Genet assignment and population structure analysis in a clonal forest-floor herb, Cardamine leucantha, using RAD-seq

To study the genetic structure of clonal plant populations, genotyping and genet detection using genetic markers are necessary to assign ramets to corresponding genets. Assignment is difficult as it involves setting a robust threshold of genetic distance for genet distinction as neighbouring genets in a plant population are often genetically related. Here, we used restriction site-associated DNA sequencing (RAD-seq) for a rhizomatous clonal herb, Cardamine leucantha [Brassicaceae] to accurately determine genet structure in a natural population. We determined a draft genome sequence of this species for the first time, which resulted in 66,617 scaffolds with N50 = 6,086 bp and an estimated genome size of approximately 253 Mbp. Using genetic distances based on the RAD-seq analysis, we successfully distinguished ramets that belonged to distinct genets even from a half-sib family. We applied these methods to 372 samples of C. leucantha collected at 1-m interval grids within a 20 × 20 m plot in a natural population in Hokkaido, Japan. From these samples, we identified 61 genets with high inequality in terms of genet size and patchy distribution. Spatial autocorrelation analyses indicated significant aggregation within 7 and 4 m at ramet and genet levels, respectively. An analysis of parallel DNA microsatellite loci (simple sequence repeats, SSR) suggested that RAD-seq can provide data that allows robust genet assignment. It remains unclear whether the large genets identified here became dominant stochastically or deterministically. Precise identification of genets will assist further study and characterization of dominant genets

Abnormal Fhit expression is an independent poor prognostic factor for cervical cancer

We analysed the expression of the fragile histidine triad (FHIT) gene in cervical cancer to evaluate its clinical relevance in relation to human papillomavirus (HPV) infection. A total of 73 women with cervical cancer of stage Ib or more advanced (67 squamous cell carcionomas, four adenocarcinomas, two adenosquamous carcinomas) were examined for Fhit expression by immunohistochemistry. They were further analysed for the presence of HPV and its subtype. Abnormal expression of Fhit (absent or reduced Fhit expression) was observed in 52 cases (71.2%). The high-risk HPV DNAs for cervical cancer, including type 16, 18, 31, 33, 51, 52, 58, 68, were identified in 63 cases (86%). The abnormal Fhit expression was not related to the clinicopathological factors including histology, tumour stage, and HPV type. Notably, the 5-year survival of patients showing the abnormal Fhit expression was significantly poorer than those showing normal Fhit expression (64 versus 87%, P=0.035). Interestingly, the mean age of the patients with the abnormal Fhit expression was significantly less than those with the normal Fhit expression (51.6 versus 58.7 years of age, P=0.027, student's t-test). These data imply that the aberrant Fhit expression could be a poor prognostic factor independent of HPV. In the light of a high incidence of abnormal Fhit expression in younger patients and HPV as a key player in cervical carcinogenesis, abnormal Fhit expression may accelerate carcinogenesis in concert with HPV

Are smoking and chlamydial infection risk factors for CIN? Different results after adjustment for HPV DNA and antibodies

To identify the risk factors for cervical intraepithelial neoplasia (CIN), we reanalysed the data from our previous case-control study by adjusting for human papillomavirus (HPV) antibodies. Unlike our previous study based only on HPV DNA, smoking and Chlamydia trachomatis infection were revealed as significant risk factors for CIN after adjustment for HPV antibodies

Frequency of D222G and Q223R Hemagglutinin Mutants of Pandemic (H1N1) 2009 Influenza Virus in Japan between 2009 and 2010

