High-yield BMP2 expression in rice cells via CRISPR and endogenous αAmy3 promoter.

Plant cells serve as versatile platforms for the production of high-value recombinant proteins. This study explored the efficacy of utilizing an endogenous αAmy3 promoter for the expression of a bioactive pharmaceutical protein, specifically the mature region of human bone morphogenetic protein 2 (hBMP2m). Utilizing a refined CRISPR/Cas9-mediated intron-targeting insertion technique, which incorporates an artificial 3' splicing site upstream of the target gene, we achieved a transformation efficiency of 13.5% in rice calli that carried the rice-codon optimized mature region of hBMP2 cDNA (rhBMP2m) in the αAmy3 intron 1. Both homozygous and heterozygous rhBMP2m knock-in rice suspension cell lines were generated. These lines demonstrated the endogenous αAmy3 promoter regulated rhBMP2m mRNA and rhBMP2m recombinant protein expression, with strongly upregulation in respond to sugar depletion. The homozygous rhBMP2m knock-in cell line yielded an impressive 21.5 µg/mL of rhBMP2m recombinant protein, accounting for 1.03% of the total soluble protein. The high-yield expression was stably maintained across two generations, indicating the genetic stability of rhBMP2m gene knock-in at the αAmy3 intron 1 locus. Additionally, the rice cell-derived rhBMP2m proteins were found to be glycosylated, capable of dimer formation, and bioactive. Our results indicate that the endogenous rice αAmy3 promoter-signal peptide-based expression system is an effective strategy for producing bioactive pharmaceutical proteins. KEY POINTS: • The endogenous αAmy3 promoter-based expression system enhanced the yield of BMP2 • The increased yield of BMP2 accounted for 1.03% of the total rice-soluble proteins • The rice-produced BMP2 showed glycosylation modifications, dimer formation, and bioactivity.

Expression of Recombinant Human Octamer-Binding Transcription Factor 4 in Rice Suspension Cells.

The rice cell suspension culture system is a good way to produce recombinant human proteins, owing to its high biosafety and low production cost. Human Octamer-binding Transcription Factor 4 (Oct4) is a fundamental transcription factor responsible for maintaining human pluripotent embryonic stem cells. Recombinant Oct4 protein has been used to induce pluripotent stem cells. In this study, recombinant Oct4 proteins are produced via a sugar starvation-inducible αAmy3/RAmy3D promoter-signal peptide-based rice recombinant protein expression system. Oct4 mRNAs accumulate in the transgenic rice suspension cells under sugar starvation. The Oct4 recombinant protein is detected in the transgenic rice suspension cells, and its highest yield is approximately 0.41% of total cellular soluble proteins after one day of sugar starvation. The rice cell-synthesized recombinant human Oct4 protein show DNA-binding activity in vitro, which implies that the protein structure is correct for enabling specific binding to the target DNA motif.

Saussurea involucrata (Snow Lotus) ICE1 and ICE2 Orthologues Involved in Regulating Cold Stress Tolerance in Transgenic Arabidopsis.

As with other environmental stresses, cold stress limits plant growth, geographical distribution, and agricultural productivity. CBF/DREB (CRT-binding factors/DRE-binding proteins) regulate tolerance to cold/freezing stress across plant species. ICE (inducer of CBF expression) is regarded as the upstream inducer of CBF expression and plays a crucial role as a main regulator of cold acclimation. Snow lotus (Saussurea involucrata) is a well-known traditional Chinese herb. This herb is known to have greater tolerance to cold/freezing stress compared to other plants. According to transcriptome datasets, two putative ICE homologous genes, SiICE1 and SiICE2, were identified in snow lotus. The predicted SiICE1 cDNA contains an ORF of 1506 bp, encoding a protein of 501 amino acids, whereas SiICE2 cDNA has an ORF of 1482 bp, coding for a protein of 493 amino acids. Sequence alignment and structure analysis show SiICE1 and SiICE2 possess a S-rich motif at the N-terminal region, while the conserved ZIP-bHLH domain and ACT domain are at the C-terminus. Both SiICE1 and SiICE2 transcripts were cold-inducible. Subcellular localization and yeast one-hybrid assays revealed that SiICE1 and SiICE2 are transcriptional regulators. Overexpression of SiICE1 (35S::SiICE1) and SiICE2 (35S::SiICE2) in transgenic Arabidopsis increased the cold tolerance. In addition, the expression patterns of downstream stress-related genes, CBF1, CBF2, CBF3, COR15A, COR47, and KIN1, were up-regulated when compared to the wild type. These results thus provide evidence that SiICE1 and SiICE2 function in cold acclimation and this cold/freezing tolerance may be regulated through a CBF-controlling pathway.

