Global transcriptomic response of bovine endometrium to blastocyst-stage embryos.

The aims of this study were (i) to investigate changes in the global transcriptome of bovine endometrial explants induced by exposure to blastocysts, (ii) to investigate if male and female blastocysts elicit a differential response in the endometrial transcriptome in vitro and (iii) to determine whether bovine endometrium responds to the presence of murine embryos. In Experiment 1, endometrial explants from the same uterus were cultured for 6 h with or without 20 in vitro-produced bovine blastocysts. In Experiment 2, endometrial explants were cultured with male or female bovine blastocysts produced in vitro by IVF either using sex-sorted semen or conventional unsorted semen followed by embryo sexing based on a biopsy. In Experiment 3, endometrial explants were cultured alone or in the presence of bovine blastocysts (n = 25) or murine blastocysts (n = 25). Following culture, explants were snap frozen and stored at -80°C until RNA extraction, qPCR or RNA-Seq. Culture with bovine blastocysts increased endometrial expression of 40 transcripts, all of which were interferon-tau induced. Culture with male or female bovine blastocysts increased transcript abundance of five classic interferon-stimulated genes (MX1, MX2, ISG15, OASY1, RSAD2) in explants; however, there was no difference in abundance of transcripts previously reported to be related to embryonic sex (IFNAR1, IFNAR2, CTGF, ARTN, SLC2A1, SLC2A5). Exposure to murine blastocysts did not elicit any detectable change in transcript abundance. These findings, coupled with our previous data, indicate that very local, interferon-tau-induced changes in endometrial gene expression occur in response to blastocysts; whether such changes play any role in subsequent pregnancy recognition remains to be established.

Insect phylogenomics.

Phylogenomics, the integration of phylogenetics with genome data, has emerged as a powerful approach to study the evolution and systematics of species. Recently, several studies employing phylogenomic tools have provided better insights into insect evolution. Next-generation sequencing methods are now increasingly used by entomologists to generate genomic and transcript sequences of various insect species and strains. These data provide opportunities for comparative genomics and large-scale multigene phylogenies of diverse lineages of insects. Phy-logenomic investigations help us to better understand systematic and evolutionary relationships of insect species that play important roles as herbivores, predators, detritivores, pollinators and disease vectors. It is important that we critically assess the prospects and limitations of phylogenomic methods. In this review, I describe the current status, outline the major challenges and remark on potential future applications of phylogenomic tools in studying insect systematics and evolution.

Urinary MCP-1 as diagnostic and prognostic marker in patients with lupus nephritis flare.

AIM OF THE STUDY: This study aimed to assess correlation of urinary monocytic chemoattractant protein-1 (UMCP-1) with severity of lupus nephritis and its role as predictor of outcome. METHOD: Twenty patients with lupus nephritis flare were included in the study. Ten patients in each group of stable systemic lupus erythematosus and non-renal flare were taken as controls. Biopsy was done to define lupus nephritis stage. UMCP-1 levels were measured in all patients at the time of entry and at four and eight weeks of follow-up. RESULTS: Mild, moderate and severe lupus nephritis flare was noted in one, five and 15 patients, respectively. UMCP-1 levels were high in patients with severe lupus nephritis flare (2.74 ± 0.95 ng/mg creatinine) as compared to patients with moderate (1.43 ± 0.46 ng/mg creatinine) and mild lupus nephritis flare (0.76 ± 0.57 ng/mg creatinine) (P = 0.0093). Baseline mean UMCP-1 levels in lupus nephritis flare, non-renal flare and stable SLE patients were 2.32 ± 1.06, 0.171 ± 0.03 and 0.213 ± 0.026 ng/mg creatinine, respectively. The difference among the three groups was very significant (P < 0.001). Also, mean UMCP-1 levels correlated significantly with severity of lupus nephritis class (P = 0.0358). During follow-up, 15 patients achieved complete or partial remission, and in these patients mean UMCP-1 levels had significant decline at eight weeks (P < 0.0001). However, mean UMCP-1 levels in the remaining five non-responders did not show significant changes at four and eight weeks (P = 0.4858). CONCLUSION: Mean UMCP-1 levels were significantly higher in the lupus nephritis flare group as compared to non-renal flare and stable patients. Baseline mean UMCP-1 levels significantly correlated with both lupus nephritis class and severity of lupus nephritis flare, hence UMCP-1 could be used as a non-invasive marker for the judgement of lupus flare and lupus nephritis class.

