Advances in ß-Diketocyclisation of Curcumin Derivatives and their Antitumor Activity.

Curcumin, derived from the popular spice turmeric, is a pharmacologically active polyphenol. Curcumin's therapeutic activity has been extensively studied in recent decades, with reports implicating curcumin in many biological activities, particularly, its significant anticancer activity. However, its potential as an oral administration product is hampered by poor bioavailability, which is associated with a variety of factors, including low water solubility, poor intestinal permeability, instability, and degradation at alkaline pH. To improve its bioavailability, modifying ß-diketone curcumin with heterocycles, such as pyrazole, isoxazole and triazole is a powerful strategy. Derivatives are synthesized while maintaining the basic skeleton of curcumin. The ß-diketone cyclized curcumin derivatives are regulators of multiple molecular targets, which play vital roles in a variety of cellular pathways. In some literatures, structurally modified curcumin derivatives have been compared with curcumin, and the former has enhanced biological activity, improved water solubility and stability. Therefore, the scope of this review is to report the most recently synthesized heterocyclic derivatives and to classify them according to their chemical structures. Several of the most important and effective compounds are reviewed by introducing different active groups into the ß-diketone position to achieve better therapeutic efficacy and bioavailability.

Expression profile analysis of a new testis-specifically expressed gene C17ORF64 and its association with cell apoptosis in MCF-7 cells.

With the increasing incidence of male infertility, identification and investigation the functions of new genes related to spermatogenesis are effective avenues to elucidate the decline of testicular function. In this study, a new gene, C17ORF64 (chromosome 17 open reading frame 64), was identified from mouse testes and its potential function was studied.RT-PCR and qRT-PCR assay showed that C17ORF64 mRNA was expressed exclusively in mouse testes and up-regulated from the 3-week old to 6-month old testes during postpartum development, which is consistent with C17ORF64 protein expression profile by western blotting analysis. Immunohistochemical analysis revealed that C17ORF64 protein was mainly localized in the cytoplasm of spermatogonia and spermatocytes, which is verified by GFP- labeled C17ORF64 gene expressed in GC-1 cells. C17ORF64 overexpression not only promoted cell apoptosis in MCF-7 cells, but also significantly decreased cell viability via MTT assay. Flow cytometric assay showed that C17ORF64 overexpression could inhibit cell cycle progression by arresting G1/S transition. Western blot and qRT-PCR analysis revealed that C17ORF64 overexpression inhibited the expression of anti-apoptotic protein bcl-2 and increased the expressions of pro-apoptotic protein caspase-3, caspase-8, caspase-9, Bax, P21 and P53. Taken together, our results confirmed C17ORF64 testis-specific expression pattern and, for the first time, demonstrated that C17ORF64 could inhibit cell viability and accelerate apoptosis in MCF-7 cells through caspase-3 regulatory pathways.

The testis-specific expressed gene Spata34 is not required for fertility in mice.

It is estimated that more than two thousand genes exhibit testis-predominant expression pattern. The functions of hundreds of these genes have been explored during mouse spermatogenesis. However, there are still many genes whose relevance to reproduction in vivo remains unexplored. Our previous studies, as well as the other documented study, have indicated that Spata34, an evolutionarily conserved gene in metazoan species, was exclusively expressed in mouse testes and involved in spermatogenesis by regulating cell cycle progression. The present study aims to determine the effect of Spata34 gene knockout on mouse reproduction in vivo by generating a Spata34 gene knockout model using CRISPR/Cas9-mediated genome editing technology. We found that the Spata34 gene KO mice had normal fertility compared with wild type mice, and no overt detectable difference was found in testis/body weight ratios, testicular histology, sperm counts and spermatozoa motility parameters between WT and Spata34 KO mice. Our report indicated that the testis-specific-expressed gene Spata34 was not required for male mouse fertility, which will help to avoid unnecessary expenditures and effort by other researchers.

Cloning of a new testis-enriched gene C4orf22 and its role in cell cycle and apoptosis in mouse spermatogenic cells.

