Distinct Nucleotide Sequences Research Articles

DNA Sequence Based Molecular Identification of Eustrongylides excisus Larvas (Nematoda) in Sander Lucioperca from Lake Eğirdir

In this study, 926-954 bp partially distinct nucleotide sequences which belong to the 18S and 28SrDNA gene loci from 3 Eustrongylides excisus samples were identified as identical and registered in GenBank (OP480437-39). Nucleotide values in 18S and 28S rDNA gene sequences of Eustrongylides excisus samples were defined respectively: 26.94% 27.26 A; 26.78%-26.84% T; 28.30-28.62% G; 17.39-17.92% C. According to the sequence dataset distance matrix obtained by the Pairwise comparison method, there was a complete agreement (0%) between the Eustrongylides excisus isolates identified in this study (OP480437-39) and the Eustrongylides excisus isolate registered in the GenBank (MK007967, MT415236, MK545494).The Eustrongylides excisus isolates were collected in the same cluster in Maximum likelihood filogram analysis. The internal transcribed spacer (ITS) region gene sequence results of the isolates confirmed the taxonomic position of Eustrongylides excisus, which was defined according to its anatomical and morphological features. More, 18S and 28S rDNA gene sequences were defined for the first time in this study on 3 samples of Eustrongylides excisus species in Lake Eğirdir, and contributions were made to the determination of genetic characteristics of the species.

Aplastic Anemia Patients Have Diverse T Cell Repertoires and Flexible T Cell Responses Similar to Healthy Individuals

Acquired aplastic anemia (AA) is a bone marrow failure disease caused by T cell-mediated autoimmune attack on hematopoietic stem and progenitor cells (HSPCs). Identifying autoreactive T cells in AA has been challenging. Advances in T cell receptor (TCR) sequencing and the discovery of Human Leukocyte Antigen Class I (HLA-I) risk alleles linked to autoantigen presentation in AA have rekindled interest in exploring T cell repertoires in AA. We hypothesized that autoreactive CD8 + T cells in AA may have convergent TCR structures allowing them to recognize AA autoantigens presented by specific HLA-I risk alleles. To test our hypothesis, we analyzed TCR repertoires and HLA-restricted CD8 + T cell reactivity in AA patients. Using TCR Vβ (TRB) sequencing, we analyzed 83 patient samples obtained at AA diagnosis or relapse. High-risk alleles HLA-B*14:02 and B*40:02 were present in 26% and 10% of patients, respectively. HLA-DR*15 was present in 24%. Patient samples were largely sorted bone marrow (BM) CD8 + T lymphocytes. Patient TRB repertoires were compared to those in 709 control samples, largely of unsorted peripheral blood (PB). To account for sampling differences, we downsampled sequences to the minimum number of clones in the matching tissue. Normalized clone size distributions were visualized on rank plots (Fig 1). TRB repertoires in both patients and controls were diverse. Highly expanded clones (>5%) were rare in either group. Patients had 1,415,069 unique CDR3 amino acid sequences (CDR3AA), of which 106,446 (7.5%) were shared by ≥2 patients. Among the 106,446 shared CDR3AA, 95.4% were found in controls, and 4.3% TCRs had reported specificity to exogenous (mostly viral) or endogenous antigens in VDJdb. 4,920 (4.6%) of the shared CDR3AA were detected only in patients (median 2 patients (2-25)). Of these, 190 exhibited TCR convergence in ≥1 patient, such that the same CDR3AA was encoded by ≥2 distinct nucleotide sequences. Clustering 4,920 patient-associated CDR3AA using GLIPH (Grouping of lymphocyte interactions by paratope hotspots) identified 43 TCR antigen convergence groups. CDR3AA in each cluster were not significantly expanded in the majority of patients and were not enriched for the high-risk alleles HLA-B*14:02 or B*40:02. Our data suggest that most CDR3AA in AA are private (unique to individual patients). Shared autoreactive CDR3AA may exist in patients but are likely to also be present in healthy individuals. As the next step towards identifying autoreactive T cells, we focused on AA patients with known HLA-I risk alleles. We tested their CD8 + T cells for reactivity with endogenously expressed antigens by stimulating T cells with hematopoietic cell lines engineered to express single HLA-I alleles matching those of the patients (Fig 2). TRB sequencing showed increased clonality and significant expansion of a subset of clonotypes after T cell stimulation. Interferon γ enzyme-linked immunospot assays confirmed the expanded T cells' responsiveness to antigens presented by HLA alleles used for T cell stimulation. Additionally, these expanded T cells demonstrated variable reactivity to cells expressing other HLA alleles from the same patient. Among the 1% of CDR3AA clonotypes most highly expanded with single HLA-I alleles (63-3000-fold), we identified several that were expanded in an HLA-restricted manner. However, several highly expanded CDR3AA were highly expanded with multiple HLA alleles. Potential reasons for expansion of some CDR3AA after stimulation with cells expressing different HLA alleles include 1) binding of the same CDR3AA to different antigens presented by distinct HLA alleles, 2) expansion in response to the same antigens presented by distinct HLA alleles, or 3) expansion in response to non-HLA antigens. In conclusion, our data show diverse and mostly private TRB repertoires in AA patients. Our in vitro findings suggest that much like T cell responses in non-AA individuals, T cell responses in AA are diverse and exhibit flexibility with respect to activation in response to various endogenous antigens in the context of different self HLA-I alleles. Given the diversity of TCR responses observed in AA patients, the tissue specificity of autoimmune attack and established association with risk HLA-I alleles in AA remain a mystery and likely require HSPC-specific autoantigen expression and efficient autoantigen presentation by individual patients' HLA-I alleles.

