger than protein-coding mRNAs. These results suggested that lncRNAs may be more sensitive to environmental change than protein-coding mRNAs. Analysis of environment-robust and environment-specific lncRNA-mRNA co-expression network between two environments revealed the characterization of lncRNAs in response to environmental change. Furthermore, candidate lncRNAs contributing to water use efficiency (WUE) identified based on the WUE-lncRNA-mRNA co-expression network suggested the roles of lncRNAs in response to environmental change. CONCLUSION: Our study provided a comprehensive understanding of expression characterization of lncRNAs in population for M. lutarioriparius under field condition, which would be useful to explore the roles of lncRNAs and could accelerate the process of adaptation in new environment for many plants.

Cotton Ascorbate Oxidase Promotes Cell Growth in Cultured Tobacco Bright Yellow-2 Cells through Generation of Apoplast Oxidation.

Ascorbate oxidase (AO) plays an important role in cell growth through the modulation of reduction/oxidation (redox) control of the apoplast. Here, a cotton (Gossypium hirsutum) apoplastic ascorbate oxidase gene (GhAO1) was obtained from fast elongating fiber tissues. GhAO1 belongs to the multicopper oxidase (MCO) family and includes a signal peptide and several transmembrane regions. Analyses of quantitative real-time polymerase chain reaction (QRT-PCR) and enzyme activity showed that GhAO1 was expressed abundantly in 15-day post-anthesis (dpa) wild-type (WT) fibers in comparison with fuzzless-lintless (fl) mutant ovules. Subcellular distribution analysis in onion cells demonstrated that GhAO1 is localized in the cell wall. In transgenic tobacco bright yellow-2 (BY-2) cells with ectopic overexpression of GhAO1, the enhancement of cell growth with 1.52-fold increase in length versus controls was indicated, as well as the enrichment of both total ascorbate in whole-cells and dehydroascorbate acid (DHA) in apoplasts. In addition, promoted activities of AO and monodehydroascorbate reductase (MDAR) in apoplasts and dehydroascorbate reductase (DHAR) in whole-cells were displayed in transgenic tobacco BY-2 cells. Accumulation of H2O2, and influenced expressions of Ca2+ channel genes with the activation of NtMPK9 and NtCPK5 and the suppression of NtTPC1B were also demonstrated in transgenic tobacco BY-2 cells. Finally, significant induced expression of the tobacco NtAO gene in WT BY-2 cells under indole-3-acetic acid (IAA) treatment appeared; however, the sensitivity of the NtAO gene expression to IAA disappeared in transgenic BY-2 cells, revealing that the regulated expression of the AO gene is under the control of IAA. Taken together, these results provide evidence that GhAO1 plays an important role in fiber cell elongation and may promote cell growth by generating the oxidation of apoplasts, via the auxin-mediated signaling pathway.

iTRAQ-Based Quantitative Proteomic Analysis Reveals Cold Responsive Proteins Involved in Leaf Senescence in Upland Cotton (Gossypium hirsutum L.).

Premature leaf senescence occurs in the ultimate phase of the plant, and it occurs through a complex series of actions regulated by stress, hormones and genes. In this study, a proteomic analysis was performed to analyze the factors that could induce premature leaf senescence in two cotton cultivars. We successfully identified 443 differential abundant proteins (DAPs) from 7388 high-confidence proteins at four stages between non-premature senescence (NS) and premature senescence (PS), among which 158 proteins were over-accumulated, 238 proteins were down-accumulated at four stages, and 47 proteins displayed overlapped accumulation. All the DAPs were mapped onto 21 different categories on the basis of a Clusters of Orthologous Groups (COG) analysis, and 9 clusters were based on accumulation. Gene Ontology (GO) enrichment results show that processes related to stress responses, including responses to cold temperatures and responses to hormones, are significantly differentially accumulated. More importantly, the enriched proteins were mapped in The Arabidopsis Information Resource (TAIR), showing that 58 proteins play an active role in abiotic stress, hormone signaling and leaf senescence. Among these proteins, 26 cold-responsive proteins (CRPs) are significantly differentially accumulated. The meteorological data showed that the median temperatures declined at approximately 15 days before the onset of aging, suggesting that a decrease in temperature is tightly linked to an onset of cotton leaf senescence. Because accumulations of H2O2 and increased jasmonic acid (JA) were detected during PS, we speculate that two pathways associated with JA and H2O2 are closely related to premature leaf senescence in cotton.