Genetic engineering of transitory starch accumulation by knockdown of OsSEX4 in rice plants for enhanced bioethanol production.

Rice straw, a common agricultural waste, is used as a potential feedstock for bioethanol production. Currently, bioethanol is made mostly from the microbial fermentation of starch-containing raw materials. Therefore, genetically engineered starch-excess rice straw through interference of starch degradation as a potential strategy to enhance bioethanol production was evaluated in this study. Arabidopsis Starch Excess 4 (SEX4) encodes a chloroplast-localized glucan phosphatase and plays a role in transitory starch degradation. Despite the identification of a SEX4 homolog in rice, OsSEX4, its biological function remains uncertain. Ectopic expression of OsSEX4 complementary DNA complemented the leaf starch-excess phenotype of the Arabidopsis sex4-4 mutant. OsSEX4-knockdown transgenic rice plants were generated using the RNA interference approach. Starch accumulation was higher in OsSEX4-knockdown suspension-cultured cells, leaves, and rice straw compared with the wild type, suggesting that OsSEX4 plays an important role in degradation of transitory starch. The OsSEX4-knockdown rice plants showed normal plant growth and no yield penalty. Starch-excess OsSEX4-knockdown rice straw used as feedstock for fermentation resulted in improved bioethanol yield, with a 50% increase in ethanol production in a vertical mass-flow type bioreactor, compared with that of the wild-type straw.

The DEAD-Box RNA Helicase AtRH7/PRH75 Participates in Pre-rRNA Processing, Plant Development and Cold Tolerance in Arabidopsis.

DEAD-box RNA helicases belong to an RNA helicase family that plays specific roles in various RNA metabolism processes, including ribosome biogenesis, mRNA splicing, RNA export, mRNA translation and RNA decay. This study investigated a DEAD-box RNA helicase, AtRH7/PRH75, in Arabidopsis. Expression of AtRH7/PRH75 was ubiquitous; however, the levels of mRNA accumulation were increased in cell division regions and were induced by cold stress. The phenotypes of two allelic AtRH7/PRH75-knockout mutants, atrh7-2 and atrh7-3, resembled auxin-related developmental defects that were exhibited in several ribosomal protein mutants, and were more severe under cold stress. Northern blot and circular reverse transcription-PCR (RT-PCR) analyses indicated that unprocessed 18S pre-rRNAs accumulated in the atrh7 mutants. The atrh7 mutants were hyposensitive to the antibiotic streptomycin, which targets ribosomal small subunits, suggesting that AtRH7 was also involved in ribosome assembly. In addition, the atrh7-2 and atrh7-3 mutants displayed cold hypersensitivity and decreased expression of CBF1, CBF2 and CBF3, which might be responsible for the cold intolerance. The present study indicated that AtRH7 participates in rRNA biogenesis and is also involved in plant development and cold tolerance in Arabidopsis.

A single-repeat MYB transcription repressor, MYBH, participates in regulation of leaf senescence in Arabidopsis.

Leaf senescence, the final stage of leaf development, is regulated tightly by endogenous and environmental signals. MYBS3, a MYB transcription factor with a single DNA-binding domain, mediates sugar signaling in rice. Here we report that an Arabidopsis MYBS3 homolog, MYBH, plays a critical role in developmentally regulated and dark-induced leaf senescence by repressing transcription. Expression of MYBH was enhanced in older and dark-treated leaves. Gain- and loss-of-function analysis indicated that MYBH was involved in the onset of leaf senescence. Plants constitutively overexpressing MYBH underwent premature leaf senescence and showed enhanced expression of leaf senescence marker genes. In contrast, the MYBH mutant line, mybh-1, exhibited a delayed-senescence phenotype. The EAR repression domain was required for MYBH-regulated leaf senescence. Overexpression and knockout of MYBH repressed and enhanced auxin-responsive gene expression, respectively. MYBH repressed the auxin-amido synthase genes DFL1/GH3.6 and DFL2/GH3.10, which regulate auxin homoeostasis, by binding directly to the TA box in each of their regulatory regions. An auxin-responsive phenotype was enhanced in MYBH overexpression lines and reduced in mybh knockout lines. Overexpression of MYBH enhanced gene expression of SAUR36, an auxin-promoted leaf senescence key regulator, and accelerated ABA- and ethylene-induced leaf senescence in transgenic Arabidopsis plants. Our results suggest that the role of MYBH in controlling auxin homeostasis accounts for its capacity to participate in regulation of age- and darkness-induced leaf senescence in Arabidopsis.