Prevalence and Molecular Genotyping of Cryptosporidium Spp. in Diarrheic Patients from Bandar Abbas City, Southern Iran

Background: Cryptosporidium species are recognized as one of the most important gastrointestinal pathogens of humans and livestock. Objectives: This study aimed to determine the prevalence and sub-genotypes of Cryptosporidium spp. among diarrheic patients in Bandar Abbas City, Iran. Methods: Diarrheic fecal samples were collected from 170 patients in three hospitals of Bandar Abbas, Iran, from October 2018 to May 2019. Initial parasitological identification of Cryptosporidium spp. was performed by modified Ziehl-Neelsen (ZN) staining. For molecular analysis, the positive specimens and the suspected ones of Cryptosporidium spp. were evaluated by sequence analysis of the 60-kDa glycoprotein gene (gp60). The collected data were analyzed using SPSS software and the relationship between the variables and the presence of Cryptosporidium spp. assessed by the chi-square test. To assess the degree of agreement between PCR and ZN staining, Cohen’s kappa-index was applied. Results: Of the 170 diarrheic patients, 98 (57.6%) were male, and 72 (42.4%) were female. Prevalence of Cryptosporidium spp. by parasitological examination was 1.8% (3/170). However, using PCR, Cryptosporidium spp. was detected in 12% (6/50) of the positive microscopically samples (3 samples) and 47 suspected specimens. Sequence analysis of the gp60 gene showed that all of the positive isolates were Cryptosporidium parvum in which all subtypes belonged to allele family IId. Two distinct nucleotide sequences obtained from this study were deposited in GenBank under the accession numbers MN820453 and MN820454. Conclusions: The predominance of C. parvum (subtype family IId) in this study emphasizes the importance of zoonotic Cryptosporidium transmission in Bandar Abbas, Southern Iran.

A Continuum of Evolving De Novo Genes Drives Protein-Coding Novelty in Drosophila

Orphan genes, lacking detectable homologs in outgroup species, typically represent 10–30% of eukaryotic genomes. Efforts to find the source of these young genes indicate that de novo emergence from non-coding DNA may in part explain their prevalence. Here, we investigate the roots of orphan gene emergence in the Drosophila genus. Across the annotated proteomes of twelve species, we find 6297 orphan genes within 4953 taxon-specific clusters of orthologs. By inferring the ancestral DNA as non-coding for between 550 and 2467 (8.7–39.2%) of these genes, we describe for the first time how de novo emergence contributes to the abundance of clade-specific Drosophila genes. In support of them having functional roles, we show that de novo genes have robust expression and translational support. However, the distinct nucleotide sequences of de novo genes, which have characteristics intermediate between intergenic regions and conserved genes, reflect their recent birth from non-coding DNA. We find that de novo genes encode more disordered proteins than both older genes and intergenic regions. Together, our results suggest that gene emergence from non-coding DNA provides an abundant source of material for the evolution of new proteins. Following gene birth, gradual evolution over large evolutionary timescales moulds sequence properties towards those of conserved genes, resulting in a continuum of properties whose starting points depend on the nucleotide sequences of an initial pool of novel genes.

PS1131 HIGH‐THROUGHPUT B‐CELL IMMUNOPROFILING AT DIAGNOSIS AND RELAPSE OFFERS FURTHER EVIDENCE OF FUNCTIONAL SELECTION THROUGHOUT THE NATURAL HISTORY OF CHRONIC LYMPHOCYTIC LEUKEMIA

