Development of a microarray for two rice subspecies: characterization and validation of gene expression in rice tissues

Background

Rice is one of the major crop species in the world helping to sustain approximately half of the global population’s diet especially in Asia. However, due to the impact of extreme climate change and global warming, rice crop production and yields may be adversely affected resulting in a world food crisis. Researchers have been keen to understand the effects of drought, temperature and other environmental stress factors on rice plant growth and development. Gene expression microarray technology represents a key strategy for the identification of genes and their associated expression patterns in response to stress. Here, we report on the development of the rice OneArray® microarray platform which is suitable for two major rice subspecies, japonica and indica.

Results

The rice OneArray® 60-mer, oligonucleotide microarray consists of a total of 21,179 probes covering 20,806 genes of japonica and 13,683 genes of indica. Through a validation study, total RNA isolated from rice shoots and roots were used for comparison of gene expression profiles via microarray examination. The results were submitted to NCBI’s Gene Expression Omnibus (GEO). Data can be found under the GEO accession number GSE50844 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE50844). A list of significantly differentially expressed genes was generated; 438 shoot-specific genes were identified among 3,138 up-regulated genes, and 463 root-specific genes were found among 3,845 down-regulated genes. GO enrichment analysis demonstrates these results are in agreement with the known physiological processes of the different organs/tissues. Furthermore, qRT-PCR validation was performed on 66 genes, and found to significantly correlate with the microarray results (R?=?0.95, p?<?0.001***).

Conclusion

The rice OneArray® 22 K microarray, the first rice microarray, covering both japonica and indica subspecies was designed and validated in a comprehensive study of gene expression in rice tissues. The rice OneArray® microarray platform revealed high specificity and sensitivity. Additional information for the rice OneArray® microarray can be found at http://www.phalanx.com.tw/index.php.     

Do We Need to Detect Isoniazid Resistance in Addition to Rifampicin Resistance in Diagnostic Tests for Tuberculosis?

Background

Multidrug-resistant tuberculosis (MDR-TB) is resistant to both rifampicin (RIF) and isoniazid (INH). Whereas many TB diagnostics detect RIF-resistance, few detect INH-monoresistance, which is common and may increase risk of acquired MDR-TB. Whether inclusion of INH-resistance in a first-line rapid test for TB would have an important impact on MDR-TB rates remains uncertain.

Methods

We developed a transmission model to evaluate three tests in a population similar to that of India: a rapid molecular test for TB, the same test plus RIF-resistance detection (“TB+RIF”), and detection of RIF and INH-resistance (“TB+RIF/INH”). Our primary outcome was the prevalence of INH-resistant and MDR-TB at ten years.

Results

Compared to the TB test alone and assuming treatment of all diagnosed MDR cases, the TB+RIF test reduced the prevalence of MDR-TB among all TB cases from 5.5% to 3.8% (30.6% reduction, 95% uncertainty range, UR: 17–54%). Despite using liberal assumptions about the impact of INH-monoresistance on treatment outcomes and MDR-TB acquisition, expansion from TB+RIF to TB+RIF/INH lowered this prevalence only from 3.8% to 3.6% further (4% reduction, 95% UR: 3–7%) and INH-monoresistant TB from 15.8% to 15.1% (4% reduction, 95% UR: (-8)-19%).

Conclusion

When added to a rapid test for TB plus RIF-resistance, detection of INH-resistance has minimal impact on transmission of TB, MDR-TB, and INH-monoresistant TB.     

HIV Incidence in a Cohort of Women at Higher Risk in Beira,Mozambique: Prospective Study 2009–2012

Background

HIV is prevalent in Sofala Province, Mozambique. To inform future prevention research, we undertook a study in the provincial capital (Beira) to measure HIV incidence in women at higher risk of HIV and assess the feasibility of recruiting and retaining them as research participants.

Methods

Women age 18–35 were recruited from schools and places where women typically meet potential sexual partners. Eligibility criteria included HIV-seronegative status and self-report of at least 2 sexual partners in the last month. History of injection drug use was an exclusion criterion, but pregnancy was not. Participants were scheduled for monthly follow-up for 12 months, when they underwent face-to-face interviews, HIV counseling and testing, and pregnancy testing.