Spermatogenesis is a complicated and dynamic cellular differentiation process mainly regulated by genes, steroid hormones and environmental factors. Although a number of genes involved in spermatogenesis have been identified, there are still a lot of genes underlying spermatogenesis remained unexplained. Here, a novel gene C4orf22, also known as 1700007G11Rik or Cfap299 was identified from mouse testis. C4orf22 protein contains 233 amino acid residues and is highly conserved in metazoan species. C4orf22 mRNA was predominantly expressed in mouse testis and increased from 2-week-old testes to 8-week-old testes during the developing testes by RT-PCR and qRT-PCR. Immunohistochemical analysis indicated that C4orf22 protein was mainly distributed in the cytoplasm of spermatogonia and primary spermatocytes, which was further confirmed by C4orf22-tagged with GFP in the GC-1 and GC-2 cells. Over-expression of pEGFP-C3-C4orf22 significantly inhibited GC-1 cells apoptosis and promoted cell cycle progression with an increase in the cell number of S and G2 phase. Conversely, small interfering RNA (siRNA) silencing C4orf22 in GC-1 cells could cause an increase in the number of apoptosis cells and the cell cycle was arrested at G2/M phase. Western blot analysis and qRT-PCR results showed that C4orf22 over-expression significantly increased the expressions of anti-apoptotic bcl-2 and decreased the expression of caspase-3, caspase-8 and Bax. Our results suggest that C4orf22 may be involved in spermatogenesis, and for the first time, unravels its potential role in regulating cell apoptosis through bcl-2 regulatory pathway in GC-1 cells.

Elevated expression of PD­1 on T cells correlates with disease activity in rheumatoid arthritis.

It is well known that lymphocytes are important in rheumatoid arthritis (RA). Programmed cell death-1 (PD­1) is one of the immunosuppressive costimulatory molecules, which mediates an inhibitory effect. However, its role in RA remains to be fully elucidated. In the present study, the expression levels of PD­1 on T cells in the peripheral blood (PB) and synovial fluid (SF) were determined using flow cytometry. In addition, the expression levels of PD­1 on T cells in the PB and SF of patients with RA were further analyzed to determine correlation with markers of the autoimmune response, inflammation and disease activity in RA. Compared with healthy controls, the expression of PD­1 on T cells in the PB was significantly elevated in patients with RA (P<0.0001). The expression of PD­1 on T cells in the SF of patients with RA was significantly increased, compared with that in the autologous PB (P<0.0001). It was also found that the expression of PD­1 on T cells in the PB of patients with RA was increased significantly in subjects with a high rheumatoid factor titer, high levels of inflammatory markers and a high disease activity score 28 (DAS28). The expression of PD­1 on T cells in the SF of patients with RA was increased significantly in subjects with a high DAS28. These data showed that the expression of PD­1 on T cells was elevated in patients with RA and was correlated with the disease activity.

Genome-wide analysis of microRNA targeting impacted by SNPs in cucumber genome.

BACKGROUND: microRNAs (miRNAs) are endogenous small RNAs that play important regulatory functions in plant development. Genetic variations in miRNAs sequences or their target-binding sites (microRNA-target interaction sites) can alter miRNA targets in animal and human. Whether these single nucleotide polymorphisms (SNPs) in plant are functional have not yet been determined. RESULTS: In this study, we constructed leaf, root, and stem-derived small libraries of cucumber (Cucumis sativus) line 9930 (cultivated China-group cucumber) and C. sativus var. hardwickii (wild India group cucumber). A total of 22 conserved miRNA families, nine less-conserved miRNA families, and 49 cucumber-specific miRNAs were identified in both line 9930 and hardwickii. We employed cucumber resequencing data to perform a genome-wide scan for SNPs in cucumber miRNA-target interaction sites, including miRNA mature sequences and miRNA-target binding sites. As a result, we identified a total of 19 SNPs in mature miRNA sequences and 113 SNPs in miRNA-target binding sites with the potential to affect miRNA-target interactions. Furthermore, we experimentally confirmed that these SNPs produced 14 9930-unique targets mRNAs and 15 hardwickii-unique targets mRNA for cucumber miRNAs. This is the first experimental validation of SNPs in miRNA-target interaction sites affecting miRNA-target binding in plants. CONCLUSIONS: Our results indicate that SNPs can alter miRNA function and produce unique miRNA targets in cultivated and wild cucumbers. Therefore, miRNA-related SNPs may have played important in events that led to the agronomic differences between domestic and wild cucumber.