Rarity of tagSNPs across transferred mtDNA inserts in human genome.

The study deals with the single nucleotide polymorphism (SNPs, HapMap data) around the mtDNA insertions in human genome. The results obtained from this study suggest that application of tagSNP approach for large scale genotyping targeting NUMT integration sites may be difficult due to lack of informative mutations around these loci. This warrants development of new approaches to tag mtDNA inserts in genome-wide association studies.

Coadaptation of isoacceptor tRNA genes and codon usage bias for translation efficiency in Aedes aegypti and Anopheles gambiae.

The transfer RNAs (tRNAs) are essential components of translational machinery. We determined that tRNA isoacceptors (tRNAs with different anticodons but incorporating the same amino acid in protein synthesis) show differential copy number abundance, genomic distribution patterns and sequence evolution between Aedes aegypti and Anopheles gambiae mosquitoes. The tRNA-Ala genes are present in unusually high copy number in the Ae. aegypti genome but not in An. gambiae. Many of the tRNA-Ala genes of Ae. aegypti are flanked by a highly conserved sequence that is not observed in An. gambiae. The relative abundance of tRNA isoacceptor genes is correlated with preferred (or optimal) and nonpreferred (or rare) codons for ∼2-4% of the predicted protein coding genes in both species. The majority (∼74-85%) of these genes are related to pathways involved with translation, energy metabolism and carbohydrate metabolism. Our results suggest that these genes and the related pathways may be under translational selection in these mosquitoes.

Correlated expression patterns of microRNA genes with age-dependent behavioural changes in honeybee.

The hive-living honeybees (Apis mellifera) show age-dependent behavioural changes; young bees usually nurse the broods in the colony and the older bees engage in foraging activities. These developmentally regulated behavioural changes were previously shown to be correlated with genome-wide transcriptional changes in the honeybee brain. The indigenous small regulatory RNA molecules, known as microRNAs (miRNAs), are potent regulators of gene expression and also are developmentally regulated. Thus, we wanted to study if there might be correlation of differential expression of miRNA genes in the brain with age-dependent behavioural changes of the bees. We determined expression patterns of a set (n= 20) of predicted miRNA genes, by quantitative real-time PCR assays, in the brains of young and old bees that were engaged in nursing or foraging activities in the colony, respectively. Our data show correlated up-regulation of miRNA-124, miRNA-14, miRNA-276, miRNA-13b, let-7 and miRNA-13a in the young nurse bees. miRNA-12, miRNA-9, miRNA-219, miRNA-210, miRNA-263, miRNA-92 and miRNA-283 showed correlated expression patterns in the old forager bees. The modular changes of miRNA genes in the young nurse and old forager bees suggest possible roles of miRNAs in age-dependent behavioural changes in bees. The correlated expression of intronic miRNA genes and their host genes as well as of miRNA genes physically clustered in the genome are also observed.

Assessment of structural variation and molecular mapping of insertion sites of Desmar-like elements in the Hessian fly genome.

The Hessian fly (Mayetiola destructor) is an agriculturally important pest of wheat. A mariner element (Desmar1) has been previously identified in the Hessian fly genome. Using Desmar1 as a probe, we isolated individual copies of Desmar-like elements from the Hessian fly genome cloned in bacterial artificial chromosomes (BACs) and studied their structural variability and flanking DNA sequences. The partial Desmar-like copies are relatively more abundant (∼64%) than full length copies (∼36%) in the Hessian fly genome. Most of the full length copies are consistently flanked by an EcoRI restriction site that occurs 32 bp from one end and 66 bp from the other end of the mariner. Using an amplified fragment length polymorphism-PCR (AFLP-PCR) based method, we identified segregating polymorphisms associated with Desmar elements in a F2 mapping population. We were able to use the segregation data to localize the chromosomal position of three Desmar elements by linkage analysis. As paternal chromosomes are eliminated in the Hessian fly during early embryogenesis, two-thirds of the AFLPs were expected to be polymorphic in the mapping population and this was observed for AFLPs anchored to full length Desmar copies but not to the partial copies. Thus, our data indicate that dead and partial Desmar-like copies are probably associated with less polymorphic regions and may represent mariner graveyards in the Hessian fly genome.