Background:Chronic lymphocytic leukemia (CLL) is divided into two broad prognostic categories, namely mutated (M) and unmutated (U) CLL, according to the somatic hypermutation (SHM) status of the clonotypic heavy chain immunoglobulin (IGHV) gene. This is perceived to remain stable over time, as evidenced by low‐throughput studies, which however precluded investigation of subclonal architecture and evolution overtime.Aims:Here, we sought to comprehensively assess the B cell receptor (BcR) IG gene repertoire at CLL diagnosis and 1st relapse after chemoimmunotherapy (FCR) by next‐generation sequencing (NGS).Methods:We studied 5 patients, of whom 1 each expressed BcR IG typical of stereotyped subset #1 (U‐CLL), subset #4 (M‐CLL) or subset #6 (U‐CLL), whereas the remaining 2 concerned non‐stereotyped U‐CLL. Genomic DNA was amplified by multiplex PCR and products were subjected to paired‐end NGS. Sequence data were processed by a validated bioinformatics pipeline performing strict quality filtering. Rearrangements with identical IGHV gene and CDR3 amino acid (aa) sequence were defined as clonotypes. The dominant clonotype was defined as major, and clonotypes with the same IGHV gene, CDR3 length, and ≤2 aa differences within the CDR3 were considered as its satellite subclones.Results:In total, we analyzed 1,682,728 filtered‐in, productive IGHV‐IGHD‐IGHJ gene rearrangements (median 195,460 rearrangements/sample). In all cases, the major clonotype (IGHV and CDR3 aa sequence) was identical to that determined by Sanger sequencing and remained the same at diagnosis and relapse. However, it consisted of multiple distinct nucleotide (nt) sequences; while most nt sequences displayed the same SHM load as with Sanger (median 73.9% of all nt sequences of the major clonotype), there were other nt sequences with higher or lower SHM load. In all cases, nt sequences with discordant SHM status were practically negligible (median frequency 0.01%). Notably, the relative frequency of the dominant nt sequence increased at relapse for all cases (median increase by 3.7%, p < 0.01). A particular note for subset #4, which by definition displays high SHM, was the emergence of major clonotype nt sequences with lower or even absent SHM at relapse. The median frequency of the major clonotype at diagnosis was 92.5% (range 86.1–93.3%) and remained stable at relapse (92.6%, range 89.9–93.8%). In all cases, the major clonotype came along with numerous satellite subclones (same IGHV gene, CDR3 length, and ≤2 aa differences within the CDR3) at both diagnosis and relapse (median n = 685 vs n = 604 respectively, p>0.05). When considering both the major clonotype and its satellite subclones, the respective cumulative frequency corresponded to nearly the entire B cell repertoire/sample (median 99.6%, range 97.6–99.8%). Subset #4 satellite sublones displayed recurrent SHM patterns within the CDR3 at relapse, which actually diverged from the major clonotype and converged towards the consensus CDR3 sequence for this subset as defined by 176 different subset #4 patients.Summary/Conclusion:Overall, our study provides insight into the evolving architecture of the BcR IG repertoire of relapsing CLL, revealing that, similar to genomic subclones, there is a universe of BcR IG subclones as well. While the possibility of technical error cannot be excluded, the identification of recurrent SHM patterns and trends at relapse strongly points towards functional selection.

Characterization of major histocompatibility complex-related molecule 1 sequence variants in non-human primates.

The major histocompatibility complex (MHC) class I-related molecule, MR1, presents vitamin B metabolites from bacteria and yeast to mucosal-associated invariant T (MAIT) cells. Despite the evolutionary conservation of MR1, we do not know whether different allele variants of MR1 exist within the nonhuman primate (NHP) populations that are commonly used for biomedical research. In this study, we identified 21 distinct MR1 nucleotide sequences representing 32 different alleles across five different NHP populations. The majority of the alleles conferring amino acid changes (allele variants) were found in or near the alpha-1 domain of the mature MR1 protein. We expressed four of the most commonly observed MR1 allele variants in 293T cells, and we found that each variant could present bacterial metabolites on the cell surface. We successfully induced cytokine production in macaque MAIT cells stimulated with 293T cells expressing the four most common MR1 allele variants, demonstrating the usefulness of these cell lines to study MAIT cell activity. Our data suggests that MR1 is not monomorphic, but that there are multiple MR1 alleles in NHPs. The materials we describe here will be valuable for characterizing differences in MR1 antigen presentation and MAIT cell function in NHPs.

Prevalence of hepatitis E virus infection among pregnant women in Zhenjiang, China

Abstract Background Most hepatitis E virus (HEV) infections are mild or subclinical, however, the infections are characteristically associated with a high incidence of symptomatic presentation among pregnant women. Objectives The aim of this study was to assess the HEV prevalence among pregnant women in Zhenjiang, China. Materials and methods 225 and 208 of serum samples collected from pregnant and healthy nonpregnant women, respectively, were used to detect anti-HEV IgG and IgM levels. IgM positive samples were further tested for HEV RNA by using RT-nested PCR (nPCR) method. Positive PCR products were sequenced and subjected to phylogenetic analysis. Results 21.8% (49/225) of samples were anti-HEV IgG positive in pregnant women, whereas 7.7% (16/208) of samples were anti-HEV IgG positive in control subjects (P Conclusions In the current study, the prevalence of HEV infection with respect to IgG and IgM levels among pregnant women was significantly higher than that in the healthy nonpregnant group. Additionally, all four HEV strains belonged to genotype 4 implying that genotype 4 is the predominant genotype in the area.