Overexpressing Ferredoxins in Chlamydomonas reinhardtii Increase Starch and Oil Yields and Enhance Electric Power Production in a Photo Microbial Fuel Cell.

Ferredoxins (FDX) are final electron carrier proteins in the plant photosynthetic pathway, and function as major electron donors in diverse redox-driven metabolic pathways. We previously showed that overexpression of a major constitutively expressed ferredoxin gene PETF in Chlamydomonas decreased the reactive oxygen species (ROS) level and enhanced tolerance to heat stress. In addition to PETF, an endogenous anaerobic induced FDX5 was overexpressed in transgenic Chlamydomonas lines here to address the possible functions of FDX5. All the independent FDX transgenic lines showed decreased cellular ROS levels and enhanced tolerance to heat and salt stresses. The transgenic Chlamydomonas lines accumulated more starch than the wild-type line and this effect increased almost three-fold in conditions of nitrogen depletion. Furthermore, the lipid content was higher in the transgenic lines than in the wild-type line, both with and without nitrogen depletion. Two FDX-overexpressing Chlamydomonas lines were assessed in a photo microbial fuel cell (PMFC); power density production by the transgenic lines was higher than that of the wild-type cells. These findings suggest that overexpression of either PETF or FDX5 can confer tolerance against heat and salt stresses, increase starch and oil production, and raise electric power density in a PMFC.

Divergence of the expression and subcellular localization of CCR4-associated factor 1 (CAF1) deadenylase proteins in Oryza sativa.

Deadenylation, also called poly(A) tail shortening, is the first, rate-limiting step in the general cytoplasmic mRNA degradation in eukaryotic cells. The CCR4-NOT complex, containing the two key components carbon catabolite repressor 4 (CCR4) and CCR4-associated factor 1 (CAF1), is a major player in deadenylation. CAF1 belongs to the RNase D group in the DEDD superfamily, and is a protein conserved through evolution from yeast to humans and plants. Every higher plant, including Arabidopsis and rice, contains a CAF1 multigene family. In this study, we identified and cloned four OsCAF1 genes (OsCAF1A, OsCAF1B, OsCAF1G, and OsCAF1H) from rice. Four recombinant OsCAF1 proteins, rOsCAF1A, rOsCAF1B, rOsCAF1G, and rOsCAF1H, all exhibited 3'-5' exonuclease activity in vitro. Point mutations in the catalytic residues of each analyzed recombinant OsCAF1 proteins were shown to disrupt deadenylase activity. OsCAF1A and OsCAF1G mRNA were found to be abundant in the leaves of mature plants. Two types of OsCAF1B mRNA transcript were detected in an inverse expression pattern in various tissues. OsCAF1B was transient, induced by drought, cold, abscisic acid, and wounding treatments. OsCAF1H mRNA was not detected either under normal conditions or during most stress treatments, but only accumulated during heat stress. Four OsCAF1-reporter fusion proteins were localized in both the cytoplasm and nucleus. In addition, when green fluorescent protein fused with OsCAF1B, OsCAF1G, and OsCAF1H, respectively, fluorescent spots were observed in the nucleolus. OsCAF1B fluorescent fusion proteins were located in discrete cytoplasmic foci and fibers. We present evidences that OsCAF1B colocalizes with AtXRN4, a processing body marker, and AtKSS12, a microtubules maker, indicating that OsCAF1B is a component of the plant P-body and associate with microtubules. Our findings provide biochemical evidence that OsCAF1 proteins may be involved in the deadenylation in rice. The unique expression patterns of each OsCAF1 were observed in various tissues when undergoing abiotic stress treatments, implying that each CAF1 gene in rice plays a specific role in the development and stress response of a plant.

