Anopheles gambiae genome reannotation through synthesis of ab initio and comparative gene prediction algorithms

Background

Complete genome annotation is a necessary tool as Anopheles gambiae researchers probe the biology of this potent malaria vector.

Results

We reannotate the A. gambiae genome by synthesizing comparative and ab initio sets of predicted coding sequences (CDSs) into a single set using an exon-gene-union algorithm followed by an open-reading-frame-selection algorithm. The reannotation predicts 20,970 CDSs supported by at least two lines of evidence, and it lowers the proportion of CDSs lacking start and/or stop codons to only approximately 4%. The reannotated CDS set includes a set of 4,681 novel CDSs not represented in the Ensembl annotation but with EST support, and another set of 4,031 Ensembl-supported genes that undergo major structural and, therefore, probably functional changes in the reannotated set. The quality and accuracy of the reannotation was assessed by comparison with end sequences from 20,249 full-length cDNA clones, and evaluation of mass spectrometry peptide hit rates from an A. gambiae shotgun proteomic dataset confirms that the reannotated CDSs offer a high quality protein database for proteomics. We provide a functional proteomics annotation, ReAnoXcel, obtained by analysis of the new CDSs through the AnoXcel pipeline, which allows functional comparisons of the CDS sets within the same bioinformatic platform. CDS data are available for download.

Conclusion

Comprehensive A. gambiae genome reannotation is achieved through a combination of comparative and ab initio gene prediction algorithms.     

Community-acquired MRSA and pig-farming

Background

Female genital tuberculosis is an uncommon disease that is rarely diagnosed in developed countries.

Case presentation

A 61-year-old postmenopausal woman who had undergone surgery and treated with adjuvant chemotherapy for infiltrating ductal carcinoma of the breast five years ago, presented with bloody vaginal discharge, fatigue, weight loss, and low grade fevers at night for two months. Histological examination of the endometrium, done based on the suspicion of a second primary cancer due to the tamoxifen therapy, revealed a granulomatous reaction. Liquid and solid mycobacterial cultures of the tissues were performed. Although the acid fast staining was negative, the liquid culture was positive for Mycobacterium tuberculosis. Involvement of other systems was not detected. The patient was treated with a three-drug antituberculosis regimen for 9 months and recovered fully.

Conclusion

Female genital tuberculosis is a rare but curable disease that should be included in the differential diagnosis of women with menstrual problems. Early diagnosis is important and may prevent unnecessary invasive procedures for the patient.     

Clonal structure and genetic diversity of three desert phreatophytes

The objective of this paper was to assess clone sizes of three perennial desert plant species with AFLP markers and to relate them to clonal and genetic diversity and to hydroecology. The study was carried out at the southern rim of the Taklamakan Desert, where sexual regeneration is only possible shortly after rare flooding events, resulting in rarely established cohorts with subsequent extensive vertical growth and horizontal clonal spread. In this environment, repeated seedling establishment is excluded. We expected decreasing clonal and genetic diversity with increasing clone size and increasing distance to the groundwater table and a common response pattern among all study species. Maximum sizes of Populus euphratica and Alhagi sparsifolia clones were 121 ha and 6.1 ha, respectively, while Tamarix ramosissima clones reached a maximum size of only 38 m(2). In P. euphratica and A. sparsifolia, clonal diversity declined with increasing clone size and increasing distance to the groundwater table, while genetic diversity remained unaffected. Tamarix ramosissima differed from the other species because of a much smaller clonality. Clone size and clonal diversity were found to be good proxy variables for clone age. Despite the considerable age of the clones, genetic diversity is maintained in the populations.     

Microphthalmia, parkinsonism, and enhanced nociception in Pitx3 416insG mice

A new spontaneous mouse mutant was characterized by closed eyelids at weaning and without apparent eyes (provisional gene name, eyeless; provisional gene symbol, eyl). The mutation follows a recessive pattern of inheritance and was mapped to the region of chromosome 19 containing Pitx3. Genetic complementation tests using Pitx3 ^ak/+ mice confirmed eyl as a new allele of Pitx3 (Pitx3 ^eyl ). Sequencing of the Pitx3 gene in eyl mutants identified an inserted G after cDNA position 416 (416insG; exon 4). The shifted open reading frame is predicted to result in a hybrid protein still containing the Pitx3 homeobox, but followed by 121 new amino acids. The novel Pitx3 ^eyl/eyl mutants expressed ophthalmological and brain defects similar to Pitx3 ^ak/ak mice: microphthalmia or anophthalmia and loss of dopamine neurons of the substantia nigra. In addition, we observed in the homozygous eyeless mutants increased extramedullary hematopoiesis in the spleen, frequently liver steatosis, and reduced body weight. There were also several behavioral changes in the homozygous mutants, including reduced forelimb grip strength and increased nociception. In addition to these alterations in both sexes, we observed in female Pitx3 ^eyl/eyl mice increased anxiety-related behavior, reduced locomotor activity, reduced object exploration, and increased social contacts; however, we observed decreased anxiety-related behavior and increased arousal in males. Most of these defects identified in the new Pitx3 mutation are observed in Parkinson patients, making the Pitx3 ^eyl mutant a valuable new model. It is the first mouse mutant carrying a point mutation within the coding region of Pitx3.     

