Determination of partial genomic sequences and development of a CAPS system of the S-RNase gene for the identification of 22 S haplotypes of apple (Malus × domestica Borkh.)

Information about self-incompatibility (S) genotypes of apple cultivars is important for the selection of pollen donors for fruit production and breeding. Although S genotyping systems using S haplotype-specific PCR of S-RNase, the pistil S gene, are useful, they are sometimes associated with false-positive/negative problems and are unable to identify new S haplotypes. The CAPS (cleaved amplified polymorphic sequences) system is expected to overcome these problems, however, the genomic sequences needed to establish this system are not available for many S-RNases. Here, we determined partial genomic sequences of eight S-RNases, and used the information to design new primer and to select 17 restriction enzymes for the discrimination of 22 S-RNases by CAPS. Using the system, the S genotypes of three cultivars were determined. The genomic sequence-based CAPS system would be useful for S genotyping and analyzing new S haplotypes of apple.     

Abundance of Zetaproteobacteria within crustal fluids in back‐arc hydrothermal fields of the Southern Mariana Trough

To extend knowledge of subseafloor microbial communities within the oceanic crust, the abundance, diversity and composition of microbial communities in crustal fluids at back‐arc hydrothermal fields of the Southern Mariana Trough (SMT) were investigated using culture‐independent molecular techniques based on 16S rRNA gene sequences. Seafloor drilling was carried out at two hydrothermal fields, on‐ and off‐ridge of the back‐arc spreading centre of the SMT. 16S rRNA gene clone libraries for bacterial and archaeal communities were constructed from the fluid samples collected from the boreholes. Phylotypes related to Thiomicrospira in the Gammaproteobacteria (putative sulfide‐oxidizers) and Mariprofundus in the Zetaproteobacteria (putative iron‐oxidizers) were recovered from the fluid samples. A number of unique archaeal phylotypes were also recovered. Fluorescence in situ hybridization (FISH) analysis indicated the presence of active bacterial and archaeal populations in the fluids. The Zetaproteobacteria accounted for up to 32% of the total prokaryotic cell number as shown by FISH analysis using a specific probe designed in this study. Our results lead to the hypothesis that the Zetaproteobacteria play a role in iron oxidation within the oceanic crust.     

Developmental capacity of Antarctic minke whale ( Balaenoptera bonaerensis ) vitrified oocytes following in vitro maturation, and parthenogenetic activation or intracytoplasmic sperm injection

The present study investigated the effects of the sexual maturity of oocyte donors on in vitro maturation (IVM) and the parthenogenetic developmental capacity of fresh minke whale oocytes. The effects of cytochalasin B (CB) pretreatment and two types of cryoprotectant solutions (ethylene glycol (EG) or ethylene glycol and dimethylsulfoxide (EG + DMSO)) on the in vitro maturation of vitrified immature whale oocytes were compared, and the developmental capacity of vitrified immature whale oocytes following IVM and intracytoplasmic sperm injection examined (ICSI). The maturation rate did not differ significantly with sexual maturity (adult, 60.9%; prepubertal, 53.1%), but the parthenogenetic activation rate of oocytes from adult donors (76.7%) was significantly higher (p < 0.05) than that of oocytes from prepubertal donors (46.4%). The maturation rates after vitrification and warming were not significantly different between the EG (22.2%) and EG + DMSO groups (30.2%), or between the CB-treated (30.4%) and non-CB-treated groups (27.3%). These results indicate that parthenogenetic activation of in vitro matured oocytes from adult minke whales was superior to that from prepubertal whales, but that the developmental capacity of the whale oocytes after parthenogenetic activation or ICSI was still low. The present study also showed that CB treatment before vitrification and two kinds of cryoprotectants did not improve the IVM rate following the vitrification of immature whale oocytes.     

Amphiphilic molecular gels from ω-aminoalkylated l-glutamic acid derivatives with unique chiroptical properties

Self-assembling amphiphiles with unique chiroptical properties were derived from l-glutamic acid through ω-aminoalkylation and double long-chain alkylation. These amphiphiles can disperse in various solvents ranging from water to n-hexane. TEM and SEM observations indicate that the improvement in dispersity is induced by the formation of tubular and/or fibrillar aggregates with nanosized diameters, which makes these amphiphiles similar to aqueous lipid membrane systems. Spectroscopic observations, such as UV–visible and CD spectroscopies indicate that the aggregates are constructed on the basis of S- and R-chirally ordered structures through interamide interactions in water and organic media, respectively, and that these chiroptical properties can be controlled thermotropically and lyotropically. It is also reported that the chiral assemblies provide specific binding sites for achiral molecules and then induce chirality for the bonded molecules. Further, the applicability of the amphiphiles to template polymerization is discussed.     

Connective tissue growth factor is a substrate of ADAM28

ADAM28, a member of the ADAM (a disintegrin and metalloproteinase) gene family, is over-expressed by carcinoma cells and the expression correlates with carcinoma cell proliferation and progression in human lung and breast carcinomas. However, information about substrates of ADAM28 is limited. We screened interacting molecules of ADAM28 in human lung cDNA library by yeast two-hybrid system and identified connective tissue growth factor (CTGF). Binding of CTGF to proADAM28 was demonstrated by yeast two-hybrid assay and protein binding assay. ADAM28 cleaved CTGF in dose- and time-dependent manners at the Ala¹⁸¹-Tyr¹⁸² and Asp¹⁹¹-Pro¹⁹² bonds in the hinge region of the molecule. ADAM28 selectively digested CTGF in the complex of CTGF and vascular endothelial growth factor₁₆₅ (VEGF₁₆₅), releasing biologically active VEGF₁₆₅ from the complex. RT-PCR and immunohistochemical analyses demonstrated that ADAM28, CTGF and VEGF are commonly co-expressed in the breast carcinoma tissues. These data provide the first evidence that CTGF is a novel substrate of ADAM28 and suggest that ADAM28 may promote VEGF₁₆₅-induced angiogenesis in the breast carcinomas by the CTGF digestion in the CTGF/VEGF₁₆₅ complex.     