23 Evidence for antigen-driven TCRB chain convergence in the tumourinfiltrating t cell repertoire of 148 research subjects with melanoma

IntroductionT cell convergence refers to the phenomenon whereby antigen-driven selection enriches for T cell receptors having a shared antigen specificity but different amino acid or nucleotide sequence. T cell recruitment and expansion within the tumour microenvironment (TME) may be directed by responses to tumour neoantigen, suggesting that elevated T cell convergence could be a general feature of the tumour infiltrating T cell repertoire. Here we evaluate evidence for T cell convergence within tumour biopsy and peripheral blood leukocytes (PBL) from a set of 148 research subjects with melanoma.Material and methodsTotal RNA from 85 tumour biopsy research samples (non-FFPE) was extracted for use in long-amplicon TCRB chain sequencing (mean amplicon length of 330 bp covering CDR1, 2 and 3) via the Oncomine TCR Beta-LR Research Assay. To evaluate T cell convergence within each biopsy, we searched for instances where TCRB chains were identical in amino acid space (shared variable gene identity and CDR3 amino acid sequence) but had distinct nucleotide sequences owing to N-addition and exonucleotide chewback within the V-D and D-J junctions of the CDR3. To provide context, we evaluated evidence for T cell convergence in PBL T cell repertoires derived from healthy donors and 63 additional individuals with melanoma.Results and discussionsSequencing of melanoma biopsy research samples yielded an average of 6029 clones per sample. 11 of 85 samples yielded fewer than 100 clones and were eliminated from downstream analysis. Convergent T cell receptors were identified in 68/74 (92%) of tumour infiltrating T cell repertoires having greater than 100 detected clones. The frequency of convergent T cell rearrangements was greater in melanoma tumour biopsies than healthy or melanoma PBL samples.ConclusionThese data suggest that T cell convergence may be a common feature of the melanoma infiltrating T cell repertoire. Convergence was more frequently observed within the TME than T cell repertoires derived from healthy and melanoma PBL, consistent with elevated antigen-driven T cell selection within the TME. The extent to which convergence is a feature of the TME in other cancers is not yet known. T cell receptor convergence may be driven by T cell responses to tumour neoantigen within the TME. In such case, in silico identification of convergent T cell receptors by long-amplicon TCRB sequencing may serve as a means for rapid identification of antigen-specific T cell receptors for future therapeutic use.

Abstract 4668: Evidence for antigen-driven TCRB chain convergence in the tumor infiltrating T cell repertoire of 85 research subjects with melanoma

Abstract Introduction T cell convergence refers to the phenomenon whereby antigen-driven selection enriches for T cell receptors having a shared antigen specificity but different amino acid or nucleotide sequence. T cell recruitment and expansion within the tumor microenvironment (TME) may be directed by responses to tumor neoantigen, suggesting that elevated T cell convergence could be a general feature of the tumor infiltrating T cell repertoire. Here we evaluate evidence for T cell convergence within tumor biopsy research samples from a set of 85 subjects with melanoma. Methods Total RNA from 85 tumor biopsy research samples (non-FFPE) was extracted for use in long-amplicon TCRB chain sequencing (mean amplicon length of 330bp covering CDR1, 2 and 3) via the Ion AmpliSeq Immune Repertoire Assay Plus, TCRB. To evaluate T cell convergence within each biopsy, we searched for instances where TCRB chains were identical in amino acid space (shared variable gene identity and CDR3 amino acid sequence) but had distinct nucleotide sequences owing to N-addition and exonucleotide chewback within the V-D and D-J junctions of the CDR3. To provide context, we evaluated evidence for T cell convergence with T cell repertoires derived from healthy donor peripheral blood leukocytes (PBL). Results Sequencing of melanoma biopsies yielded an average of 6029 clones per sample. 11 of 85 samples yielded fewer than 100 clones and were eliminated from downstream analysis. Convergent T cell receptors were identified in 68/74 (92%) of tumor infiltrating T cell repertoires having greater than 100 detected clones. The frequency of convergent rearrangements was approximately 50-fold greater in the melanoma-infiltrating T cell repertoire than healthy PBL samples (p<.001). Conclusions These data suggest that T cell convergence may be a common feature of the melanoma infiltrating T cell repertoire. Convergence was more frequently observed within the TME than T cell repertoires derived from healthy PBL, consistent with elevated antigen-driven T cell selection within the TME. The extent to which convergence is a feature of the TME in other cancers is not yet known. T cell receptor convergence may be driven by T cell responses to tumor neoantigen within the TME. In such case, in silico identification of convergent T cell receptors by long-amplicon sequencing may serve as an approach for rapid identification of antigen-specific T cell receptors for future therapeutic use. For research use only. Citation Format: Timothy J. Looney, Sean Glenn, Sarabjot Pabla, Jeff Conroy, Carl Morrison, Alice Zheng, Lauren Miller, Elizabeth Linch, Denise Topacio, Geoff Lowman, Fiona Hyland, Mark Anderson. Evidence for antigen-driven TCRB chain convergence in the tumor infiltrating T cell repertoire of 85 research subjects with melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4668.