RNA-based detection of genetically modified plants via current-voltage characteristic measurement.

The widespread adoption of genetically modified (GM) crops has escalated concerns about their safety and ethical implications, underscoring the need for efficient GM crop detection methods. Conventional detection methods, such as polymerase chain reaction, can be costly, lab-bound, and time-consuming. To overcome these challenges, we have developed RapiSense, a cost-effective, portable, and sensitive biosensor platform. This sensor generates a measurable voltage shift (0.1-1 V) in the system's current-voltage characteristics, triggered by an increase in membrane's negative charge upon hybridization of DNA/RNA targets with a specific DNA probe. Probes designed to identify the herbicide resistance gene hygromycin phosphotransferase show a detection range from ∼1 nM to ∼10 µM and can discriminate between complementary, non-specific, and mismatched nucleotide targets. The incorporation of a small membrane sensor to detect fragmented RNA samples substantially improve the platform's sensitivity. In this study, RapiSense has been effectively used to detect specific DNA and fragmented RNA in transgenic variants of Arabidopsis, sweet potato, and rice, showcasing its potential for rapid, on-site GM crop screening.

Sugar starvation- and GA-inducible calcium-dependent protein kinase 1 feedback regulates GA biosynthesis and activates a 14-3-3 protein to confer drought tolerance in rice seedlings.

Germination followed by seedling growth constitutes two essential steps in the initiation of a new life cycle in plants, and in cereals, completion of these steps is regulated by sugar starvation and the hormone gibberellin. A calcium-dependent protein kinase 1 gene (OsCDPK1) was identified by differential screening of a cDNA library derived from sucrose-starved rice suspension cells. The expression of OsCDPK1 was found to be specifically activated by sucrose starvation among several stress conditions tested as well as activated transiently during post-germination seedling growth. In gain- and loss-of-function studies performed with transgenic rice overexpressing a constitutively active or RNA interference gene knockdown construct, respectively, OsCDPK1 was found to negatively regulate the expression of enzymes essential for GA biosynthesis. In contrast, OsCDPK1 activated the expression of a 14-3-3 protein, GF14c. Overexpression of either constitutively active OsCDPK1 or GF14c enhanced drought tolerance in transgenic rice seedlings. Hence, our studies demonstrated that OsCDPK1 transduces the post-germination Ca(2+) signal derived from sugar starvation and GA, refines the endogenous GA concentration and prevents drought stress injury, all essential functions to seedling development at the beginning of the life cycle in rice.

Improving pharmaceutical protein production in Oryza sativa.

Application of plant expression systems in the production of recombinant proteins has several advantages, such as low maintenance cost, absence of human pathogens, and possession of complex post-translational glycosylation capabilities. Plants have been successfully used to produce recombinant cytokines, vaccines, antibodies, and other proteins, and rice (Oryza sativa) is a potential plant used as recombinant protein expression system. After successful transformation, transgenic rice cells can be either regenerated into whole plants or grown as cell cultures that can be upscaled into bioreactors. This review summarizes recent advances in the production of different recombinant protein produced in rice and describes their production methods as well as methods to improve protein yield and quality. Glycosylation and its impact in plant development and protein production are discussed, and several methods of improving yield and quality that have not been incorporated in rice expression systems are also proposed. Finally, different bioreactor options are explored and their advantages are analyzed.

Overexpression of ferredoxin, PETF, enhances tolerance to heat stress in Chlamydomonas reinhardtii.

Reactive oxygen species (ROS) produced by plants in adverse environments can cause damage to organelles and trigger cell death. Removal of excess ROS can be achieved through the ascorbate scavenger pathway to prevent plant cell death. The amount of this scavenger can be regulated by ferredoxin (FDX). Chloroplastic FDXs are electron transfer proteins that perform in distributing photosynthetic reducing power. In this study, we demonstrate that overexpression of the endogenous photosynthetic FDX gene, PETF, in Chlamydomonas reinhardtii could raise the level of reduced ascorbate and diminish H2O2 levels under normal growth conditions. Furthermore, the overexpressing PETF transgenic Chlamydomonas lines produced low levels of H2O2 and exhibited protective effects that were observed through decreased chlorophyll degradation and increased cell survival under heat-stress conditions. The findings of this study suggest that overexpression of PETF can increase the efficiency of ROS scavenging in chloroplasts to confer heat tolerance. The roles of PETF in the downregulation of the ROS level offer a method for potentially improving the tolerance of crops against heat stress.

