Asian population frequencies and haplotype distribution of killer cell immunoglobulin-like receptor (KIR) genes among Chinese, Malay, and Indian in Singapore

Killer cell immunoglobulin-like receptors (KIR) gene frequencies have been shown to be distinctly different between populations and contribute to functional variation in the immune response. We have investigated KIR gene frequencies in 370 individuals representing three Asian populations in Singapore and report here the distribution of 14 KIR genes (2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1) with two pseudogenes (2DP1, 3DP1) among Singapore Chinese (n = 210); Singapore Malay (n = 80), and Singapore Indian (n = 80). Four framework genes (KIR3DL3, 3DP1, 2DL4, 3DL2) and a nonframework pseudogene 2DP1 were detected in all samples while KIR2DS2, 2DL2, 2DL5, and 2DS5 had the greatest significant variation across the three populations. Fifteen significant linkage patterns, consistent with associations between genes of A and B haplotypes, were observed. Eighty-four distinct KIR profiles were determined in our populations, 38 of which had not been described in other populations. KIR haplotype studies were performed using nine Singapore Chinese families comprising 34 individuals. All genotypes could be resolved into corresponding pairs of existing haplotypes with eight distinct KIR genotypes and eight different haplotypes. The haplotype A2 with frequency of 63.9% was dominant in Singapore Chinese, comparable to that reported in Korean and Chinese Han. The A haplotypes predominate in Singapore Chinese, with ratio of A to B haplotypes of approximately 3:1. Comparison with KIR frequencies in other populations showed that Singapore Chinese shared similar distributions with Chinese Han, Japanese, and Korean; Singapore Indian was found to be comparable with North Indian Hindus while Singapore Malay resembled the Thai. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biological Filtration Limits Carbon Availability and Affects Downstream Biofilm Formation and Community Structure

Carbon removal strategies have gained popularity in the mitigation of biofouling in water reuse processes, but current biofilm-monitoring practices based on organic-carbon concentrations may not provide an accurate representation of the in situ biofilm problem. This study evaluated a submerged microtiter plate assay for direct and rapid monitoring of biofilm formation by subjecting the plates to a continuous flow of either secondary effluent (SE) or biofilter-treated secondary effluent (BF). This method was very robust, based on a high correlation (R² = 0.92) between the biomass (given by the A₆₀₀ in the microtiter plate assay) and the biovolume (determined from independent biofilms developed on glass slides under identical conditions) measurements, and revealed that the biomasses in BF biofilms were consistently lower than those in SE biofilms. The influence of the organic-carbon content on the biofilm community composition and succession was further evaluated using molecular tools. Terminal restriction fragment length polymorphism analysis of 16S rRNA genes revealed a group of pioneer colonizers, possibly represented by Sphingomonadaceae and Caulobacter organisms, to be common in both SE and BF biofilms. However, differences in organic-carbon availabilities in the two water samples eventually led to the selection of distinct biofilm communities. Alphaproteobacterial populations were confirmed by fluorescence in situ hybridization to be enriched in SE biofilms, while Betaproteobacteria were dominant in BF biofilms. Cloning analyses further demonstrated that microorganisms adapted for survival under low-substrate conditions (e.g., Aquabacterium, Caulobacter, and Legionella) were preferentially selected in the BF biofilm, suggesting that carbon limitation strategies may not achieve adequate biofouling control in the long run.     

Field evaluation of cotton near-isogenic lines introgressed with QTLs for productivity and drought related traits

Quantitative trait loci (QTLs) for yield and drought related physiological traits, osmotic potential (OP), carbon isotope ratio (δ¹³C, an indicator of water use efficiency), and leaf chlorophyll content (Chl), were exchanged via marker-assisted selection (MAS) between elite cultivars of the two cotton species Gossypium barbadense cv. F-177 and G. hirsutum cv. Siv’on. The resulting near isogenic lines (NILs) were examined in two field trials, each with two irrigation regimes, in order to (1) evaluate the potential to improve cotton drought resistance by MAS and (2) test the role of physiological traits in plant productivity. NILs introgressed with QTLs for high yield rarely exhibited an advantage in yield relative to the recipient parent, whereas a considerable number of NILs exhibited the expected phenotype in terms of lower OP (5 out of 9), higher δ¹³C (4 out of 6) or high Chl (2 out of 3). Several NILs exhibited considerable modifications in non-targeted traits including leaf morphology, stomatal conductance and specific leaf weight (SLW). In G. barbadense genotypes, yield was correlated negatively with δ¹³C and OP and positively with stomatal conductance, SLW and Chl, whereas in G. hirsutum yield was negatively correlated with δ¹³C, SLW and Chl. This dissimilarity suggests that each of the respective species has evolved different mechanisms underlying plant productivity. We conclude that the improvement of drought related traits in cotton NILs may lead to improved drought resistance via MAS, but that conventional breeding may be necessary to combine the introduced QTL(s) with high yield potential.     