Transcriptome-wide characterization of candidate genes for improving the water use efficiency of energy crops grown on semiarid land.

Understanding the genetic basis of water use efficiency (WUE) and its roles in plant adaptation to a drought environment is essential for the production of second-generation energy crops in water-deficit marginal land. In this study, RNA-Seq and WUE measurements were performed for 78 individuals of Miscanthus lutarioriparius grown in two common gardens, one located in warm and wet Central China near the native habitats of the species and the other located in the semiarid Loess Plateau, the domestication site of the energy crop. The field measurements showed that WUE of M. lutarioriparius in the semiarid location was significantly higher than that in the wet location. A matrix correlation analysis was conducted between gene expression levels and WUE to identify candidate genes involved in the improvement of WUE from the native to the domestication site. A total of 48 candidate genes were identified and assigned to functional categories, including photosynthesis, stomatal regulation, protein metabolism, and abiotic stress responses. Of these genes, nearly 73% were up-regulated in the semiarid site. It was also found that the relatively high expression variation of the WUE-related genes was affected to a larger extent by environment than by genetic variation. The study demonstrates that transcriptome-wide correlation between physiological phenotypes and expression levels offers an effective means for identifying candidate genes involved in the adaptation to environmental changes.

On the Flow of a Cement Suspension: The Effects of Nano-Silica and Fly Ash Particles.

Additives such as nano-silica and fly ash are widely used in cement and concrete materials to improve the rheology of fresh cement and concrete and the performance of hardened materials and increase the sustainability of the cement and concrete industry by reducing the usage of Portland cement. Therefore, it is important to study the effect of these additives on the rheological behavior of fresh cement. In this paper, we study the pulsating Poiseuille flow of fresh cement in a horizontal pipe by considering two different additives and when they are combined (nano-silica, fly ash, combined nano-silica, and fly ash). To model the fresh cement suspension, we used a modified form of the power-law model to demonstrate the dependency of the cement viscosity on the shear rate and volume fraction of cement and the additive particles. The convection-diffusion equation was used to solve for the volume fraction. After solving the equations in the dimensionless forms, we conducted a parametric study to analyze the effects of nano-silica, fly ash, and combined nano-silica and fly ash additives on the velocity and volume fraction profiles of the cement suspension. According to the parametric study presented here, larger nano-silica content results in lower centerline velocity of the cement suspension and larger non-uniformity of the volume fraction. Compared to nano-silica, fly ash exhibits an opposite effect on the velocity. Larger fly ash content results in higher centerline velocity, while the effect of the fly ash on the volume fraction is not obvious. For cement suspension containing combined nano-silica and fly ash additives, nano-silica plays a dominant role in the flow behavior of the suspension. The findings of the study can help the design and operation of the pulsating flow of fresh cement mortars and concrete in the 3D printing industry.

Macrophage-membrane-coated hybrid nanoparticles with self-supplied hydrogen peroxide for enhanced chemodynamic tumor therapy.

Addressing the challenges of chemodynamic therapies (CDTs) relying on Fenton reactions in malignant tumors is an active research area. Here, we report a method to develop pH-responsive hybrid nanoparticles for enhanced chemodynamic tumor treatment. Reactive CaO2 nanoparticles (core) are isolated by biocompatible ZIF-8 doped with Fe2+ (shell), and then encapsulated by macrophage membranes (symbolized as CaO2@Fe-ZIF-8@macrophage membrane or CFZM), thus endowed with high stability under normal physiological conditions. Our design features active tumor-homing by the macrophage-membrane coating, tumor microenvironment (TME)-responsive cargo release, and self-supplied hydrogen peroxide for promotion of the Fenton reaction. We demonstrate the improved delivery/tumor cell uptake of CFZM, the efficient production of toxic ˙OH with self-supplied H2O2 in CFZM, and high-efficacy tumor ablation on BALB/c mice bearing CT26 tumor cells. This offers a translational strategy to develop active tumor-targeting and TME-responsive nanotherapeutics with enhanced CDT against malignant tumors.