Production of mouse granulocyte-macrophage colony-stimulating factor by gateway technology and transgenic rice cell culture.

To establish a production platform for recombinant proteins in rice suspension cells, we first constructed a Gateway-compatible binary T-DNA destination vector. It provided a reliable and effective method for the rapid directional cloning of target genes into plant cells through Agrobacterium-mediated transformation. We used the approach to produce mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) in a rice suspension cell system. The promoter for the αAmy3 amylase gene, which is induced strongly by sugar depletion, drove the expression of mGM-CSF. The resulting recombinant protein was fused with the αAmy3 signal peptide and was secreted into the culture medium. The production of rice-derived mGM-CSF (rmGM-CSF) was scaled up successfully in a 2-L bioreactor, in which the highest yield of rmGM-CSF was 24.6 mg/L. Due to post-translational glycosylation, the molecular weight of rmGM-CSF was larger than that of recombinant mGM-CSF produced in Escherichia coli. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60.

Applications of CRISPR/Cas9 in a rice protein expression system via an intron-targeted insertion approach.

The sugar starvation-inducible rice αAmy3 promoter and signal peptide are widely used to produce valuable recombinant proteins in rice suspension culture cells. Conventionally, the recombinant gene expression cassette is inserted into the genome at random locations by Agrobacterium- or particle bombardment-mediated transformation. CRISPR/Cas9 gene editing enables gene insertion at a precise target site in the genome. In this study the CRISPR/Cas9 approach was modified for intron-targeted insertion by adding an artificial 3' splicing site upstream of the recombinant gene. Knock-in transgenic rice cell lines containing the recombinant GFP gene inserted in intron 1 of αAmy3 were generated. The endogenous αAmy3 promoter regulated recombinant gene expression and the αAmy3 signal peptide directed secretion of the recombinant GFP protein into the culture medium. In addition, the recombinant GFP protein was localized in amyloplasts, identical to the subcellular localization of endogenous αAmy3 reported previously. This modified CRISPR/Cas9 knock-in approach is simple and highly efficient, and the recombinant gene insertion frequency attained 12.5%. The approach can be applied in the production of pharmaceutical proteins in rice suspension cell cultures. The high efficiency of the GFP reporter gene knock-in method and the maintenance of target gene behavior also make the strategy applicable to endogenous gene functional studies in rice.

Knockdown expression of a MYB-related transcription factor gene, OsMYBS2, enhances production of recombinant proteins in rice suspension cells.

BACKGROUND: Transgenic plant suspension cells show economic potential for the production of valuable bioproducts. The sugar starvation-inducible rice αAmy3 promoter, together with its signal peptide, is widely applied to produce recombinant proteins in rice suspension cells. The OsMYBS2 transcription factor was shown recently to reduce activation of the αAmy3 promoter by competing for the binding site of the TA box of the αAmy3 promoter with the potent OsMYBS1 activator. In this study, rice suspension cells were genetically engineered to silence OsMYBS2 to enhance the production of recombinant proteins. RESULTS: The mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) gene was controlled by the αAmy3 promoter and expressed in OsMYBS2-silenced transgenic rice suspension cells. Transcript levels of the endogenous αAmy3 and the transgene mGM-CSF were increased in the OsMYBS2-silenced suspension cells. The highest yield of recombinant mGM-CSF protein attained in the OsMYBS2-silenced transgenic suspension cells was 69.8 µg/mL, which is 2.5-fold that of non-silenced control cells. The yield of recombinant mGM-CSF was further increased to 118.8 µg/mL in cultured cells derived from homozygous F5 seeds, which was 5.1 times higher than that of the control suspension cell line. CONCLUSIONS: Our results demonstrate that knockdown of the transcription factor gene OsMYBS2 increased the activity of the αAmy3 promoter and improved the yield of recombinant proteins secreted in rice cell suspension cultures.

[Expression and Significance of BLIMP-1 in Regulatory T Cells of Children with Aplastic Anemia].