Functional marker development of miR1511-InDel and allelic diversity within the genus Glycine.

BACKGROUND: Single-stranded non-protein coding small RNAs, 18-25 nucleotides in length, are ubiquitous throughout plants genomes and are involved in post-transcriptional gene regulation. Several types of DNA markers have been reported for the detection of genetic diversity or sequence variation in soybean, one of the most important legume crops in worldwide for seed protein and oil content. Recently, with the available of public genomic databases, there has been a shift from the labor-intensive development of PCR-based markers to sequence-based genotyping and the development of functional markers within genes, often coupled with the use of RNA information. But thus far miRNA-based markers have been only developed in rice and tobacco. Here we report the first functional molecular miRNA marker, miR1511-InDel, in soybean for a specific single copy locus used to assess genetic variation in domesticated soybean (Glycine max [L.] Merr) and its wild progenitor (Glycine soja Sieb. & Zucc.). RESULTS: We genotyped a total of 1,669 accessions of domesticated soybean (G. max) and its wild progenitor G. soja which are native throughout the China and parts of Korea, Japan and Russia. The results indicate that the miR1511 locus is distributed in cultivated soybean and has three alleles in annual wild soybean. Based on this result, we proposed that miR-InDel marker technology can be used to assess genetic variation. The inclusion of geo-reference data with miR1511-InDel marker data corroborated that accessions from the Yellow River basin (Huanghuai) exhibited high genetic diversity which provides more molecular evidence for gene diversity in annual wild soybean and domestication of soybean. CONCLUSIONS: These results provide evidence for the use of RNA marker, miRNA1511-InDel, as a soybean-specific functional maker for the study of genetic diversity, genotyping of germplasm and evolution studies. This is also the first report of functional marker developed from soybean miRNA located within the functional region of pre-miRNA1511.

Qip gene in Fusarium oxysporum is required for normal hyphae morphology and virulence.

Ribonucleic acid (RNA)-silencing mechanisms exist in many eukaryotes to regulate a variety of biological processes. The known molecular components are related to Dicers, Argonautes and RNA-dependent RNA polymerases. Previous biochemical studies have also suggested that Qip, with an exonuclease domain, facilitates the conversion of duplex small interfering RNAs into single strands. In our study, the Qip gene in Fusarium oxysporum was disrupted using homologous recombination technology. The deletion of the Qip gene resulted in a decrease in colony growth rates but increased the number of branches. Additionally, the ΔQip mutant had a reduced pathogenicity in cabbage. Our results show Qip gene in F. oxysporum is required for normal hyphae morphology and virulence. The mutant will be useful for elucidating the relationship between the RNA-silencing mechanism and hyphal growth and development in F. oxysporum.

A high efficiency gene disruption strategy using a positive-negative split selection marker and electroporation for Fusarium oxysporum.

The Fusarium oxysporum species complex consists of fungal pathogens that cause serial vascular wilt disease on more than 100 cultivated species throughout the world. Gene function analysis is rapidly becoming more and more important as the whole-genome sequences of various F. oxysporum strains are being completed. Gene-disruption techniques are a common molecular tool for studying gene function, yet are often a limiting step in gene function identification. In this study we have developed a F. oxysporum high-efficiency gene-disruption strategy based on split-marker homologous recombination cassettes with dual selection and electroporation transformation. The method was efficiently used to delete three RNA-dependent RNA polymerase (RdRP) genes. The gene-disruption cassettes of three genes can be constructed simultaneously within a short time using this technique. The optimal condition for electroporation is 10µF capacitance, 300Ω resistance, 4kV/cm field strength, with 1µg of DNA (gene-disruption cassettes). Under these optimal conditions, we were able to obtain 95 transformants per µg DNA. And after positive-negative selection, the transformants were efficiently screened by PCR, screening efficiency averaged 85%: 90% (RdRP1), 85% (RdRP2) and 77% (RdRP3). This gene-disruption strategy should pave the way for high throughout genetic analysis in F. oxysporum.

[Diagnostic value of anti-alpha-fodrin antibody testing for Sjögren's syndrome: a meta-analysis].