In Search for Pheromone Receptors: Certain Members of the Odorant Receptor Family in the Desert Locust Schistocerca gregaria (Orthoptera: Acrididae) Are Co-expressed with SNMP1.

Under given environmental conditions, the desert locust (Schistocera gregaria) forms destructive migratory swarms of billions of animals, leading to enormous crop losses in invaded regions. Swarm formation requires massive reproduction as well as aggregation of the animals. Pheromones that are detected via the olfactory system have been reported to control both reproductive and aggregation behavior. However, the molecular basis of pheromone detection in the antennae of Schistocerca gregaria is unknown. As an initial step to disclose pheromone receptors, we sequenced the antennal transcriptome of the desert locust. By subsequent bioinformatical approaches, 119 distinct nucleotide sequences encoding candidate odorant receptors (ORs) were identified. Phylogenetic analyses employing the identified ORs from Schistocerca gregaria (SgreORs) and OR sequences from the related species Locusta migratoria revealed a group of locust ORs positioned close to the root, i.e. at a basal site in a phylogenetic tree. Within this particular OR group (termed basal or b-OR group), the locust OR sequences were strictly orthologous, a trait reminiscent of pheromone receptors from lepidopteran species. In situ hybridization experiments with antennal tissue demonstrated expression of b-OR types from Schistocerca gregaria in olfactory sensory neurons (OSNs) of either sensilla trichodea or sensilla basiconica, both of which have been reported to respond to pheromonal substances. More importantly, two-color fluorescent in situ hybridization experiments showed that most b-OR types were expressed in cells co-expressing the “sensory neuron membrane protein 1” (SNMP1), a marker indicative of pheromone-sensitive OSNs in insects. Analyzing the expression of a larger number of SgreOR types outside the b-OR group revealed that only a few of them were co-expressed with SNMP1.In summary, we have identified several candidate pheromone receptors from Schistocerca gregaria that could mediate responses to pheromones implicated in controlling reproduction and aggregation behavior.

Molecular epidemiology of rotavirus gastroenteritis in Central Kenya before vaccine introduction, 2009-2014

Between July 2009 and June 2014, a total of 1,546 fecal specimens were collected from children <5 years of age with acute gastroenteritis admitted to Kiambu County Hospital, Central Kenya. The specimens were screened for group A rotavirus (RVA) using ELISA, and RVA-positive specimens were subjected to semi-nested RT-PCR to determine the G and P genotypes. RVA was detected in 429/1,546 (27.5%) fecal specimens. RVA infections occurred in all age groups <59 months, with an early peak at 6-17 months. The infections persisted year-round with distinct seasonal peaks depending on the year. G1P[8] (28%) was the most predominant genotype, followed by G9P[8] (12%), G8P[4] (7%), G1P[4] (5%), G9P[4] (4%), and G12P[6] (3%). In the yearly change of G and P genotypes, a major shift from G9P[8] to G1P[8] was found in 2012. Phylogenetic analysis of the nucleotide sequences of the VP7 and VP4 genes of seven strains with unusual G8 or P[6] showed that the VP7 nucleotide sequences of G8 were clustered in lineage 6 in which African strains are included, and that there are at least two distinct VP4 nucleotide sequences of P[6] strains. These results represent basic data on RVA strains circulating in this region before vaccine introduction. J. Med. Virol. 89:809-817, 2017. © 2016 Wiley Periodicals, Inc.

Molecular detection and genome characterization of porcine circovirus type 2 in rats captured on commercial swine farms.