Transfers of mitochondrial DNA to the nuclear genome in the wasp Nasonia vitripennis.

Many organisms carry nuclear sequences of mitochondrial origin (NUMTs). We have identified 76 NUMTs in 25 genomic locations in the jewel wasp Nasonia vitripennis. The total amount of NUMTs in Nasonia is 42 972 bp exceeding over four-fold that found in Tribolium castaneum and almost fifty-fold that found in Drosophila melanogaster, whereas Apis mellifera has an even larger number of NUMTs in its genome (over 230 kb). The Nasonia NUMTs were inserted by multiple independent events and frequently involved large fragments spanning multiple mitochondrial genes. Most of the NUMTs are recent transfers that occurred less than one million years ago after the speciation of N. vitripennis. Duplications and rearrangements in the nucleus have also occurred. Data suggest that NUMTs may be more common in hymenoptera than in other insect genomes.

Comparative analysis of nuclear tRNA genes of Nasonia vitripennis and other arthropods, and relationships to codon usage bias.

Using bioinformatics methods, we identified a total of 221 and 199 tRNA genes in the nuclear genomes of Nasonia vitripennis and honey bee (Apis mellifera), respectively. We performed comparative analyses of Nasonia tRNA genes with honey bee and other selected insects to understand genomic distribution, sequence evolution and relationship of tRNA copy number with codon usage patterns. Many tRNA genes are located physically close to each other in the form of small clusters in the Nasonia genome. However, the number of clusters and the tRNA genes that form such clusters vary from species to species. In particular, the Ala-, Pro-, Tyr- and His-tRNA genes tend to accumulate in clusters in Nasonia but not in honey bee, whereas the bee contains a long cluster of 15 tRNA genes (of which 13 are Gln-tRNAs) that is absent in Nasonia. Though tRNA genes are highly conserved, contrasting patterns of nucleotide diversity are observed among the arm and loop regions of tRNAs between Nasonia and honey bee. Also, the sequence convergence between the reconstructed ancestral tRNAs and the present day tRNAs suggests a common ancestral origin of Nasonia and honey bee tRNAs. Furthermore, we also present evidence that the copy number of isoacceptor tRNAs (those having a different anticodon but charge the same amino acid) is correlated with codon usage patterns of highly expressed genes in Nasonia.

Spectrum of kidney diseases in patients with preeclampsia-eclampsia.

AIM: The aim of this study was to analyse the clinical spectrum of renal manifestation of preeclampsia in pregnant women. METHOD: Diagnosis of preeclampsia was made using two cardinal feature of the disease after 20th weeks of gestation in previously normotensive and nonproteinuric women: (1) Blood pressure > 140/90 mm Hg and (2) urinary protein excretion of > 300 mg/24 hour. The patients with renal manifestations were followed up to 12 weeks postpartum or till death whichever was earlier. RESULT: Of 1805 pregnant women, preeclampsia was diagnosed in 106 (5.87%) patients. Primiparity constitutes 53.77% of total patients. Hypertension and proteinuria were observed in all patients. Hyperuricemia was observed in 93.65% of cases. Acute renal failure occurred in 22 patients. Dialysis support was needed in only four cases of ARF with complete recovery of renal function in 82% of cases. HELLP syndrome was seen in 16 (preeclampsia 5; eclampsia 11) patients. Sixty six patients (Death 13 and lost to follow up 27) were followed for 12 weeks. The renal parameters (Hypertension, Proteinuria and renal function) returned to normal in all except in two patients. Renal biopsy in these two cases revealed FSGS and MPGN in one each. CONCLUSION: The incidence of preeclampsia was 5.87%. Nephrotic syndrome was observed in 11.32% of patients. Acute renal failure occurred in 20.8% of patients. Hypertension, proteinuria and renal function resolved to normal over a average period of 35.8 days in all survivors. The overall mortality was 12%. Neurological complication, pulmonary edema and multiple organ failure were the causes of death.

Polymorphisms flanking the mariner integration sites in the rice gall midge (Orseolia oryzae Wood-Mason) genome are biotype-specific.