OBJECTIVE: To study the expression of B lymphocyte-induced mature protein-1 (BLIMP-1) in regulatory T cells (Tregs) of children with aplastic anemia (AA), and analyze its correlation with the number of Tregs and the levels of inhibitory cytokines interleukin (IL)-10 and transforming growth factor (TGF)-ß in plasma. METHODS: The peripheral blood samples of 10 newly diagnosed AA children and 10 healthy children were collected for experiment. qPCR was used to detect FOXP3 and PRDM1 mRNA expression levels. Flow cytometry was used to detect the proportion of Tregs, the expression of BLIMP-1 in Tregs, and the levels of cytokines such as IL-2, IL-17A, IL-6, interferon (IFN)-γ, IL-10 and TGF-ß in plasma. Pearson correlation model was used to evaluate the relationship between the expression of BLIMP-1 in Treg and the number of Tregs, as well as the levels of IL-10 and TGF-ß in plasma. RESULTS: Compared with control group, the proportion of Tregs in peripheral blood of AA children was decreased significantly (P<0.001); The plasma levels of proinflammatory cytokines IL-2, IL-6 and IFN-γ in AA children were increased significantly (P=0.033, P=0.031, P=0.006), and IL-17A also was increased but the difference was not statistically significant (P=0.052), while anti-inflammatory cytokines IL-10 and TGF-ß were significantly reduced (P=0.048, P=0.002). The relative expressions level of FOXP3 and PRDM1 mRNA in AA children were significantly lower than those in control group (P=0.037, P=0.016). The expression of BLIMP-1 protein in Tregs of AA children was significantly lower than that in control group (P<0.001). The expression level of BLIMP-1 protein in Tregs was positively correlated with the percentage of Tregs in lymphocytes (r=0.671, P=0.001), and was also positively correlated with the levels of IL-10 and TGF-ß in plasma (r=0.500, P=0.029; r=0.486, P=0.030). CONCLUSION: The expression of BLIMP-1 in Tregs of AA children is impaired, and the low expression of BLIMP-1 is related to the decrease of the number in Tregs and IL-10 and TGF-ß expressions.

A DEAD-box protein, AtRH36, is essential for female gametophyte development and is involved in rRNA biogenesis in Arabidopsis.

DEAD-box RNA helicases are involved in RNA metabolism, including pre-mRNA splicing, ribosome biogenesis, RNA decay and gene expression. In this study, we identified a homolog of the RH36 gene, AtRH36, which encodes a DEAD-box protein in Arabidopsis thaliana. The gene was expressed ubiquitously throughout the plant. The AtRH36 fused to green fluorescent protein was localized in the nucleus. Homozygosity for the Arabidopsis atrh36 mutants, atrh36-1 and atrh36-2, could not be obtained. Progeny of selfed Arabidopsis atrh36 heterozygote plants were obtained at a heterozygote to wild-type ratio of 1 : 1, which suggested that the AtRH36 gene was involved in gametogenesis. Therefore, we performed a reciprocal cross to determine whether AtRH36 was involved in female gametophyte development. Female gametogenesis was delayed in atrh36-1, and asynchronous development of the female gametophytes was found within a single pistil. Knock-down of AtRH36 gave a pleiotropic phenotype and led to the accumulation of unprocessed 18S pre-rRNA. These results suggest that AtRH36 is essential for mitotic division during female gametogenesis and plays an important role in rRNA biogenesis in Arabidopsis.

[An analysis of opportunistic infection in 762 inpatients with human immunodeficiency virus infection in Guangdong areas].

