Common-path multimodal three-dimensional fluorescence and phase imaging system.

A stable multimodal system is developed by combining two common-path digital holographic microscopes (DHMs): coherent and incoherent, for simultaneous recording and retrieval of three-dimensional (3-D) phase and 3-D fluorescence imaging (FI), respectively, of a biological specimen. The 3-D FI is realized by a single-shot common-path off-axis fluorescent DHM developed recently by our group. In addition, we accomplish, the phase imaging by another single-shot, highly stable common-path off-axis DHM based on a beam splitter. In this DHM configuration, a beam splitter is used to divide the incoming object beam into two beams. One beam serves as the object beam carrying the useful information of the object under study, whereas another beam is spatially filtered at its Fourier plane by using a pinhole and it serves as a reference beam. This DHM setup, owing to a common-path geometry, is less vibration-sensitive and compact, having a similar field of view but with high temporal phase stability in comparison to a two-beam Mach-Zehnder-type DHM. The performance of the proposed common-path DHM and the multimodal system is verified by conducting various experiments on fluorescent microspheres and fluorescent protein-labeled living cells of the moss Physcomitrella patens. Moreover, the potential capability of the proposed multimodal system for 3-D live fluorescence and phase imaging of the fluorescent beads is also demonstrated. The obtained experimental results corroborate the feasibility of the proposed multimodal system and indicate its potential applications for the analysis of functional and structural behaviors of a biological specimen and enhancement of the understanding of physiological mechanisms and various biological diseases.

PD-1 Imposes Qualitative Control of Cellular Transcriptomes in Response to T Cell Activation.

Targeted blockade of programmed cell death 1 (PD-1), an immune-checkpoint receptor that inhibits T cell activation, provides clinical benefits in various cancers. However, how PD-1 modulates gene expression in T cells remains enigmatic. Here we investigated how PD-1 affects transcriptome changes induced by T cell receptor (TCR) activation. Intriguingly, we identified a huge variance in PD-1 sensitivity among TCR-inducible genes. When we quantified the half maximal effective concentration (EC50) as the relationship between change in gene expression and TCR signal strength, we found that genes associated with survival and proliferation were efficiently expressed upon TCR activation and resistant to PD-1-mediated inhibition. Conversely, genes encoding cytokines and effector molecules were expressed less efficiently and sensitive to PD-1-mediated inhibition. We further demonstrated that transcription factor binding motifs and CpG frequency in the promoter region affect EC50 and thus the PD-1 sensitivity of genes. Our findings explain how PD-1, dependent on the TCR signal strength, calibrates cellular transcriptomes to shape functional properties of T cell populations.

MicroRNA-140 mediates RB tumor suppressor function to control stem cell-like activity through interleukin-6.

We established an in vitro cell culture system to determine novel activities of the retinoblastoma (Rb) protein during tumor progression. Rb depletion in p53-null mouse-derived soft tissue sarcoma cells induced a spherogenic phenotype. Cells retrieved from Rb-depleted spheres exhibited slower proliferation and less efficient BrdU incorporation, however, much higher spherogenic activity and aggressive behavior. We discovered six miRNAs, including mmu-miR-18a, -25, -29b, -140, -337, and -1839, whose expression levels correlated tightly with the Rb status and spherogenic activity. Among these, mmu-miR-140 appeared to be positively controlled by Rb and to antagonize the effect of Rb depletion on spherogenesis and tumorigenesis. Furthermore, among genes potentially targeted by mmu-miR-140, Il-6 was upregulated by Rb depletion and downregulated by mmu-mir-140 overexpression. Altogether, we demonstrate the possibility that mmu-mir-140 mediates the Rb function to downregulate Il-6 by targeting its 3'-untranslated region. Finally, we detected the same relationship among RB, hsa-miR-140 and IL-6 in a human breast cancer cell line MCF-7. Because IL-6 is a critical modulator of malignant features of cancer cells and the RB pathway is impaired in the majority of cancers, hsa-miR-140 might be a promising therapeutic tool that disrupts linkage between tumor suppressor inactivation and pro-inflammatory cytokine response.

[Effects of Influent C/N Ratios on Denitrifying Phosphorus Removal Performance Based on ABR-MBR Combined Process].