A super water-resistant MXene sponge flexible sensor for bifunctional sensing of physical and chemical stimuli.

Flexible wearable sensors with multifunctional features have attracted great interest in various applications such as disease diagnosis, environmental detection and healthcare monitoring. However, it is still a challenge to achieve a multifunctional sensor with super water resistance without compromising the overall performance of the sensing material. Here, we developed a 3D bifunctional flexible sensor based on an MXene melamine sponge (MS) through a simple and effective ultrasonic mixing process and a further vacuum annealing process. The sensor is able to show excellent response to different stimuli, including pressure and humidity. The thermal annealing treatment allows MXene to adhere more firmly to the internal skeleton of the sponge, which does not easily fall off and improves the water resistance, thus achieving wearability and high sensitivity over a wide area. The T-MXene@MS sensor has a sensitivity of 9.97 kPa-1 in the 5-15 kPa range, a fast response time (180 ms), and good stability at 4000 cycles, enabling accurate monitoring of human movement. The sensor has a rich porous structure while maintaining its inherent flexibility, which allows for long term testing of human respiration as well as the ability to respond quickly to dynamic changes in humidity, demonstrating excellent long-term stability for 40 days of humidity detection.

Efficient clearance of periodontitis pathogens by S. gordonii membrane-coated H2O2 self-supplied nanocomposites in a "Jenga" style.

As a key pathogen of periodontitis, P. gingivalis requires support of the initial colonizing bacterium (S. gordonii preferably) to form symbiotic biofilms on gingival tissues with enhanced antibiotic resistance. Here, we report a new strategy to treat periodontitis biofilms with S. gordonii membrane-coated H2O2 self-supplied nanocomposites (ZnO2/Fe3O4@MV NPs) in a "Jenga" style. Integration of our special MV coatings enables selectively enhanced internalization of the cargos in S. gordonii, thus inducing severe damage to the foundational bacterial layer and collapse/clearance of symbiotic biofilms consequently. This strategy allows us to clear the symbiotic biofilms of S. gordonii and P. gingivalis with active hydroxyl radicals (˙OH) derived from ZnO2-Fe3O4@MV NPs in a H2O2 self-supplied, nanocatalyst-assisted manner. This "Jenga-style" treatment provides a cutting-edge proof of concept for the removal of otherwise robust symbiotic biofilms of periodontitis where the critical pathogens are difficult to target and have antibiotic resistance.

Hypoxia-targeted and spatial-selective tumor suppression by near infrared nanoantenna sensitized engineered bacteria.