OBJECTIVE: To analyze the characteristics of opportunistic infection (OI) in patients with HIV/AIDS in Guangdong and the relationship between OI and the change in blood CD4+ T lymphocyte count (CD4+). METHODS: Seven hundred and sixty two patients with HIV/AIDS admitted were analyzed. RESULTS: Among all the 762 patients, 704 (92.39%) had more than one kind of OI, with 1428 episodes totally. Etiologically, fungus infection (38.38%) was most common, followed by bacteria (36.20%), and virus (7.77%) infection. Most OI occurred in the lungs (33.05%), mouth (26.89%), skin (10.29%) and gastro-intestine (8.96%). Septicemia and other systemic disseminated diseases accounted for 6.58% and 9.94% respectively. The incidence of OI in patients with CD4+≥200/µl (103/136, 75.74%) was significantly lower than that in patients with CD4+<200/µl (601/626, 96.01%), P<0.01. All the AIDS defining OI were found in patients with CD4+<200/µl. Among them, 81.97% of patients with pneumonia carinii pneumonia (PCP), 71.43% of patients with cytomegalovirus retinitis and all the patients with cryptococcal meningitis, disseminated cryptococcosis, disseminated histoplasmosis, mycobacterium avium intracellular complex (MAC), disseminated penicilliosis marneffei and toxoplasma cerebritis had the CD4+ less than 50/µl. CONCLUSIONS: The most common OI in patients with AIDS in Guangdong area are fungi, bacterial and viral infections. Lung, mouth, skin, gastro-intestine and systemic disseminated infections are the most prevalent infections. As the CD4+ decreased, the incidence of OI especially AIDS defining OI increased. Dynamic detection of CD4+ will be of great help for the prediction, prevention, early diagnosis and treatment of OI in patients with AIDS.

Novel Homogeneous Anion Exchange Membranes for Reproducible and Sensitive Nucleic Acid Detection via Current-Voltage Characteristic Measurement.

One-pot synthesis of novel hydrogel-based anion exchange membranes (AEMs), with only a single-phase monomer mixture, was used to eliminate surface heterogeneity and generate reproducible electroconvective microvortices in the over-limiting region of the current-voltage characteristic (CVC) curves. Diallyldimethylammonium chloride (DDA) was used as the main component to provide the cation charge groups, and 2-hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethyl acrylate (EGDMA) were used as the auxiliary structure monomers. The uniform membrane structure allowed reproducible and sensitive DNA detection and quantification, as probe-target surface complexes can gate the ion flux and produce large voltage shifts in the over-limiting region. Suppressed membrane curvature due to controlled swelling is a crucial part to avoid the reduction of depletion region for maintaining the influence of target gene hybridization. Fourier-transform infrared (FTIR) spectroscopy verified the synthesized membrane structure, with a residual vinyl group that allows easy carboxylation via additional photografting reaction. Consequently, a significantly higher DNA probe functionalization efficiency is obtained on the homogeneous AEMs, evidenced by the increasing nitrogen element content and bonding via X-ray photoelectron spectroscopy (XPS). The DDA content was optimized to provide a sufficient coulomb force between AEM and nucleic acid backbone to promote the specific binding efficiency but without high dimensional swelling which might change the surface geometry and restrict the voltage shifting for sensing in the over-limiting region, and the optimal DDA/HEMA ratio was found to be 4/10. The synthesized AEM sensor for recombinant 35S promoter sequence identification exhibited a reproducible calibration standard curve with dynamic range between 30 fM and 1 µM and high selectivity with only 0.01 V shift for 1 µM nontarget oligo.

Effects of the multiple polyadenylation signal AAUAAA on mRNA 3'-end formation and gene expression.

Polyadenylation (poly(A)) of eukaryotic mRNA is a critical step for gene expression. In plants, poly(A) signals leading to the formation of polyadenosine tails after mRNAs include the far upstream elements, the AAUAAA-like signals, and the mRNA cleavage sites for poly(A). Multiple AAUAAA signals leading to alternative polyadenosine formation have been found in many genes, but the effects of each AAUAAA signal on gene expression remain to be uncovered. A DNA fragment, whose transcript contains two canonical AAUAAA signals from the 3'-untranslation region of endochitinase gene of tobacco (Nicotiana tabacum L. cv. W38), was mutated and constructed into the downstream of beta-glucuronidase (GUS) coding region. Transient expression of GUS gene from these constructs indicated that the distal AAUAAA signal from the stop codon was more important than the proximal one in stimulating gene expression. Also, the sequence rather than the distance between the stop codon and the AAUAAA signal region was critical for gene expression. Transgenic tobaccos with these constructs were also generated, and the position of the polyadenosine tail formation in this region was mapped. Results revealed that both AAUAAA signals were functional, and that polyadenosine tails of most transcripts were directed by the distal AAUAAA signal. Finally, the RNA stabilities of these variants in transgenic plants were measured. RNAs from the variants with the functional distal AAUAAA signal were more stable than those with the functional proximal one only. The possible secondary structure in this poly(A) signal region was predicted and discussed.