Evolutionary Genetics of Human Enterovirus 71: Origin,Population Dynamics,Natural Selection,and Seasonal Periodicity of the VP1 Gene

Human enterovirus 71 (EV-71) is one of the major etiologic causes of hand, foot, and mouth disease (HFMD) among young children worldwide, with fatal instances of neurological complications becoming increasingly common. Global VP1 capsid sequences (n = 628) sampled over 4 decades were collected and subjected to comprehensive evolutionary analysis using a suite of phylogenetic and population genetic methods. We estimated that the common ancestor of human EV-71 likely emerged around 1941 (95% confidence interval [CI], 1929 to 1952), subsequently diverging into three genogroups: B, C, and the now extinct genogroup A. Genealogical analysis revealed that diverse lineages of genogroup B and C (subgenogroups B1 to B5 and C1 to C5) have each circulated cryptically in the human population for up to 5 years before causing large HFMD outbreaks, indicating the quiescent persistence of EV-71 in human populations. Estimated phylogenies showed a complex pattern of spatial structure within well-sampled subgenogroups, suggesting endemicity with occasional lineage migration among locations, such that past HFMD epidemics are unlikely to be linked to continuous transmission of a single strain of virus. In addition, rises in genetic diversity are correlated with the onset of epidemics, driven in part by the emergence of novel EV-71 subgenogroups. Using subgenogroup C1 as a model, we observe temporal strain replacement through time, and we investigate the evidence for positive selection at VP1 immunogenic sites. We discuss the consequences of the evolutionary dynamics of EV-71 for vaccine design and compare its phylodynamic behavior with that of influenza virus.Enterovirus 71 (EV-71) is a member of the genus Enterovirus in the family Picornaviridae. Classified as human enterovirus species A (HEV-A) along with some group A coxsackieviruses (CV-A), EV-71 is a small, nonenveloped, positive-stranded RNA virus with a genome approximately 7,400 bases long and is genetically most related to CV-A16. EV-71 is divided into three major genogroups (denoted A, B, and C), and various subgenogroups within genogroups B and C.Since its first isolation in the United States in 1969 (71), EV-71 has been identified worldwide as a common cause of hand, foot, and mouth disease (HFMD) in young children and infants. Large EV-71-associated HFMD outbreaks have been reported in the United States, Europe, Australia, and Asia and constitute a significant and emerging threat to global public health (9, 50, 62, 63). Although EV-71 infection manifests most frequently as a mild, self-limited febrile illness characterized by papulovesicular lesions on the hands, feet, oropharyngeal mucosa, and buttocks, a small proportion of acute infections are associated with fatal neurological symptoms, including brain stem encephalitis, aseptic meningitis, and poliomyelitis-like paralysis (4, 28, 47). Such cases of neurological disease with a high case fatality rate were first reported in Bulgaria in 1975 (21) and Hungary in 1978 (52). However, large HFMD epidemics with high mortality rates resurfaced 2 decades later, in Malaysia in 1997 (2, 13, 16, 43) and Taiwan in 1998 (33, 42). Following these outbreaks, the Asia-Pacific region has experienced more frequent large-scale EV-71-associated HFMD epidemics—most with a high incidence of neurotropic infections and significant case fatality rates—and the virus has attracted global attention (3, 5, 14, 15, 18, 37, 46, 48, 55, 57, 74, 81, 82). Intriguingly, almost all outbreaks reported in the Asia-Pacific region during the last decade were caused by previously undefined EV-71 subgenogroups, raising questions about their origin, genetic complexity, and epidemiological behavior.The icosahedral particles of EV-71, which are structurally similar to those of other members of the Picornaviridae, consist of structural proteins (capsid proteins VP1 to VP4) assembled as pentameric subunits (66). The VP1 protein is highly exposed and usually targeted by host neutralizing antibodies, predisposing the VP1 gene to constant immune selective pressure. This selection may drive the adaptive evolution of the capsid region of many enteroviruses, possibly resulting in amino acid fixations in virus populations (19, 45, 79). Because the VP1 gene of enteroviruses is thought to play an important role in viral pathogenesis and virulence (10, 12, 30), understanding the tempo and mode of evolution of the capsid protein can provide new insights into the epidemiological dynamics of EV-71 that may be useful in predicting the genetic basis and periodicity of future EV-71 epidemics and in facilitating the development of an effective EV-71 vaccine candidate.In this study, we investigated the evolutionary dynamics and genetic history of EV-71. We estimate the dates of emergence of various subgenogroups identified in recent HFMD outbreaks. Using recently developed Bayesian methods of evolutionary analysis, we estimate the divergence time of EV-71 from its closely related ancestor CV-A16, thereby providing a date of origin for EV-71. We also reconstruct the global population dynamics of EV-71 over the past 40 years, revealing temporal trends in genetic diversity within and between major epidemics. Finally, despite finding little evidence of positive selection in the VP1 capsid protein, we observed a pattern of continuous strain and lineage replacement through time, with strong selective pressure detected at several potentially immunogenic sites. The impact of EV-71 evolution on the development of an EV-71 vaccine is also discussed.