BACKGROUND: In April 2009, a novel swine-derived influenza A virus (H1N1pdm) emerged and rapidly spread around the world, including Japan. It has been suggested that the virus can bind to both 2,3- and 2,6-linked sialic acid receptors in infected mammals, in contrast to contemporary seasonal H1N1 viruses, which have a predilection for 2,6-linked sialic acid. METHODS/RESULTS: To elucidate the existence and transmissibility of α2,3 sialic acid-specific viruses in H1N1pdm, amino acid substitutions within viral hemagglutinin molecules were investigated, especially D187E, D222G, and Q223R, which are related to a shift from human to avian receptor specificity. Samples from individuals infected during the first and second waves of the outbreak in Japan were examined using a high-throughput sequencing approach. In May 2009, three specimens from mild cases showed D222G and/or Q223R substitutions in a minor subpopulation of viruses infecting these individuals. However, the substitutions almost disappeared in the samples from five mild cases in December 2010. The D187E substitution was not widespread in specimens, even in May 2009. CONCLUSIONS: These results suggest that α2,3 sialic acid-specific viruses, including G222 and R223, existed in humans as a minor population in the early phase of the pandemic, and that D222 and Q223 became more dominant through human-to-human transmission during the first and second waves of the epidemic. These results are consistent with the low substitution rates identified in seasonal H1N1 viruses in 2008

Jaw Laterality and Related Handedness in the Hunting Behavior of a Scale-Eating Characin, Exodon paradoxus

BACKGROUND: Asymmetry in animal bodies and behavior has evolved several times, but our knowledge of their linkage is limited. Tanganyikan scale-eating cichlids have well-known antisymmetry in their bodies and behavior; individuals open their mouths leftward (righty) or rightward (lefty), and righties always attack the right flank of the prey, whereas lefties attack the left. This study analyzed the morphological asymmetry in a scale-eating characiform, Exodon paradoxus, and its behavioral handedness. METHODOLOGY/PRINCIPAL FINDINGS: Each eight E. paradoxus was observed for 1-h with a prey goldfish in an aquarium to detect the behavioral handedness. Following the experiment, the lateral differences in the mandibles and head-inclination of these eight and ten additional specimens were analyzed. Both measurements on the morphology showed a bimodal distribution, and the laterality identified by these two methods was always consistent within a given individual, indicating that the characin has morphological antisymmetry. Furthermore, this laterality significantly corresponded to behavioral handedness; that is, lefties more often rasped scales from the right flank of the prey and vice versa. However, the correlation between laterality and handedness is the opposite of that in the cichlids. This is due to differences in the feeding apparatus and technique. The characin has cuspids pointing forward on the external side of the premaxilla, and it thrusts its dominant body side outward from its body axis on the flank of the prey to tear off scales. By contrast, the cichlids draw their dominant body side inward toward the axis or rotate it to scrape or wrench off scales with the teeth lined in the opened mouth. CONCLUSIONS/SIGNIFICANCE: This study demonstrated that the antisymmetry in external morphology and the corresponding behavioral handedness have evolved in two lineages of scale-eating fishes independently, and these fishes adopt different utilization of their body asymmetry to tear off scales

Extreme genetic fragility of the HIV-1 capsid

Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies

A possible involvement of aberrant expression of the FHIT gene in the carcinogenesis of squamous cell carcinoma of the uterine cervix

To investigate involvement of an aberrant expression of the FHIT (fragile histidine triad) gene in the process of carcinogenesis and progression in cervical carcinoma, we examined its expression by the reverse transcriptase polymerase chain reaction (RT-PCR) and cDNA sequence method in 32 cervical invasive carcinomas (25 squamous cell carcinomas and seven adeno- or adenosquamous carcinomas) and 18 of its precursor lesions [four low-grade and 14 high-grade cervical intraepithelial neoplasias (CINs)]. We also examined a link between the occurrence of the aberrant expression and human papillomavirus (HPV). We detected the aberrant FHIT transcripts in 11 of 25 (44%) cervical invasive squamous cell carcinomas and in 5 of 14 (36%) high-grade CINs (CIN 2 or 3), whereas they were not found in seven non-squamous type and four low-grade CINs (CIN 1). The alteration patterns of the FHIT gene expression in high-grade CINs were virtually similar to those found in invasive carcinomas, such that the exons 5–7 were consistently deleted associated or unassociated with loss of the exon 4 and/or 8. The incidence of the aberrant expression was not related to the presence of HPV and its type. These data indicate that the aberrant expression of the FHIT gene is observed in precursor lesions of cervical carcinoma as well as invasive carcinomas, with its incidence not increasing with advance of clinical stage. Given the squamous cell type dominant expression, the aberrant expression may play a critical role in the generation of squamous cell carcinoma of the uterine cervix, but not the consequence of the progression of the cancer. © 1999 Cancer Research Campaig