An ABR-MBR integrated reactor based on a combination of the anaerobic baffled reactor (ABR) with the microbial phase separation and membrane bioreactor (MBR) with high-effect entrapment was constructed and the circulation and interactivity of the combined process were examined by adding nitrate recycling and sludge reflux. By increasing the influent COD to adjust the COD/TN ratio, the influence of the mechanism on the denitrifying phosphorus removal performance under the condition of continuous-flow was investigated. The results showed that the average effluent concentration of soluble phosphorus under different influent C/N conditions were 0.22, 0.34, 0.39, 0.42, and 2.45 mg·L-1 and the low influent C/N ratio was beneficial to phosphate removal. When the influent C/N was 4.8-6.0, the average removal rates of COD, TN, and soluble PO43--P were more than 87%, 76%, and 93%. In addition, when the influent C/N ratio was 3.6-6.0, the removal of TN was proportional to the anoxic phosphorus uptake of ABR and conducive to the removal of TN after increasing the influent COD concentration. Higher C/N ratios of the influent improved the removal of TN at this stage. Finally, the C/N ratio of 6 was suggested to achieve the simultaneous removal of nitrogen and phosphorus.

[Quick Start-up of Anaerobic Ammonium Oxidation Process].

In order to study the quick start-up process of anaerobic ammonium oxidation (ANAMMOX), two types of reactors with different hydraclic flow state inoculated with aerobic nitrifying sludge were investigated, the membrane bioreactor (MBR) and anaerobic baffled reactor (ABR), respectively. The result showed that both reactors successfully started up ANAMMOX process. The start-up period of the MBR (90 d) was 20% shorter than the ABR (111 d). During the stable operation, the average nitrogen (NH4+-N+NO2--N) removal capacity of 0.098 kg·(m3·d)-1 in the MBR was also higher than that of 0.089 kg·(m3·d)-1 in the ABR. In addition, the differences of sludge morphology were obvious in the two reactors; flocculent sludge was developed in the MBR while ANAMMOX granular sludge was mainly formed in the first compartment of the ABR. Moreover, the quantitative relationship analysis between NH4+-N, NO2--N and NO3--N showed that the MBR system contained more kinds of bacteria with nitrogen removal function, compared to the ABR, and it was therefore more conducive to the removal of nitrogen. MBR exhibited a more excellent performance for ANAMMOX start-up.

[Optimization of Denitrifying Phosphorus Removal Performance Based on ABR-MBR Combined Process].

An integrated process based on combination of the anaerobic baffled reactor(ABR)-membrane bioreactor(MBR) was adopted to treat domestic sewage with low C/N ratio. In order to realize the function of highly efficient denitrifying phosphorus removal, nitrate recycling ratio and sludge recycle ratio were optimized in this study. The results indicated that the optimized denitrifying phosphorus removal efficiency was achieved under the conditions of organic loading rate of 2.0 kg·(m3·d)-1 in ABR, total hydraulic retention time (HRT) of ABR-MBR at 9 h, the SRT at 15 d, sludge reflux ratio of 100%, and nitrate recycling ratios set to 300%. The average removal rates of TN and soluble PO43--P were 84% and 94%, the amount of phosphorus removed by denitrifying accounted for 87% of the total phosphorus removed, and the average effluent concentration for TN and soluble phosphorus were 12.98 mg·L-1 and 0.43 mg·L-1 respectively.

Loss of function mutations in the rice chromomethylase OsCMT3a cause a burst of transposition.

Methylation patterns of plants are unique as, in addition to the methylation at CG dinucleotides that occurs in mammals, methylation also occurs at non-CG sites. Genes are methylated at CG sites, but transposable elements (TEs) are methylated at both CG and non-CG sites. The role of non-CG methylation in transcriptional silencing of TEs is being extensively studied at this time, but only very rare transpositions have been reported when non-CG methylation machineries have been compromised. To understand the role of non-CG methylation in TE suppression and in plant development, we characterized rice mutants with changes in the chromomethylase gene, OsCMT3a. oscmt3a mutants exhibited a dramatic decrease in CHG methylation, changes in the expression of some genes and TEs, and pleiotropic developmental abnormalities. Genome resequencing identified eight TE families mobilized in oscmt3a during normal propagation. These TEs included tissue culture-activated copia retrotransposons Tos17 and Tos19 (Lullaby), a pericentromeric clustered high-copy-number non-autonomous gypsy retrotransposon Dasheng, two copia retrotransposons Osr4 and Osr13, a hAT-tip100 transposon DaiZ, a MITE transposon mPing, and a LINE element LINE1-6_OS. We confirmed the transposition of these TEs by polymerase chain reaction (PCR) and/or Southern blot analysis, and showed that transposition was dependent on the oscmt3a mutation. These results demonstrated that OsCMT3a-mediated non-CG DNA methylation plays a critical role in development and in the suppression of a wide spectrum of TEs. These in planta mobile TEs are important for studying the interaction between TEs and the host genome, and for rice functional genomics.