Aqueous Cytokine Changes Associated with Posner-Schlossman Syndrome with and without Human Cytomegalovirus

Aim

To study the differences in aqueous cytokines in Posner-Schlossman Syndrome (PSS) patients with and without human cytomegalovirus (CMV) DNA in the aqueous humor.

Methods

This is a prospective study. Fifty-three uveitis patients with clinical signs of PSS were enrolled and aqueous humor samples were collected. Fourteen PSS patients were positive of CMV DNA in the aqueous by polymerase chain reaction (PCR) analysis. These eyes were negative of common ocular pathogens such as herpes simplex virus, varicella-zoster virus, rubella virus and toxoplasma. Twenty-five otherwise healthy cataract patients were enrolled as controls. Cytokine concentration was measured by a magnetic color-bead-based multiplex assay and analyzed using statistical and classification approaches.

Results

The average age of 53 PSS patients was 48.74±13.43 years (yrs) (mean ± standard deviation) and 66.3±15.0 yrs for the controls. The median CMV viral DNA copy number was 26000/mL aqueous (range 1400 to 85000 copies/mL) in 14 CMV positive patients as determined by quantitative PCR. PSS aqueous had significantly higher Interleukin (IL)-8 (CXCL8), monocyte chemotactic protein-1 (CCL2), macrophage inhibitory protein 1-β (CCL4), granulocyte colony-stimulating factor (GCSF) and transforming growth factor-β (TGF-β) levels than controls after adjusted by age and gender. IL-2, IL-12, tumor necrosis factor-α (TNF-α) and interferon-α (IFN-α) levels were significantly lower in PSS aqueous than controls. No difference between CMV positive PSS and CMV negative PSS aqueous was observed. Over 97% of PSS samples were distinguished from controls by elevated CXCL10 (>500 ng/mL), CXCL8 (>30 ng/mL) and CCL2 (>60 ng/mL) levels.

Conclusion

PSS eyes were characterized by elevated aqueous chemokine concentration. The presence of CMV viral DNA was not associated with significant change of the type of cytokine expression in PSS patients.     

Comparative study of the T cell response to two allelic forms of a malarial vaccine candidate protein.

T cell responses to two allelic forms of the merozoite surface Ag 2 (MSA2) of Plasmodium falciparum were mapped in mice using the rMSA2 proteins, Ag 1609 which has the sequence of the FCQ27/PNG strain and Ag 1615 which has the sequence of the Indochina 1 strain. Lymph node cells of BL/10 and B10.BR mice immunized with either Ag 1609 or Ag 1615 responded to both Ag in in vitro proliferation assays. Lymph node cells of BALB/c mice did not respond. The T cell determinants recognized by the responder strains were mapped to conserved and variant regions of these Ag using overlapping synthetic peptides. The determinants recognized by each mouse strain were distinct. Marked difference in sequence between the central regions of the two rMSA2 proteins did not affect antigenic processing of the conserved N and C terminal regions. Hence lymph node cells of BL/10 mice immunized with either Ag 1615 or Ag 1609 recognized an immunodominant T cell determinant at the highly conserved N terminal end within the sequence YSNTFINNAYNMSIR (peptide 3b) and B10.BR mice similarly immunized recognized an immunodominant determinant at the highly conserved C terminal within the sequence CTDGNKENCGAATSL (peptide 23). Several peptides identified as containing immunodominant T cell determinants specific to BL/10 mice induced peptide-specific T cells in both BL/10 and B10.BR mouse strains when used as immunogens. However, the ability of the peptide-primed T cells to proliferate in response to the rMSA2 proteins was confined to BL/10 mice. An example of this was observed with peptides 3b and N (KNESKYSNTFINNAYNMSIRRSMAN). Peptide N was able to prime B10.BR and BL/10 mice for an enhanced antibody response when these mice were subsequently immunized with Ag 1615 even though Ag 1615-specific T cell proliferation was not detected in B10.BR mice primed with N. The study concluded that 1) conserved sequences such as peptide N when used in vaccines may give rise to MSA2-specific memory Th cells amenable to boosting by subsequent exposure to all parasite strains and 2) peptide priming may be a useful pathway for inducing defined memory Th cells in a wider population and for preferentially inducing T dependent over T independent responses to some malarial Ag.     

