BRCA1 mutation screening using restriction endonuclease fingerprinting-single-strand conformation polymorphism in an automated capillary electrophoresis system

Efficient mutation scanning techniques are needed for the rapid detection of novel disease-associated mutations and rare-sequence variants of putative importance. The large size of the breast cancer 1 gene (BRCA1) and the many mutations found throughout its entire coding sequence make screening for mutations in this gene particularly challenging. We have developed a method for screening exon 11 of the BRCA1 gene based on restriction enzyme digestion of fluorescence-labeled polymerase chain reaction (PCR) products followed by single-strand conformation polymorphism (SSCP) using an automated capillary electrophoresis system, denoted capillary restriction endonuclease fingerprinting (REF)-SSCP electrophoresis. Using this strategy on a control set of samples, we were able to detect 17 of 18 known sequence alterations. The method was then applied to screen 73 Norwegian females with family histories of breast and/or ovarian cancer. A total of 172 sequence alterations were detected, including substitutions, insertions, and deletions. One novel substitution of unknown function was identified. Sequencing of all samples negative in the capillary REF-SSCP system gave no additional mutations confirming the high sensitivity of the described methodology. Capillary REF-SSCP electrophoresis appeared as a technically convenient technique, requiring amplification of fewer PCR fragments than traditional SSCP. The novel strategy allows high-throughput mutation scanning without radioactive labeling and polyacrylamide gel electrophoresis (PAGE).     

Assessing calves as carriers of Cryptosporidium and Giardia with zoonotic potential on dairy and beef farms within a water catchment area by mutation scanning

In the present study, we undertook a molecular epidemiological survey of Cryptosporidium and Giardia in calves on three dairy and two beef farms within an open drinking water catchment area (Melbourne, Australia). Faecal samples (n = 474) were collected from calves at two time points (5 months apart) and tested using a PCR‐based mutation scanning‐targeted sequencing phylogenetic approach, employing regions within the genes of small subunit (SSU) of ribosomal RNA (designated partial SSU), 60 kDa glycoprotein (pgp60) and triose phosphate isomerase (ptpi) as genetic markers. Using partial SSU, the C. bovis, C. parvum, C. ryanae and a new genotype of Cryptosporidium were characterised from totals of 74 (15.6%), 35 (7.3%), 37 (7.8%) and 9 (1.9%) samples, respectively. Using pgp60, C. parvum genotype IIa subgenotype A18G3R1 was detected in 29 samples. Using ptpi, G. duodenalis assemblages A and E were detected in totals of 10 (2.1%) and 130 (27.4%) samples, respectively. The present study showed that a considerable proportion of dairy and beef calves in this open water catchment region excreted Cryptosporidium (i.e. subgenotype IIaA18G3R1) and Giardia (e.g. assemblage A) that are consistent with those infecting humans, inferring that they are of zoonotic importance. Future work should focus on exploring, in a temporal and spatial way, whether these parasites occur in the environment and water of the catchment reservoir.     

Restriction of Intramolecular Motion(RIM): Investigating AIE Mechanism from Experimental and Theoretical Studies

Mechanistic studies promote scientific development from phenomena to theories.Aggregation-induced emission(AIE),as an unusual photophysical phenomenon,builds the bridge between molecular science and aggregate mesoscience.With the twenty-year development of AIE,restriction of intramolecular motion(RIM)has been verified as the working mechanism of AIE effect.In this review,these mechanistic works about RIM are summarized from experimental and theoretical perspectives.Thereinto,the experimental studies are introduced from three parts:external rigidification,structural modification and structural characterization.In the theoretical part,calculations on the low-frequency motion of AIEgens have been performed to prove the RIM mechanism.By virtue of the theoretical calculations,some new mechanisms are proposed to supplement the RIM,such as restriction of access to conical intersection,suppression of Kasha transition,restriction of access to dark state,etc.It is foreseeable that the RIM mechanism will unify the photophysical theories for both molecules and aggregates,and inspire more progress in aggregate science.     

Highly sensitive non‐isotopic restriction endonuclease fingerprinting of nucleotide variability in the gp60 gene within Cryptosporidium species,genotypes and subgenotypes infective to humans,and its implications

The high‐resolution analysis of genetic variation has major implications for the identification of parasites and micro‐organisms to species and subspecies as well as for population genetic and epidemiological studies. In this study, we critically assessed the effectiveness of a PCR‐based restriction endonuclease fingerprinting (REF) method for the detection of mutations in the 60 kDa glycoprotein gene (gp60) of Cryptosporidium, a genus of parasitic protists of major human and animal health importance globally. This gene displays substantial intraspecific variability in sequence, particularly in a TCA (perfect and imperfect) microsatellite region, is present as a single copy in the nuclear genome and is used widely as a marker in molecular epidemiological studies of Cryptosporidium hominis and C. parvum, the two predominant species that infect humans. The results of this study demonstrated an exquisite capacity of REF to detect nucleotide variability in the gp60 gene within each of the two species. The differentiation of genotypes/subgenotypes based on REF analysis was supported by targeted sequencing, allowing the detection of levels of variation as low as a single‐nucleotide transversion for amplicons of ∼1 kb in size. The high‐throughput potential and relatively low‐cost of REF make it a particularly useful tool for large‐scale genetic analyses of C. hominis and C. parvum. REF could also be utilized for comparative surveys of genetic variability across large nuclear genomic regions. Such analyses of Cryptosporidium in clinical and environmental samples by REF have important implications for identifying sources of infection, modes of transmission and/or possible infectivity to humans, thus assisting in the surveillance and control of cryptosporidiosis. Given its excellent mutation detection capacity, REF should find broad applicability to various single‐copy genes as well as a wide range of other protozoan and metazoan parasites. (The nucleotide sequences reported in this article are available in the GenBank database under accession numbers GU214343–GU214371).     