Porcine circovirus type 2 (PCV2) is considered the major etiological pathogen of porcine circovirus-associated diseases (PCVADs) in pigs. Recently, PCV2 was also found in non-porcine animals such as cattle, rats, and mice. However, there was no record of PCV2 in rats in China. The goal of this study was to investigate whether PCV2 was present in rats (Rattus norvegicus, RN) on three swine farms, using molecular tools. PCR results showed that 30 of 95 (31.6%) rat samples were positive for PCV2. Moreover, further genotype analysis suggested that 10 of 30 (33.3%) were positive for PCV2a, 19 of 30 (63.3%) were positive for PCV2b, and only one sample (1/30, 3.33%) was co-infected by PCV2a and PCV2b. To determine the possible origin of PCV2, 60 serum samples were also collected from weaned pigs on those swine farms, and 23 out of 60 samples were positive for PCV2. In addition, two distinct RN-origin and two distinct porcine-origin PCV2 full-length nucleotide sequences were obtained from the farms. Sequence and phylogenetic analysis indicated that they had the highest nucleotide similarity and closest genetic relationships to each other. In this study, we report the infection and genome characterization of PCV2 in rats and compare RN-origin and porcine-origin PCV2 sequences obtained from the same pig farm, revealing possible cross-species transmission of PCV2.

Intraspecific and interspecific genetic variation of Gongylonema pulchrum and two rodent Gongylonema spp. (G. aegypti and G. neoplasticum), with the proposal of G. nepalensis n. sp. for the isolate in water buffaloes from Nepal.

The gullet worm (Gongylonema pulchrum) has been recorded from a variety of mammals worldwide. In an earlier study, we demonstrated two separate transmission cycles in cattle (Bos taurus) and wild mammals in Japan based on nucleotide sequences of the ribosomal RNA gene (rDNA) and cytochrome c oxidase subunit I (cox-1) region of mitochondrial DNA of multiple isolates of different origins. Our earlier study additionally demonstrated two major cox-1 haplotypes of G. pulchrum prevalent in cattle in Japan. In the present study, we collected G. pulchrum from cattle and goats (Capra hircus) in Alashan League, Inner Mongolia, China; Gongylonema aegypti from spiny mice (Acomys dimidiatus) in the Sinai Peninsula, Egypt; and Gongylonema neoplasticum from a black rat (Rattus rattus) in Okinawa Island, Japan, to analyze their genetic relationships with G. pulchrum in Japan. The gullet worms from Alashan League had almost identical rDNA nucleotide sequences and two cox-1 haplotypes as seen in G. pulchrum from the cattle in Japan. The two rodent Gongylonema spp. had distinct rDNA nucleotide sequences compared with those of G. pulchrum; only the 18S and 5.8S rDNA sequences showed high identities at 97.2-98.7%, while the remaining sequences were less than 75% identical. The 18S, 5.8S, and 28S rDNA sequences of the two rodent Gongylonema spp. showed nucleotide identities of 99.8% (1811/1814), 100% (158/158), and 98.9% (3550/3590), respectively. The cox-1 regions showed 91.6% (338/369)-92.1% (340/369) identities, with completely identical amino acid sequences. The genetic diversities of three distinct Gongylonema spp. and their possible intraspecific genetic variation may allow us to resolve the taxonomic position of Gongylonema spp. which display few obvious morphological differences from their congeners. Consequently, the Gongylonema isolate from water buffaloes (Bubalus bubalis) in Nepal reported in our previous study is concluded to be a new species, and Gongylonema nepalensis n. sp. is erected for it.

Global distribution of malaria-resistant MHC-HLA alleles: the number and frequencies of alleles and malaria risk.

BackgroundThe major histocompatibility complex (MHC) is the most polymorphic genetic region in vertebrates, but the origin of such genetic diversity remains unresolved. Several studies have demonstrated at the within-population level that individuals harbouring particular alleles can be less or more susceptible to malaria, but these do not allow strong generalization.MethodsHere worldwide data on the frequencies of several hundred MHC alleles of the human leucocyte antigen (HLA) system in relation to malaria risk at the between-population level were analysed in a phylogenetic framework, and results for different alleles were quantitatively summarized in a meta-analysis.ResultsThere was an overall positive relationship between malaria pressure and the frequency of several HLA alleles indicating that allele frequencies increase in countries with strong malaria pressure. Nevertheless, considerable heterogeneity was observed across alleles, and some alleles showed a remarkable negative relationship with malaria risk. When heterogeneities were partitioned into different organization groups of the MHC, the strongest positive relationships were detected for alleles of the HLA-A and HLA-B loci, but there were also differences between MHC supertypes that constitute functionally distinct nucleotide sequences. Finally, the number of MHC alleles that are maintained within countries was also related to malaria risk.ConclusionTherefore, malaria represents a key selection pressure for the human MHC and has left clear evolutionary footprints on both the frequencies and the number of alleles observed in different countries.Electronic supplementary materialThe online version of this article (doi:10.1186/1475-2875-13-349) contains supplementary material, which is available to authorized users.