[Shortcut Nitrosation-Denitrifying Phosphorus Removal Based on High-quality Carbon Source in Combined Process of CAMBR].

The volatile fatty acids (VFAs) are high-quality carbon source which can be utilized in the process of denitrifying phosphorus removal. Therefore, a combined process of anaerobic baffled reactor (ABR) with microbial phase separation and membrane bioreactor (MBR), i. e. CAMBR, with high-quality effluent was investigated in this experiment for highly efficient denitrifying phosphorus removal through providing high-quality carbon source by optimizing operational conditions such as hydraulic retention time (HRT) in the ABR. The results indicated that sufficient high-quality carbon source VFA could be acquired when the HRT of ABR was 4.8 h. The consumption of VFA was 56. 1 mg · L⁻¹ while the release of phosphorus was 10.43 mg · L⁻¹ showing that 5.38 mg of VFA was required to release 1 mg PO4³â»-P. Meanwhile, the uptake of phosphorus was 12. 35 mg · L⁻¹, while the uptake of phosphorus of MBR tank was 1.33 mg · L⁻¹. In the process of shortcut nitrification, 0.62 mg NOx⁻-N was needed when 1 mg PO4³â»-P was taken up, and 1.67 to 2.04 mg NO2⁻-N was required when 1 mg PO4³â»-P was taken up, and the combined system achieved stable effluent quality. The average removal rates of COD, TN and soluble PO4³â»-P were 91%, 84% and 93%, respectively, correspondingly the average effluent concentrations were 30, 7.15 and 0.55 mg · L⁻¹, respectively. The results stated that the CAMBR achieved stable and highly efficient denitrifying phosphorus removal in domestic sewage treatment.

The Selaginella genome identifies genetic changes associated with the evolution of vascular plants.

Vascular plants appeared ~410 million years ago, then diverged into several lineages of which only two survive: the euphyllophytes (ferns and seed plants) and the lycophytes. We report here the genome sequence of the lycophyte Selaginella moellendorffii (Selaginella), the first nonseed vascular plant genome reported. By comparing gene content in evolutionarily diverse taxa, we found that the transition from a gametophyte- to a sporophyte-dominated life cycle required far fewer new genes than the transition from a nonseed vascular to a flowering plant, whereas secondary metabolic genes expanded extensively and in parallel in the lycophyte and angiosperm lineages. Selaginella differs in posttranscriptional gene regulation, including small RNA regulation of repetitive elements, an absence of the trans-acting small interfering RNA pathway, and extensive RNA editing of organellar genes.

Study of laser noises corresponding to different feedback configurations for blue-light optical pickup heads.

Based on the technology of real-time laser noise measurement which had been developed for the red-light DVD optical storage system before, this study investigates the laser noises corresponding to different feedback configurations by using the optical system of a blue-light HD-DVD optical pickup head (OPH) with the numerical aperture (NA) of 0.65 and the laser wavelength of 405 nm. It combines with a digital servo controller for dynamically measuring the blue laser noise while operated in an OPH. Experimental results show the variations of relative intensity noise (RIN) values corresponding to focusing errors, lens off-axis errors, disk rotation speeds, and laser emitting powers, respectively.

Phylogenetic analysis of Oryza rufipogon strains and their relations to Oryza sativa strains by insertion polymorphism of rice SINEs.

Oryza rufipogon, the progenitor of the cultivated rice species Oryza sativa, is known by its wide intraspecific variation. In this study, we performed phylogenetic analyses of O. rufipogon strains and their relationships to O. sativa strains by using 26 newly identified p-SINE1 members from O. rufipogon strains, in addition to 23 members previously identified from O. sativa strains. A total of 103 strains of O. rufipogon and O. sativa were examined for the presence and absence of each of the p-SINE1 members at respective loci by PCR with a pair of primers that hybridize to the regions flanking each p-SINE1 member. A phylogenetic tree constructed on the basis of the insertion polymorphism of p-SINE1 members showed that O. rufipogon and O. sativa strains are classified into three groups. The first group consisted of O. rufipogon perennial strains mostly from China and O. sativa ssp. japonica strains, which included javanica strains forming a distinct subgroup. The second group consisted of almost all the O. rufipogon annual strains, a few O. rufipogon perennial strains and O. sativa ssp. indica strains. These groupings, in addition to other results, support the previous notion that annual O. rufipogon originated in the O. rufipogon perennial population, and that O. sativa originated polyphyletically in the O. rufipogon populations. The third group consisted of the other perennial strains and intermediate-type strains of O. rufipogon, in which the intermediate-type strains are most closely related to a hypothetical ancestor with no p-SINE1 members at the respective loci and to those belonging to the other rice species with the AA genome. This suggests that O. rufipogon perennial strains are likely to have originated from the O. rufipogon intermediate-ecotype population.