OBJECTIVE: To evaluate the diagnostic accuracy of anti-alpha-fodrin antibody for Sjögren's syndrome (SS). METHODS: Qualified literatures on evaluation of anti-alpha-fodrin antibody in diagnosis of SS in English and Chinese published between January 1997 and December 2007 were retrieved from the Cochrane Library, Medline, Embase, and China National Knowledge Infrastructure (CNKI) databases, etc. Two reviewers independently assessed the methodological quality of each study with the tool QUADAS (quality assessment of diagnostic accuracy studies). Statistical analysis was performed by employing MATLAB, Review Manager 4.2 and Meta-Disc1.4. A meta-analysis of the reported sensitivity and specificity of each study and Summary Receiver Operating Characteristic (SROC) curve was performed. RESULTS: Eighteen literatures were included at last. After testing the heterogeneity of the included articles, proper effect model was selected to calculate the pooled weighted sensitivity and specificity with 95% confidence interval: for anti-alpha-fodrin antibody IgG, the sensitivity was 0.40 [95%CI (0.37-0.43)] and the specificity was 0.82 [95%CI (0.79-0.84)], the area under the curve (AUC) of SROC was 0.8029 (SE=0.0580). Eight studies tested anti-alpha-fodrin antibody IgA, the pooled weighted sensitivity and specificity with 95% confidence interval were 0.34 [95%CI (0.30-0.38)] and 0.83 [95%CI (0.79-0.86)] respectively, the AUC of SROC was 0.6374 (SE=0.1841), the synthesis data all showed heterogeneity. The subgroups were analyzed to identify the sources of heterogeneity according to age, race, assay method, agent source, diagnostic criteria, and country. There was homogeneity among the 4 studies from China, and the 6 studies from Japan, the AUC of SROC were 0.7343 (SE=0.0448) and 0.9273 (SE=0.0394), respectively. CONCLUSION: Diagnostic criteria, agent source, assay method, age, race, and country are the important sources of heterogeneity. Anti-alpha-fodrin antibodies IgG and IgA have relatively low pooled sensitivity and relatively high pooled specificity. Negative anti-alpha-fodrin antibody has not important value in excluding SS, but positive anti-alpha-fodrin antibody may be a useful parameter in clinical diagnosis of SS.

[Diagnostic value of anti-cyclic citrullinated peptide antibody for rheumatoid arthritis: a meta-analysis].

OBJECTIVE: To evaluate the diagnostic value of anti-cyclic citrullinated peptide (CCP) antibody for rheumatoid arthritis. METHODS: Data about RA from January 2000 to December 2005 were retrieved through Cochrane Library, Pubmed database, Excerpta Medica Database (EMBASE), OVID database, and China National Knowledge Infrastructure (CNKI), especially through the Annul of the Rheumatic Disease and relevant gray literatures, by entering the words "cyclic citrullinated peptides", "rheumatoid arthritis", "sensitivity", and "specificity". The inclusion of qualified literatures was based on the criteria for diagnostic research recommended by the Cochrane Methods Group on Screening and Diagnostic Test. Statistical analysis wes performed by employing the softwares of MATLAB and Review Manager 4, 2, and summary receiver operation characteristic (SROC) curve method. RESULTS: Twenty-two articles, 15 in English and 7 in Chinese, were extracted. The reported sensitivity of anti-CCP for the diagnosis of RA ranged from 39.2% to 84.6%, and the reported specificity ranged from 90% to 97.9%. The heterogeneity of the included articles was tested, a proper effect model was selected to calculate the pooled weighted sensitivity and specificity with 95% confidence interval for anti-CCP antibody as 77.3% (63.1%, 89.2%) and 93.85% (85.5%, 98.1%), and the positive and negative likelihood ratios as 12.0 and 0.24 respectively. The area under the curve of SROC was 0.8976, and the Q value was 0.87. The sensitivity of the patients with the duration of illness < 1 year was 43%, significantly lower than that of the patients with a duration of illness > 1 year (70.2%, P < 0.01); and the specificity of the patients with the duration of illness < 1 year was 94.2%, not significantly different from that of the patients with the duration of illness > 1 year (95.2%, P = 0.94). CONCLUSION: With relatively high sensitivity and specificity, anti-CCP antibody may be a useful parameter in the clinical diagnosis pf RA.