CELL NUCLEAR ALTERATIONS IN MYOTONIC DYSTROPHY

In the cell nucleus, genes are transcribed, and the primary transcripts undergo molecular processing which generates mature RNAs to be exported to the cytoplasm. The events leading to the formation of mature RNAs are chronologically and spatially ordered, and they mostly occur on distinct ribonucleoprotein (RNP)-containing structures. Defects in the RNA maturation pathways have been related to diseases leading to muscle dystrophy: in myotonic dystrophy type 1 (DM1) and type 2 (DM2), the characteristic multisystemic features (e.g., myotonia, muscular dystrophy, dilated cardiomyopathy, cardiac conduction defects, cataracts, insulin-resistance, and disease-specific serological abnormalities) are caused by the expansion of two distinct nucleotide sequences: (CTG)n in the 3’ untranslated region of the DMPK gene on chromosome 19q13 in DM1, and (CCTG)n in the first intron of the ZNF9 gene on chromosome 3q21 in DM2. Combining biomolecular and cytochemical techniques, it has been demonstrated that the basic mechanisms of both DMs reside in the nuclear sequestration of the expanded RNAs: CUG- and CCUG-containing transcripts accumulate in intranuclear foci in DM1 and DM2 cells respectively, and alter the regulation and intranuclear localization of the RNA-binding proteins CUGBP1 and MBLN, which are necessary for the physiological processing of pre-mRNA. Using immunocytochemical techniques at light and electron microscopy, we have demonstrated that MBNL1-containing foci in DM2 cells also sequester snRNPs and hnRNPs, splicing factors involved in the early phases of transcript processing; this strengthens the hypothesis that the multifactorial phenotype of dystrophic patients could be due to a general alteration of the pre-mRNA post-transcriptional pathway. Interestingly, we also demonstrated that, in skeletal muscles of DM1 and DM2 patients, splicing and cleavage factors accumulate in myonuclei, suggesting an impairment of pre-mRNA processing reminiscent of the nuclear alterations typical of sarcopenia (i.e. the loss of muscle mass and function physiologically occurring during ageing). Moreover, in an in vitro study, we observed that satellite-cell-derived DM2 myoblasts show cell senescence alterations and impairment of the pre-mRNA maturation pathways earlier than the myoblasts from healthy patient. These results suggest possible common cellular mechanisms responsible for skeletal muscle wasting in different pathologies.

Skeletal muscle features in myotonic dystrophy and sarcopenia: do similar nuclear mechanisms lead to skeletal muscle wasting?

In the cell nucleus, the gene primary transcripts undergo molecular processing to generate mature RNAs, which are finally exported to the cytoplasm. These mRNA maturation events are chronologically and spatially ordered, and mostly occur on distinct ribonucleoprotein (RNP)-containing structures. Defects in the mRNA maturation pathways have been demonstrated in myotonic dystrophy type 1 (DM1) and type 2 (DM2) whose characteristic multisystemic features are caused by the expansion of two distinct nucleotide sequences: (CTG)n in the DMPK gene on chromosome 19q13 in DM1, and (CCTG)n in the ZNF9 gene on chromosome 3q21 in DM2. By combining biomolecular and cytochemical techniques, it has been shown that the basic mechanisms of DMs reside in the accumulation of CUG- or CCUG-containing transcripts in intranuclear foci where several RNA-binding proteins necessary for the physiological processing of pre-mRNA are sequestered. Moreover, a nucleoplasmic accumulation of splicing and cleavage factors has been found in DMs. This suggests that the dystrophic phenotype could depend on a general alteration of the pre-mRNA post-transcriptional pathway. Interestingly, the accumulation of pre-mRNA processing factors in the myonuclei of DM1 and DM2 patients is reminiscent of the nuclear alterations typical of sarcopenia, i.e., the loss of muscle mass and function which physiologically occurs during ageing. Consistently, in an in vitro study, we observed that satellite-cell-derived DM2 myoblasts show cell senescence alterations and impairment of the pre-mRNA maturation pathways earlier than the myoblasts from healthy patient. These results suggest possible common cellular mechanisms responsible for skeletal muscle wasting in sarcopenia and in myotonic dystrophy.

Seroprevalence and molecular detection of hepatitis E virus in Yunnan Province, China

In this study, the prevalence and characteristics of hepatitis E virus (HEV) in pigs and the general population in the Yunnan province, China, were evaluated. Nine hundred sixty sera, 95 liver and 60 feces samples were randomly collected from pig farms and abattoirs, in addition 173 human sera were sampled in the provincial capital city for a serological survey and an RT-nPCR assay. The screening results showed that among 621 samples collected from five pig farms, the HEV-specific IgG positive rate ranged from 73.2% to 83.5%, and the overall seroprevalence was 78.9% (490/621). A further analysis revealed that the seroprevalence increased with age. The positive rate of human serum samples was 39.9% (69/173). HEV RNA was detected in five swine feces, six swine liver and one anti-HEV-IgM-positive human serum sample by RT-nPCR. Sequence and alignment of the 348-nt PCR-amplified products of 12 HEV strains identified nine distinct nucleotide sequences. Phylogenetic and molecular evolutionary analysis revealed that these nine sequences shared 84.2% to 100.0% nucleotide sequence identity with each other, with all isolates belonging to genotype 4 HEV and clustering with other Chinese swine and human HEV sequences determined earlier. This study results suggest that the prevalence of genotype 4 HEV is serious, both in pig herds and in the human population, and authorities should pay more attention to the prevalence of HEV in southwest China.