Results

387 women were eligible and contributed follow-up data. Most were from 18–24 years old (median 21). Around one-third of participants (33.8%) reported at least one new sexual partner in the last month. Most women (65.5%) reported not using a modern method of contraception at baseline. Twenty-two women seroconverted for a prospective HIV incidence of 6.5 per 100 woman-years (WY; 95% confidence interval (CI): 4.1–9.9). Factors associated with HIV seroconversion in the multivariable analysis were: number of vaginal sex acts without using condoms with partners besides primary partner in the last 7 days (hazard ratio (HR) 1.7; 95% CI: 1.2–2.5) and using a form of contraception at baseline other than hormonal or condoms (vs. no method; HR 25.3; 95% CI: 2.5–253.5). The overall retention rate was 80.0% for the entire follow-up period.

Conclusions

We found a high HIV incidence in a cohort of young women reporting risky sexual behavior in Beira, Mozambique. HIV prevention programs should be strengthened. Regular HIV testing and condom use should be encouraged, particularly among younger women with multiple sexual partners.     

Dynamic stability and compensatory stepping responses during anterior gait–slip perturbations in people with chronic hemiparetic stroke

To examine the control of dynamic stability and characteristics of the compensatory stepping responses to an unexpected anterior gait slip induced under the non-involved limb in people with hemi-paretic stroke (PwHS) and to examine any resulting adaptive changes in these on the second slip due to experience from prior slip exposure. Ten PwHS experienced overground slip (S1) during walking on the laboratory walkway after 5–8 regular walking (RW) trials followed by a second consecutive slip trial (S2). The slip outcome (backward loss of balance, BLOB and no loss of balance, NLOB) and COM state (i.e. its COM position and velocity) stability were examined between the RW and S1 and S1 and S2 at touchdown (TD) of non-involved limb and at liftoff (LO) of the contralateral limb. At TD there was no difference in stability between RW and S1, however at LO, subjects demonstrated a lower stability on S1 than RW resulting in a 100% backward loss of balance (BLOB) with compensatory stepping response (recovery step, RS, 4/10 or aborted step, AS, 6/10). On S2, although there was no change in stability at TD, there was a significant improvement in stability at LO with a 40% decrease in BLOB. There was also a change in step strategy with a decrease in AS response (60% to 35%, p<0.05) which was replaced by an increase in the ability to step (increased compensatory step length, p<0.05) either via a recovery step or a walkover step. PwHS have the ability to reactively control COM state stability to decrease fall-risk upon a novel slip; prior exposure to a slip did not significantly alter feedforward control but improved the ability to use such feedback control for improved slip outcomes.     

A reinforced merging methodology for mapping unique peptide motifs in members of protein families

Background  

Members of a protein family often have highly conserved sequences; most of these sequences carry identical biological functions and possess similar three-dimensional (3-D) structures. However, enzymes with high sequence identity may acquire differential functions other than the common catalytic ability. It is probable that each of their variable regions consists of a unique peptide motif (UPM), which selectively interacts with other cellular proteins, rendering additional biological activities. The ability to identify and localize such UPMs is paramount in recognizing the characteristic role of each member of a protein family.     

Dual binding sites for translocation catalysis by Escherichia coli glutathionylspermidine synthetase

Most organisms use glutathione to regulate intracellular thiol redox balance and protect against oxidative stress; protozoa, however, utilize trypanothione for this purpose. Trypanothione biosynthesis requires ATP-dependent conjugation of glutathione (GSH) to the two terminal amino groups of spermidine by glutathionylspermidine synthetase (GspS) and trypanothione synthetase (TryS), which are considered as drug targets. GspS catalyzes the penultimate step of the biosynthesis-amide bond formation between spermidine and the glycine carboxylate of GSH. We report herein five crystal structures of Escherichia coli GspS in complex with substrate, product or inhibitor. The C-terminal of GspS belongs to the ATP-grasp superfamily with a similar fold to the human glutathione synthetase. GSH is likely phosphorylated at one of two GSH-binding sites to form an acylphosphate intermediate that then translocates to the other site for subsequent nucleophilic addition of spermidine. We also identify essential amino acids involved in the catalysis. Our results constitute the first structural information on the biochemical features of parasite homologs (including TryS) that underlie their broad specificity for polyamines.     