Types, levels and patterns of low-copy DNA sequence divergence, and phylogenetic implications, for Gossypium genome types

To explore types, levels and patterns of genetic divergence among diploid Gossypium (cotton) genomes, 780 cDNA, genomic DNA and simple sequence repeat (SSR) loci were re-sequenced in Gossypium herbaceum (A1 genome), G. arboreum (A2), G. raimondii (D5), G. trilobum (D8), G. sturtianum (C1) and an outgroup, Gossypioides kirkii. Divergence among these genomes ranged from 7.32 polymorphic base pairs per 100 between G. kirkii and G. herbaceum (A1) to only 1.44 between G. herbaceum (A1) and G. arboreum (A2). SSR loci are least conserved with 12.71 polymorphic base pairs and 3.77 polymorphic sites per 100 base pairs, whereas expressed sequence tags are most conserved with 3.96 polymorphic base pairs and 2.06 sites. SSR loci also exhibit the highest percentage of 'extended polymorphisms' (spanning multiple consecutive nucleotides). The A genome lineage was particularly rapidly evolving, with the D genome also showing accelerated evolution relative to the C genome. Unexpected asymmetry in mutation rates was found, with much more transition than transversion mutation in the D genome after its divergence from a common ancestor shared with the A genome. This large quantity of orthologous DNA sequence strongly supports a phylogeny in which A-C divergence is more recent than A-D divergence, a subject that is of much importance in view of A-D polyploid formation being key to the evolution of the most productive and finest-quality cottons. Loci that are monomorphic within A or D genome types, but polymorphic between genome types, may be of practical importance for identifying locus-specific DNA markers in tetraploid cottons including leading cultivars.     

HLA class I-associated diseases with a suspected autoimmune etiology: HLA-B27 subtypes as a model system

Although most autoimmune diseases are connected to major histocompatibility complex (MHC) class II alleles, a small number of these disorders exhibit a variable degree of association with selected MHC class I genes, like certain human HLA-A and HLA-B alleles. The basis for these associations, however, has so far remained elusive. An understanding might be obtained by comparing functional, biochemical, and biophysical properties of alleles that are minimally distinct from each other, but are nevertheless differentially associated to a given disease, like the HLA-B*27:05 and HLA-B*27:09 antigens, which differ only by a single amino acid residue (Asp116His) that is deeply buried within the binding groove. We have employed a number of approaches, including X-ray crystallography and isotope-edited infrared spectroscopy, to investigate biophysical characteristics of the two HLA-B27 subtypes complexed with up to ten different peptides. Our findings demonstrate that the binding of these peptides as well as the conformational flexibility of the subtypes is greatly influenced by interactions of the C-terminal peptide residue. In particular, a basic C-terminal peptide residue is favoured by the disease-associated subtype HLA-B*27:05, but not by HLA-B*27:09. This property appears also as the only common denominator of distinct HLA class I alleles, among them HLA-B*27:05, HLA-A*03:01 or HLA-A*11:01, that are associated with diseases suspected to have an autoimmune etiology. We postulate here that the products of these alleles, due to their unusual ability to bind with high affinity to a particular peptide set during positive T cell selection in the thymus, are involved in shaping an abnormal T cell repertoire which predisposes to the acquisition of autoimmune diseases.     