Exhaustive T-cell repertoire sequencing of human peripheral blood samples reveals signatures of antigen selection and a directly measured repertoire size of at least 1 million clonotypes

Massively parallel sequencing is a useful approach for characterizing T-cell receptor diversity. However, immune receptors are extraordinarily difficult sequencing targets because any given receptor variant may be present in very low abundance and may differ legitimately by only a single nucleotide. We show that the sensitivity of sequence-based repertoire profiling is limited by both sequencing depth and sequencing accuracy. At two timepoints, 1 wk apart, we isolated bulk PBMC plus naïve (CD45RA+/CD45RO-) and memory (CD45RA-/CD45RO+) T-cell subsets from a healthy donor. From T-cell receptor beta chain (TCRB) mRNA we constructed and sequenced multiple libraries to obtain a total of 1.7 billion paired sequence reads. The sequencing error rate was determined empirically and used to inform a high stringency data filtering procedure. The error filtered data yielded 1,061,522 distinct TCRB nucleotide sequences from this subject which establishes a new, directly measured, lower limit on individual T-cell repertoire size and provides a useful reference set of sequences for repertoire analysis. TCRB nucleotide sequences obtained from two additional donors were compared to those from the first donor and revealed limited sharing (up to 1.1%) of nucleotide sequences among donors, but substantially higher sharing (up to 14.2%) of inferred amino acid sequences. For each donor, shared amino acid sequences were encoded by a much larger diversity of nucleotide sequences than were unshared amino acid sequences. We also observed a highly statistically significant association between numbers of shared sequences and shared HLA class I alleles.

Transcriptional inhibition of progressive renal disease by gene silencing pyrrole–imidazole polyamide targeting of the transforming growth factor-β1 promoter

Pyrrole-imidazole (PI) polyamides are small synthetic molecules that recognize and attach to the minor groove of DNA, thereby inhibiting gene transcription by blocking transcription factor binding. These derivatives can act as gene silencers inhibiting target gene expression under stimulatory conditions such as disease. To evaluate PI polyamides as treatments for the progression of renal diseases, we examined morphological effects, pharmacological properties, and the specificity of PI polyamides targeted to the transforming growth factor (TGF)-β1 promoter during salt-induced hypertensive nephrosclerosis in Dahl salt-sensitive rats. The targeted PI polyamide markedly reduced glomerulosclerosis and interstitial fibrosis without side effects. PI polyamide significantly decreased expression of TGF-β1 and extracellular matrix in the renal cortex. Microarray analysis found that only 3% of the transcripts were affected by PI polyamide, but this included decreased expression of extracellular matrix, TGF-β1-related cytokines, angiogenic, and cell stabilizing factors, proteinases, and renal injury-related factors. Thus, targeted PI polyamides are potential gene silencers for diseases not treatable by current remedies.

Analysis of genomic DNA of DcACS1, a 1-aminocyclopropane-1-carboxylate synthase gene, expressed in senescing petals of carnation (Dianthus caryophyllus) and its orthologous genes in D. superbus var. longicalycinus

Carnation (Dianthus caryophyllus) flowers exhibit climacteric ethylene production followed by petal wilting, a senescence symptom. DcACS1, which encodes 1-aminocyclopropane-1-carboxylate synthase (ACS), is a gene involved in this phenomenon. We determined the genomic DNA structure of DcACS1 by genomic PCR. In the genome of 'Light Pink Barbara', we found two distinct nucleotide sequences: one corresponding to the gene previously shown as DcACS1, designated here as DcACS1a, and the other novel one designated as DcACS1b. It was revealed that both DcACS1a and DcACS1b have five exons and four introns. These two genes had almost identical nucleotide sequences in exons, but not in some introns and 3'-UTR. Analysis of transcript accumulation revealed that DcACS1b is expressed in senescing petals as well as DcACS1a. Genomic PCR analysis of 32 carnation cultivars showed that most cultivars have only DcACS1a and some have both DcACS1a and DcACS1b. Moreover, we found two DcACS1 orthologous genes with different nucleotide sequences from D. superbus var. longicalycinus, and designated them as DsuACS1a and DsuACS1b. Petals of D. superbus var. longicalycinus produced ethylene in response to exogenous ethylene, accompanying accumulation of DsuACS1 transcripts. These data suggest that climacteric ethylene production in flowers was genetically established before the cultivation of carnation.