In an effort to study genome diversity within and between the Indian biotypes of the Asian rice gall midge, Orseolia oryzae, a major insect pest of rice, we made use of mariner transposable element integration site polymorphisms. Using degenerate primers, the design of which is based on mariner sequences, we amplified a ca. 450 bp mariner sequence from the rice gall midge. The mariner sequence showed homology with that of a mariner element isolated from the Hessian fly, Mayetiola destructor, a major dipteran pest of wheat. Southern hybridization, using this mariner fragment as a probe, revealed that the mariner elements are moderately to highly repetitive in the rice gall midge genome. Based on the sequence information of this 450-bp PCR-amplified fragment, outward-directed primers were designed and used in an inverse PCR (iPCR) to amplify the DNA flanking the conserved regions. To study the regions flanking the mariner integration sites, we employed a novel PCR-based approach: a combination of sequence specific amplification polymorphism (SSAP) and amplified fragment length polymorphism (AFLP). The outward-directed mariner-specific primer was used in combination with adapter-specific primers with 1-3 selective nucleotides at their 3' ends. The amplification products were resolved on an agarose gel, Southern-transferred onto nylon membranes, and probed with the iPCR fragment. Results revealed biotype-specific polymorphisms in the regions flanking the mariner integration sites, suggesting that mariner elements in the rice gall midge may be fixed in a biotype-specific manner. The implications of these results are discussed in the context of biotype differentiation.

An AFLP marker that differentiates biotypes of the Asian rice gall midge (Orseolia oryzae, Wood-Mason) is sex-linked and also linked to avirulence.

In an attempt to identify a specific marker for biotype 2 of the Asian rice gall midge (Orseolia oryzae, Wood-Mason), we used AFLP (amplified fragment length polymorphism) fingerprinting. We identified an AFLP marker that is specifically amplified in biotypes 1, 2 and 5 of the rice gall midge, but not in biotype 4. Biotypes 1, 2 and 5 are avirulent to hosts bearing the Gm2 resistance gene (found in rice variety Phalguna), whereas biotype 4 is virulent to Gm2. Based on the sequence of this AFLP marker, SCAR (sequence characterized amplified region) primers were designed and used in combination with previously developed SCAR primers to distinguish effectively all five biotypes in a multiplex PCR-based assay. The inheritance pattern of this marker in the progenies of inter-biotype crosses between biotypes 1, 2 and 4 shows that the marker can be amplified by PCR from all F1 females, irrespective of the biotype status of their parents. However, the marker is present only in those male progenies whose mother was of a Gm2 avirulent biotype. The specific amplification of this marker in the avirulent biotypes and its pattern of inheritance show that avirulence with respect to carriers of the Gm2 gene in rice gall midge is sex-linked.

Wolbachia in the Asian rice gall midge, Orseolia oryzae (Wood-Mason): correlation between host mitotypes and infection status.

Using a PCR-based method, we detected Wolbachia in the Asian rice gall midge. Furthermore, results showed that all females across all biotypes are infected with Wolbachia. However, all male flies are not infected and show different infection frequency in different biotypes. We have also identified three mitotypes, in the rice gall midge, based on DraI restriction pattern of a portion of the 12S rRNA gene that was PCR amplified using primers specific to this gene. All the females and infected male flies had type 1 mtDNA while uninfected males showed only type 2 or 3 mtDNA. Inheritance patterns of mtDNA revealed the existence of a correlation between mtDNA type and Wolbachia infection in the Asian rice gall midge. Evidence for paternal inheritance of mtDNA in Wolbachia-free gall midge is also presented.

Differentiation of Asian rice gall midge, Orseolia oryzae (Wood-Mason), biotypes by sequence characterized amplified regions (SCARs).

We developed a polymerase chain reaction (PCR)-based assay that distinguished five different biotypes of the Asian gall midge (Orseolia oryzae), a major insect pest of rice. A total of 400 random primers were screened using random amplified polymorphic DNAs (RAPDs). Five diagnostic PCR products were isolated, cloned, sequenced and converted to sequence characterized amplified regions (SCARs). Primers specific to these SCARs were able to amplify specific DNA fragments from genomic DNAs of five biotypes of gall midge in a multiplexed-PCR-based assay. The amplified DNA fragments were used as diagnostic markers to identify different biotypes of gall midge. The SCAR primers were also capable of differentiating the Asian from the African rice gall midge (Orseolia oryzivora) as well as detecting a variant of biotype 5 which caused an outbreak in Kerala, India. Unlike the use of plant host differentials and midge feeding behaviour for identifying biotypes, this assay is fast, reliable and unaffected by environmental factors.