Epigenetic regulation of the rice retrotransposon Tos17.

Transposable elements are major components of plant genomes. Their activity seems to be epigenetically regulated by gene silencing systems. Here we report epigenetic variation in the retrotransposon Tos17 activity in rice varieties. Of the two copies of Tos17 present in chromosome 7 (Tos17 (chr.7)) and chromosome 10 (Tos17 (chr.10)), Tos17 (chr.7) is strongly activated by tissue culture in most varieties including Nipponbare except for Moritawase, despite the identity of the DNA sequences in Moritawase and Nipponbare. Tos17 (chr.7) activity correlated with its methylation status, and Tos17 (chr.7 )in Moritawase was heavily methylated and activated by treatment of 5-azacytidine (5-azaC), a DNA methylation inhibitor. Although the original copies of Tos17 are methylated to some extent in all varieties examined, the transposed copies in calli mostly are not methylated. When plants were regenerated from calli, the degree of methylation of the Tos17 DNA increased gradually with the growth of plants, and a significant progress of DNA methylation occurred in the next generation after a completed reproductive cycle. With increasing DNA methylation, the transcription of transposed and original Tos17 copies driven by its own as well as by a flanking gene promoter were suppressed. We conclude that Tos17 DNA methylation controls the transpositional activity of Tos17, and modulates the activity of neighboring genes. Based on the analysis of the inactive Tos17 (chr.10), we propose that another mechanism, called transcriptional interference, is involved in the control of Tos17 activity.

Polyphyletic origin of cultivated rice: based on the interspersion pattern of SINEs.

The wild rice species Oryza rufipogon with wide intraspecific variation is thought to be the progenitor of the cultivated rice species Oryza sativa with two ecotypes, japonica and indica. To determine the origin of cultivated rice, subfamily members of the rice retroposon p-SINE1, which show insertion polymorphism in the O. sativa -O. rufipogon population, were identified and used to "bar code" each of 101 cultivated and wild rice strains based on the presence or absence of the p-SINE1 members at the respective loci. A phylogenetic tree constructed based on the bar codes given to the rice strains showed that O. sativa strains were classified into two groups corresponding to japonica and indica, whereas O. rufipogon strains were in four groups, in which annual O. rufipogon strains formed a single group, differing from the perennial O. rufipogon strains of the other three groups. Japonica strains were closely related to the O. rufipogon perennial strains of one group, and the indica strains were closely related to the O. rufipogon annual strains, indicating that O. sativa has been derived polyphyletically from O. rufipogon. The subfamily members of p-SINE1 constitute a powerful tool for studying the classification and relationship of rice strains, even when one has limited knowledge of morphology, taxonomy, physiology, and biochemistry of rice strains.

Evolutionary relationships among rice species with AA genome based on SINE insertion analysis.

Previous studies based on morphological and molecular markers indicated that there are two cultivated and five wild rice species within the Oryza genus with the AA genome. In the cultivated rice species, Oryza sativa, a retroposon named p-SINE1 has been identified. Some of the p-SINE1 members characterized previously showed interspecific insertion polymorphisms in the species with the AA genome. In this study, we identified new p-SINE1 members showing interspecific insertion polymorphisms from representative strains of four wild rice species with the AA genome: O. barthii, O. glumaepatula, O. longistaminata, and O. meridionalis. Some of these members were present only in strains of one species, whereas the others were present in strains of two or more species. The p-SINE1 insertion patterns in the strains of the Asian and African cultivated rice species O. sativa and O. glaberrima were very similar to those of the Asian and African wild rice species O. rufipogon and O. barthii, respectively. This is consistent with the previous hypothesis that O. sativa and O. glaberrima are derived from specific wild rice species. Phylogenetic analysis based on the p-SINE1 insertion patterns showed that the strains of each of the five wild rice species formed a cluster. The strains of O. longistaminata appear to be distantly related to those of O. meridionalis. The strains of these two species appear to be distantly related to those of three other species, O. rufipogon, O. barthii and O. glumaepatula. The latter three species are closely related to one another with O. barthii and O. glumaepatula being most closely related. A phylogenetic tree including a hypothetical ancestor with all loci empty for p-SINE1 insertion showed that the strains of O. longistaminata are related most closely to the hypothetical ancestor. This indicates that O. longistaminata and O. meridionalis diverged early on, whereas the other species diverged relatively recently, and suggests that the Oryza genus with AA genome might have originated in Africa, rather than in Asia.