It is an active research area in the development of engineered bacteria to address the bottleneck issue of hypoxic tumors, which otherwisely possess resistance to chemotherapies, radiotherapies, and photodynamic therapies. Here we report a new method to ablate hypoxic tumors with NIR-nanoantenna sensitized engineered bacteria (NASEB) in a highly effective and dual selective manner. It features engineered E. coli MG1655 (EB) with coatings of lanthanide upconversion nanoparticles (UCNPs) as external antennas on bacterial surface (MG1655/HlyE-sfGFP@UCNP@PEG), enabling NIR laser-switchable generation/secretion of HlyE perforin to kill cancer cells. We have demonstrated that NASEB enrichment on hypoxic tumor sites via their innate chemotactic tendency, in assistance of localized NIR laser irradiation, can suppress tumors with improved efficacy and selectivity, thus minimizing potential side effects in cancer treatment. The NIR-responsive nanoantenna sensitized switching in engineering bacteria is distinct from the previous reports, promising conceptually new development of therapeutics against hypoxic tumors. STATEMENT OF SIGNIFICANCE: Tumor hypoxia exacerbates tumor progression, but also reduces the efficacy of conventional chemotherapies, radiotherapies, or photodynamic therapies. Here we develop near infrared Nano Antenna Sensitized Engineered Bacteria (NASEB) to treat hypoxic tumors. NASEB can accumulate and proliferate on hypoxic tumor sites via their innate chemotactic tendency. After receiving NIR laser signals, the upconversion nanoparticles on NASEB surface as antennas can transduce them to blue light for activation of HlyE perforin in the protein factory of EB. Our method features dual selectivity on the tumor sites, contributed by hypoxic tumor homing of anaerobic bacteria and spatial confinement through selective NIR laser irradiation. The concept of NASEB promises to address the challenges of tumor hypoxia for cancer therapies.

Single-Cell Enzymatic Screening for Epithelial Mesenchymal Transition with an Ultrasensitive Superwetting Droplet-Array Microchip.

The phenotypic changes of circulating tumor cells (CTCs) during the epithelial-mesenchymal transition (EMT) have been a hot topic in tumor biology and cancer therapeutic development. Here, an integrated platform of single-cell fluorescent enzymatic assays with superwetting droplet-array microchips (SDAM) for ultrasensitive functional screening of epithelial-mesenchymal sub-phenotypes of CTCs is reported. The SDAM can generate high-density, volume well-defined droplet (0.66 nL per droplet) arrays isolating single tumor cells via a discontinuous dewetting effect. It enables sensitive detection of MMP9 enzyme activities secreted by single tumor cells, correlating to their epithelial-mesenchymal sub-phenotypes. In the pilot clinical double-blind tests, the authors have demonstrated that SDAM assays allow for rapid identification and functional screening of CTCs with different epithelial-mesenchymal properties. The consistency with the clinical outcomes validates the usefulness of single-cell secreted MMP9 as a biomarker for selective CTC screening and tumor metastasis monitoring. Convenient addressing and recovery of individual CTCs from SDAM have been demonstrated for gene mutation sequencing, immunostaining, and transcriptome analysis, revealing new understandings of the signaling pathways between MMP9 secretion and the EMT regulation of CTCs. The SDAM approach combined with sequencing technologies promises to explore the dynamic EMT plasticity of tumors at the single-cell level.

Microsatellites for Tetracentron sinense (Trochodendraceae), a Tertiary relict endemic to East Asia.

PREMISE OF THE STUDY: Tetracentron sinense (Trochodendraceae) is a Tertiary relict endemic to East Asia. Microsatellite markers were developed and characterized to investigate the population genetics of the species. METHODS AND RESULTS: Microsatellite markers were isolated from the genome of T. sinense using the protocol of Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO). Eight polymorphic microsatellite markers were assessed in 44 samples collected from three wild populations. The number of alleles observed for each locus ranged from two to five. The observed and expected heterozygosities ranged from 0.0000 to 0.9375 and 0.0000 to 0.7681, respectively. CONCLUSIONS: The microsatellite markers will be helpful in further studies of the population genetics and phylogeography of T. sinense.

Microsatellite markers for the relictual dove tree, Davidia involucrata (Cornaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for the dove tree, Davidia involucrata (Cornaceae), a Tertiary relict currently endemic to China, to investigate its population genetics and phylogeography. METHODS AND RESULTS: Using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol, nine polymorphic microsatellite loci were identified and screened in 44 individuals from three wild populations of D. involucrata. The number of alleles per locus ranged from two to three, while the observed heterozygosity and expected heterozygosity ranged from 0.0000 to 0.6000 and from 0.0000 to 0.6323, respectively. CONCLUSIONS: These new microsatellite loci will facilitate further studies of the population genetics and phylogeography of D. involucrata, as well as of the evolutionary history of the plant and other Tertiary relicts endemic to East Asia.