An effective sequence characterized amplified region‐PCR method derived from restriction site‐amplified polymorphism for the identification of female Schistosoma japonicum of zoonotic significance

In the present study, restriction site‐amplified polymorphism (RSAP) markers were used to examine the genetic variability of Schistosoma japonicum isolates from different endemic provinces in mainland China. Of the 45 pairs of primers screened, 10 RSAP markers showed a clear banding pattern with good resolution; however, only six exhibited a polymorphism among different isolates. Among six RSAP markers, one pair of primers (R8+R10) was able to differentiate male and female parasites, and amplified one constant specific band for female S. japonicum isolates. The specific band was recovered, re‐amplified and sequenced, and a sequence of 162 bp was obtained. Based on this sequence, a pair of specific primers was designed and used to develop sequence characterized amplified region (SCAR)‐PCR assay for identification and differentiation of female S. japonicum isolates. The SCAR‐PCR assay allowed the specific identification of female S. japonicum, with no amplicons being amplified from male S. japonicum, Fasciola hepatica, Clonorchis sinensis, S. mansoni (male and female parasite). DNA sequencing confirmed the identity of the amplified products. The minimum amount of DNA detectable using SCAR‐PCR assay was 0.3 ng for female S. japonicum. The SCAR‐PCR was able to differentiate effectively the male and female S. japonicum worms collected from 12 geographical origins in eight endemic provinces, the gender of which was known based on the morphological and biological features. These results showed that SCAR‐PCR provides an effective tool for the sex differentiation studies of S. japonicum, identification of female S. japonicum, diagnosis and epidemiological survey of S. japonicum infections in animals and human.     

Geodesic restrictions for the Casimir operator

We consider the hyperbolic Casimir operator C defined on the tangent sphere bundle SY of a compact hyperbolic Riemann surface Y. We prove a non-trivial bound on the L²-norm of the restriction of eigenfunctions of C to certain natural hypersurfaces in SY. The result that we obtain goes beyond known (sharp) local bounds of L. Hörmander.     

α-三Ⅰ约束问题的形式解

提出了α-三Ⅰ约束问题,并在逻辑系统L~*和Luk中给出了这些问题的形式解,建立了这两个逻辑系统中三Ⅰ约束算法的形式推理机制,为α-三Ⅰ约束算法建立了严格的理论基础。     

Highly sensitive restriction enzyme assay and analysis: a review

Biological assays at the single molecule level are crucial to fundamental studies of DNA-protein mechanisms. In order to cater for high throughput applications, one area of immense research potential is single-molecule bioassays where miniaturized devices are developed to perform rapid and effective biological reactions and analyses. With the success of various emerging technologies for engineering miniaturized structures down to the nanoscale level, supported by specialized equipment for detection, many investigations in the field of life science that were once thought impossible can now be actively explored. In this review, the significance of downscaling to the single-molecule level is firstly presented in selected examples, with the focus placed on restriction enzyme assays. To determine the effectiveness of single-molecule restriction enzyme reactions, simple and direct analytical methods based on DNA stretching have often been reliably employed. DNA stretching can be realized based on a number of working principles related to the physical forces exerted on the DNA samples. We then discuss two examples of a nanochannel system and a microchamber system where single-molecule restriction enzyme digestion and DNA stretching have been integrated, which possess prospective capabilities of developing into highly sensitive and high-throughput restriction enzyme assays. Finally, we take a brief look at the general trends in technological development in this field by comparing the advantages and disadvantages of performing assays at bulk, microscale and single-molecule levels. Figure Minaturization of Restriction Enzyme Assays and DNA Stretching     

Restrictions of bounded linear operators: Closed range

Let be a bounded linear operator on a Banach space and let be a subspace of which is a Banach space and invariant. Denote by the restriction of to This paper explores the questions:

If the range of is closed, under what conditions is the range of closed?

If the range of is closed, under what conditions is the range of closed?

     

2[(N-乙基)-1-吩噻嗪基]腙的合成及聚集荧光增强性能

以氮-氮单键连接2个吩噻嗪环构成共轭结构, 合成了2[(N-乙基)-1-吩噻嗪基]腙, 并对其进行了结构表征. 该化合物在四氢呋喃溶剂中呈分散态时无荧光; 在四氢呋喃/水混合溶剂中呈现聚集荧光增强. 荧光增强是由于聚集态中分子内单键旋转受分子堆积效应的阻碍, 氮-氮单键连接的2个吩噻嗪环趋于平面化以及J-聚集体形成的协同作用使非辐射跃迁减少所致. 同时, 利用这种聚集荧光增强性质考察了该化合物对血红蛋白的探针识别性能.