The alarmin Mrp8/14 as regulator of the adaptive immune response during allergic contact dermatitis

Mrp8 and Mrp14 are endogenous alarmins amplifying inflammation via Toll‐like receptor‐4 (TLR‐4) activation. Due to their pro‐inflammatory properties, alarmins are supposed to enhance adaptive immunity via activation of dendritic cells (DCs). In contrast, analysing a model of allergic contact dermatitis (ACD) we observed a more severe disease outcome in Mrp8/14‐deficient compared to wild‐type mice. This unexpected phenotype was associated with an enhanced T‐cell response due to an accelerated maturation of DCs in Mrp8/14‐deficient mice. Accordingly, Mrp8, the active component of the heterocomplex, inhibits early DC maturation and antigen presentation in a TLR‐4‐dependent manner. Transfer of DCs purified from the local lymph nodes of sensitized Mrp8/14‐deficient to wild‐type mice determined the outcome of ACD. Our results link a pro‐inflammatory role of the endogenous TLR‐4 ligand Mrp8/14 to a regulatory function in adaptive immunity, which shows some similarities with the ‘hygiene hypothesis’ regarding continuous TLR‐4 stimulation and decreased risk of allergy.     

Development of anti-CD20 therapy for multiple sclerosis

The therapeutic utility of the targeting of B lymphocytes is currently being evaluated in a range of autoimmune diseases that include multiple sclerosis (MS). For MS, even though intrathecal immunoglobulin production is a hallmark of multiple sclerosis (MS), T cells have long been considered as the main effectors of pathogenesis. Recognition of the roles of autoreactive B cells has changed this conventional view of the disease and also provided a rationale for studies of anti-CD20 therapy in MS. Recent trials suggest that this approach may provide clinical benefits in some MS patients that equal or surpass currently approved approaches, yet not all patients may benefit. In this review we provide an overview on recent progress on these trials.     

Interaction studies between the chloroplast signal recognition particle subunit cpSRP43 and the full-length translocase Alb3 reveal a membrane-embedded binding region in Alb3 protein

Posttranslational targeting of the light-harvesting chlorophyll a,b-binding proteins depends on the function of the chloroplast signal recognition particle, its receptor cpFtsY, and the translocase Alb3. The thylakoid membrane protein Alb3 of Arabidopsis chloroplasts belongs to the evolutionarily conserved YidC/Oxa1/Alb3 protein family; the members of this family facilitate the insertion, folding, and assembly of membrane proteins in bacteria, mitochondria, and chloroplasts. Here, we analyzed the interaction sites of full-length Alb3 with the cpSRP pathway component cpSRP43 by using in vitro and in vivo studies. Bimolecular fluorescence complementation and Alb3 proteoliposome studies showed that the interaction of cpSRP43 is dependent on a binding domain in the C terminus of Alb3 as well as an additional membrane-embedded binding site in the fifth transmembrane domain (TMD5) of Alb3. The C-terminal binding domain was mapped to residues 374-388, and the binding domain within TMD5 was mapped to residues 314-318 located close to the luminal end of TMD5. A direct binding between cpSRP43 and these binding motifs was shown by pepspot analysis. Further studies using blue-native gel electrophoresis revealed that full-length Alb3 is able to form dimers. This finding and the identification of a membrane-embedded cpSRP43 binding site in Alb3 support a model in which cpSRP43 inserts into a dimeric Alb3 translocation pore during cpSRP-dependent delivery of light-harvesting chlorophyll a,b-binding proteins.     

Mechanistic insights into an engineered riboswitch: a switching element which confers riboswitch activity

While many different RNA aptamers have been identified that bind to a plethora of small molecules only very few are capable of acting as engineered riboswitches. Even for aptamers binding the same ligand large differences in their regulatory potential were observed. We address here the molecular basis for these differences by using a set of unrelated neomycin-binding aptamers. UV melting analyses showed that regulating aptamers are thermally stabilized to a significantly higher degree upon ligand binding than inactive ones. Regulating aptamers show high ligand-binding affinity in the low nanomolar range which is necessary but not sufficient for regulation. NMR data showed that a destabilized, open ground state accompanied by extensive structural changes upon ligand binding is important for regulation. In contrast, inactive aptamers are already pre-formed in the absence of the ligand. By a combination of genetic, biochemical and structural analyses, we identified a switching element responsible for destabilizing the ligand free state without compromising the bound form. Our results explain for the first time the molecular mechanism of an engineered riboswitch.     

Nitrogen Forms Influence Microcystin Concentration and Composition via Changes in Cyanobacterial Community Structure

The eutrophication of freshwaters is a global health concern as lakes with excess nutrients are often subject to toxic cyanobacterial blooms. Although phosphorus is considered the main element regulating cyanobacterial biomass, nitrogen (N) concentration and more specifically the availability of different N forms may influence the overall toxicity of blooms. In this study of three eutrophic lakes prone to cyanobacterial blooms, we examined the effects of nitrogen species and concentrations and other environmental factors in influencing cyanobacterial community structure, microcystin (MC) concentrations and MC congener composition. The identification of specific MC congeners was of particular interest as they vary widely in toxicity. Different nitrogen forms appeared to influence cyanobacterial community structure leading to corresponding effects on MC concentrations and composition. Total MC concentrations across the lakes were largely explained by a combination of abiotic factors: dissolved organic nitrogen, water temperature and ammonium, but Microcystis spp. biomass was overall the best predictor of MC concentrations. Environmental factors did not appear to affect MC congener composition directly but there were significant associations between specific MC congeners and particular species. Based on redundancy analyses (RDA), the relative biomass of Microcystis aeruginosa was associated with MC-RR, M. wesenbergii with MC-LA and Aphanizomenon flos-aquae with MC-YR. The latter two species are not generally considered capable of MC production. Total nitrogen, water temperature, ammonium and dissolved organic nitrogen influenced the cyanobacterial community structure, which in turn resulted in differences in the dominant MC congener and the overall toxicity.