The family 21 carbohydrate-binding module of glucoamylase from Rhizopus oryzae consists of two sites playing distinct roles in ligand binding

The starch-hydrolysing enzyme GA (glucoamylase) from Rhizopus oryzae is a commonly used glycoside hydrolase in industry. It consists of a C-terminal catalytic domain and an N-terminal starch-binding domain, which belong to the CBM21 (carbohydrate-binding module, family 21). In the present study, a molecular model of CBM21 from R. oryzae GA (RoGACBM21) was constructed according to PSSC (progressive secondary structure correlation), modified structure-based sequence alignment, and site-directed mutagenesis was used to identify and characterize potential ligand-binding sites. Our model suggests that RoGACBM21 contains two ligand-binding sites, with Tyr32 and Tyr67 grouped into site I, and Trp47, Tyr83 and Tyr93 grouped into site II. The involvement of these aromatic residues has been validated using chemical modification, UV difference spectroscopy studies, and both qualitative and quantitative binding assays on a series of RoGACBM21 mutants. Our results further reveal that binding sites I and II play distinct roles in ligand binding, the former not only is involved in binding insoluble starch, but also facilitates the binding of RoGACBM21 to long-chain soluble polysaccharides, whereas the latter serves as the major binding site mediating the binding of both soluble polysaccharide and insoluble ligands. In the present study we have for the first time demonstrated that the key ligand-binding residues of RoGACBM21 can be identified and characterized by a combination of novel bioinformatics methodologies in the absence of resolved three-dimensional structural information.     

Metabolic complementarity and genomics of the dual bacterial symbiosis of sharpshooters

Mutualistic intracellular symbiosis between bacteria and insects is a widespread phenomenon that has contributed to the global success of insects. The symbionts, by provisioning nutrients lacking from diets, allow various insects to occupy or dominate ecological niches that might otherwise be unavailable. One such insect is the glassy-winged sharpshooter (Homalodisca coagulata), which feeds on xylem fluid, a diet exceptionally poor in organic nutrients. Phylogenetic studies based on rRNA have shown two types of bacterial symbionts to be coevolving with sharpshooters: the gamma-proteobacterium Baumannia cicadellinicola and the Bacteroidetes species Sulcia muelleri. We report here the sequencing and analysis of the 686,192–base pair genome of B. cicadellinicola and approximately 150 kilobase pairs of the small genome of S. muelleri, both isolated from H. coagulata. Our study, which to our knowledge is the first genomic analysis of an obligate symbiosis involving multiple partners, suggests striking complementarity in the biosynthetic capabilities of the two symbionts: B. cicadellinicola devotes a substantial portion of its genome to the biosynthesis of vitamins and cofactors required by animals and lacks most amino acid biosynthetic pathways, whereas S. muelleri apparently produces most or all of the essential amino acids needed by its host. This finding, along with other results of our genome analysis, suggests the existence of metabolic codependency among the two unrelated endosymbionts and their insect host. This dual symbiosis provides a model case for studying correlated genome evolution and genome reduction involving multiple organisms in an intimate, obligate mutualistic relationship. In addition, our analysis provides insight for the first time into the differences in symbionts between insects (e.g., aphids) that feed on phloem versus those like H. coagulata that feed on xylem. Finally, the genomes of these two symbionts provide potential targets for controlling plant pathogens such as Xylella fastidiosa, a major agroeconomic problem, for which H. coagulata and other sharpshooters serve as vectors of transmission.     

Simulated movement termination for balance recovery: can movement strategies be sought to maintain stability in the presence of slipping or forced sliding?

Slipping during various kinds of movement often leads to potentially dangerous incidents of falling. The purpose of this study was to determine whether there was evidence to support the theory that movement strategies could be used by individuals to regain stability during an episode of slipping and whether forced sliding from a moving platform accurately simulated the effect of slipping on stability and balance. A single-link-plus-foot biomechanical model was used to mathematically simulate base of support (BOS) translation and body segment rotation during movement termination in sagittal plane. An optimization routine was used to determine region of stability [defined at given COM locations as the feasible range of horizontal velocities of the center of mass (COM) of human subject that can be reduced to zero with respect to the BOS while still allowing the COM to traverse within the BOS limits]. We found some 30% overlap in the region of stability for slipping and non-slipping conditions. This finding supports the theory that movement strategies can be sought for restoring stability and balance even if slipping unexpectedly occurs. We also found that forced sliding produces effects on stability that are similar to those of slipping, indicated by over 50% overlap in the regions of stability for the two conditions. In addition, forced sliding has distinctive effects on stability, including a "shift" of the region of stability extended beyond the BOS in the direction of sliding. These findings may provide quantifiable guidance for balance training aimed at reducing fall incidents under uncertain floor surface conditions.