The cysteine-rich sprouty translocation domain targets mitogen-activated protein kinase inhibitory proteins to phosphatidylinositol 4,5-bisphosphate in plasma membranes

Sprouty (Spry) proteins have been revealed as inhibitors of the Ras/mitogen-activated protein kinase (MAPK) cascade, a pathway crucial for developmental processes initiated by activation of various receptor tyrosine kinases. In COS-1 and Swiss 3T3 cells, all Spry isoforms translocate to the plasma membrane, notably ruffles, following activation. Here we show that microinjection of active Rac induced the translocation of Spry isoforms, indicating that the target of the Spry translocation domain (SpryTD) is downstream of active Rac. Targeted disruption of actin polymerization revealed that the SpryTD target appeared upstream of cytoskeletal rearrangements. Accumulated evidence indicated that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is the likely SpryTD target. Human Spry2TD (hSpry2TD) binds to PtdIns(4,5)P(2) in vesicle-binding assays. hSpry2TD colocalizes with the pleckstrin homology domain of phospholipase Cdelta, which binds PtdIns(4,5)P(2). The plasma membrane localization of hSpry2TD was abolished in ionomycin-treated MDCK cells or when PtdIns(4,5)P(2) was specifically dephosphorylated by overexpression of an engineered, green fluorescent protein-tagged inositol 5-phosphatase. Similarly, Spred, a novel Ras/MAPK inhibitor recently found to contain the conserved cysteine-rich SpryTD, also translocated to peripheral membranes and bound to PtdIns(4,5)P(2). Alignment of the Spry and Spred proteins led us to identify a translocation-defective point mutant, hSpry2 D252. Targeting of hSpry2 to PtdIns(4,5)P(2) was shown to be essential for the down-regulation of Ras/MAPK signaling.     

Symptomatic Dengue Disease in Five Southeast Asian Countries: Epidemiological Evidence from a Dengue Vaccine Trial

Dengue incidence has increased globally, but empirical burden estimates are scarce. Prospective methods are best-able to capture all severities of disease. CYD14 was an observer-blinded dengue vaccine study conducted in children 2–14 years of age in Indonesia, Malaysia, Thailand, the Philippines, and Vietnam. The control group received no vaccine and resembled a prospective, observational study. We calculated the rates of dengue according to different laboratory or clinical criteria to make inferences about dengue burden, and compared with rates reported in the passive surveillance systems to calculate expansion factors which describe under-reporting. Over 6,933 person-years of observation in the control group there were 319 virologically confirmed dengue cases, a crude attack rate of 4.6%/year. Of these, 92 cases (28.8%) were clinically diagnosed as dengue fever or dengue hemorrhagic fever by investigators and 227 were not, indicating that most symptomatic disease fails to satisfy existing case definitions. When examining different case definitions, there was an inverse relationship between clinical severity and observed incidence rates. CYD14’s active surveillance system captured a greater proportion of symptomatic dengue than national passive surveillance systems, giving rise to expansion factors ranging from 0.5 to 31.7. This analysis showed substantial, unpredictable and variable under-reporting of symptomatic dengue, even within a controlled clinical trial environment, and emphasizes that burden estimates are highly sensitive to case definitions. These data will assist in generating disease burden estimates and have important policy implications when considering the introduction and health economics of dengue prevention and control interventions.     

Identification and distribution of new insertion sequences in the genome of the extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831.

Six kinds of new insertion sequences (ISs), IS667 to IS672, a group II intron (Oi.Int), and an incomplete transposon (Tn852loi) were identified in the 3,630,528-bp genome of the extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831. Of 19 ISs identified in the HTE831 genome, 7 were truncated, indicating the occurrence of internal rearrangement of the genome. All ISs except IS669 generated a 4- to 8-bp duplication of the target site sequence, and these ISs carried 23- to 28-bp inverted repeats (IRs). Sequence analysis revealed that four ISs (IS669, IS670, IS671, and IS672) were newly identified as belonging to separate IS families (IS200/IS605, IS30, IS5, and IS3, respectively). IS667 and IS668 were also characterized as new members of the ISL3 family. Tn8521oi, which belongs to the Tn3 family as a new member, generated a 5-bp duplication of the target site sequence and carried complete 38-bp IRs. Of the eight protein-coding sequences (CDSs) identified in Tn8521oi, three CDSs (OB481, OB482, and OB483) formed a ger gene cluster, and two other paralogous gene clusters were found in the HTE831 genome. Most of the ISs and the group II intron widely distributed throughout the genome were inserted in noncoding regions, while two ISs (IS667-08 and IS668-02) and Oi.Int-04 were inserted in the coding regions.